xvEPA
   United States
   Environmental Protection
   Agency
   MICROBIAL LABORATORY GUIDANCE MANUAL
   FOR THE FINAL LONG TERM 2 ENHANCED
   SURFACE WATER TREATMENT RULE
   Office of Water (4601M)
   EPA815-R06-006
   February 2006

-------
     U.S. Environmental Protection Agency
            Office of Water (4601M)
        1200 Pennsylvania Avenue NW
            Washington, DC 20460
                EPA815-R-06-006
http://www.epa.gov/safewater/disinfection/lt2/compliance.html
                February 2006
                             Printed on Recycled Paper

-------
                                        Disclaimer

The Standards and Risk Management Division, of the Office of Ground Water and Drinking Water, has
reviewed and approved this guidance for publication. Neither the United States Government nor any of its
employees, contractors, or their employees make any warranty, expressed or implied, or assumes any
legal liability or responsibility for any third party's use of or the results of such use of any information,
apparatus, product, or process discussed in this report, or represents that its use by such party would not
infringe on privately owned rights. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.

Contact for technical inquiries regarding Method 1623 and laboratory approval:

        Carrie Moulton
        U.S. Environmental Protection Agency
        Office of Ground Water and Drinking Water
        Technical Support Center, MCI40
        26 West Martin Luther King Drive
        Cincinnati, OH 45268-1320
        (513)569-7919
        (513)569-7191  (fax)
        mouiton.carriefecpa.gov

Contact for technical inquires regarding sampling and analysis for E. coli:

        Jennifer Best
        U.S. Environmental Protection Agency
        Office of Ground Water and Drinking Water
        Technical Support Center, MC 140
        26 West Martin Luther King Drive
        Cincinnati, OH 45268
        513-569-7012
        513-569-7191 (fax)
        bcst.Jcnnifcrfficpa.gov

All other inquiries should be addressed to:

        Sean Conley
        U.S. Environmental Protection Agency
        Office of Ground Water and Drinking Water
        Mail Code 4607M
        USEPA Headquarters, EPA East
        1200 Pennsylvania Ave., NW
        Washington, DC 20460
        202-564-1781
        202-564-3767 (fax)
        conley.sean@epa.gov

-------
                             TABLE OF  CONTENTS


Section 1: Introduction	1
  1.1      Background	2
  1.2      LT2 Rule Microbial Monitoring Requirements	2
  1.3      Use of Cryptosporidium Data	4
    1.3.1      Cryptosporidium Monitoring Sample Data	4
    1.3.2      Cryptosporidium Matrix Spike Data	5
  1.4      Use of,/?, coll and Turbidity Data	6
Section 2: General Microbial Laboratory Quality Assurance	7
  2.1      Quality Assurance Plans	7
  2.2      Sample Temperature Monitoring	10
Section 3: Guidance for Cryptosporidium Laboratories	13
  3.1      LT2 Rule Cryptosporidium Sample Analysis Requirements	13
       .1     Approved Laboratories	13
       .2     Revised Cryptosporidium Method	13
       .3     Minimum Sample Volume Analysis Requirements	14
       .4     Spiking Suspensions Requirements for Spiked Quality Control Samples	15
       .5     Acceptable Sample Results	15
    3.1.6     Cryptosporidium Oocyst Counts to Report	15
  3.2      Laboratory Quality Assurance Evaluation Program	16
    3.2.1     Application	16
    3.2.2     Personnel Qualifications and Training	17
    3.2.3     Initial Proficiency Testing	18
    3.2.4     On-Site Evaluation	19
    3.2.5     Approval and Ongoing Proficiency Testing	19
    3.2.6     Additional Training	20
    3.2.7     Notifying Utilities of Change in Laboratory Status	20
  3.3      Cryptosporidium Method Quality Control	20
    3.3.1     Cryptosporidium Spiking Materials	22
    3.3.2     Initial Precision and Recovery Test	23
    3.3.3     Method Blank Test	24
    3.3.4     Ongoing Precision and Recovery Test	24
    3.3.5     Ongoing Precision and Recovery Control Charts	25
    3.3.6     Quality Control Batches	26
    3.3.7     Holding Time Requirements	26
    3.3.8     Staining Controls	27
    3.3.9     Examination Preparation	28
    3.3.10    Verification of Analyst Performance	28
    3.3.11    Acceptance Criteria for Receipt of Field Samples	29
    3.3.12    Matrix Spike Samples	29
    3.3.13    QC Guidance for Method Modifications and Use of Multiple Method Variations	31
    3.3.14    Guidance on QC for Different Sample Volumes	34
  3.4      Sample Collection Procedures	34
  3.5      Recordkeeping	36
  3.6      Calculations for EPA Methods 1622/1623	39
    3.6.1     Calculating Oocyst Concentrations	39
    3.6.2     Matrix Spike Recovery Calculations	40
    3.6.3     OPR Sample Calculations	42
  3.7      Electronic Data Reporting	42
    3.7.1     Data Entry/Upload	43
    3.7.2     PWS Data Review	44
                                                  i                                    February 2006

-------
    3.7.3     EPA/State Review	45
  3.8     Data Archiving	45
    3.8.1     HardcopyData	45
    3.8.2     Slides	45
  3.9     Equipment, Supplies, Reagents, and Standards	46
Section 4: Guidance for E. coli Laboratories	47
  4.1      Laboratory Certification Program	48
  4.2     Summary of LT2 Rule E. coli Methods	49
    4.2.1     Most Probable Number (MPN) Methods	49
    4.2.2     Membrane Filtration (MF) Methods	50
  4.3     Quality Assurance/Quality Control for E. coli Analyses	51
    4.3.1     Quality Control Specifications Applicable to LT2 Samples	52
    4.3.2     Quality Control Specifications for E. coli Methods	52
  4.4     Sample Collection Procedures	53
  4.5      Sample Volume and Dilution Guidance	54
    4.5.1     Sample Volume and Dilution Guidance for Multiple-Well Methods	54
    4.5.2     Sample Volume and Dilution Guidance for Multiple-Tube Methods	54
    4.5.3     Sample Volume and Dilution Guidance for Membrane Filtration	54
  4.6     E. coli Data Recording and Calculations	55
    4.6.1     Multiple-Well Data	56
    4.6.2     Membrane Filtration Data	58
    4.6.3     Multiple-Tube Data	62
  4.7     Electronic Data Reporting	65
    4.7.1     Data Entry/Upload	65
    4.7.2     PWS Data Review	66
    4.7.3     EPA/State Review	66
  4.8     Data Archiving	67
Section 5: References	69
                                                                                       February 2006

-------
                                           TABLES
Table 1-1.    Summary of LT2 Rule Monitoring Requirements	3
Table 1-2.    Bin Classifications for Filtered Systems	4
Table 3-1.    Suggested Laboratory QA Program Personnel Qualifications	17
Table 3-2.    Summary of Routine QC Requirements	22
Table 3-3.    Method 1622/1623 Holding Times	27
Table 3-4.    Recommended QC for Different Types of Changes to EPA Method 1622/1623	32
Table 3-5.    Sample Receipt Data Elements to Record in the Laboratory	37
Table 3-6.    Primary Data Elements to Record for Calculations	38
Table 3-7.    Key QC Data Elements to Record	38
Table 3-8.    LT2/Stage 2 Data Collection and Tracking System Data Entry, Review, and Transfer	43
Table 4-1.    Approved E. coli Methods for LT2 Rule	48
Table 4-2.    Incubation Time and Temperature Specifications for MPN Methods	53
Table 4-3.    Incubation Time and Temperature Specifications for Membrane Filter Methods	53
Table 4-4.    Recommended Minimum Record for Quanti-tray 2000® Colilert® and Colilert-18® Analyses	56
Table 4-5.    Recommended Data to Record for Quanti-Tray® Colilert® and Colilert-18® Analyses	57
Table 4-6.    Recommended Data Elements for Record for Membrane Filtration Analyses	59
Table 4-7.    Recommended Data Elements to Record for 15-Tube MPN Methods (Colilert®)	62
Table 4-8.    Recommended Data Elements to Record for 15-Tube Fermentation Methods	63
Table 4-9.    Examples of Different Combinations of Positive Tubes	64
                                          FIGURES
Figure 3-1.    Process for Receiving and Monitoring Laboratory Approval	21
Figure 3-2.    Ongoing Precision and Recovery Control Chart Example	25
                                               in
February 2006

-------
                                  APPENDICES
Appendix A	Laboratory Biosafety Guidelines
Appendix B	EPA Method 1622 for Ctyptosporidium (December 2005)
Appendix C	EPA Method 1623 for Cryptosporidlum and Giardia (December 2005)
Appendix D	Cryptosporidium Sample Results Acceptability Checklist for the LT2 Rule
Appendix E	Cryptosporidium Laboratory QA Program Application
Appendix F	Cryptosporidium Laboratory QA Program Audit Checklist
Appendix G	EPA Method 1622/1623 Bench Sheet
Appendix H	EPA Method 1622/1623 Cryptosporidium Slide Examination Form
                                          iv                                February 2006

-------
                                     ACRONYMS
CPU
CNFG
DAPI
DIC
DCTS
EPA
FA
FITC
GWUDI
ICR
IDC
IESWTR
IFA
IMS
IPR
IPT
L
LT2 Rule
LT2ESWTR
mL
MPC
MPN
MS
MS/MSD
NA-MUG
NELAC
nm
NPDWR
NTU
OPR
OPT
PBMS
PT
PWS
QA
QAP
QC
RSD
SDWA
SOP
TNTC
UV
Colony-forming unit
Confluent growth
4, 6-diamidino-2-phenylindole
Differential interference contrast
LT2/Stage 2 Data Collection and Tracking System
United States Environmental Protection Agency
Fluorescence assay
Fluorescein isothiocyanate
Ground water under the direct influence [of surface water]
Information Collection Rule
Initial demonstration of capability
Interim Enhanced Surface Water Treatment Rule
Immunofluorescence assay
Immunomagnetic separation
Initial precision and recovery
Initial proficiency testing
Liter
Long Term 2 Enhanced Surface Water Treatment Rule
Long Term 2 Enhanced Surface Water Treatment Rule
Milliliter
Magnetic particle concentrator
Most probable number
Matrix spike
Matrix spike/matrix spike duplicate
Micrometer
Nutrient agar (NA) with 4-methylumbelliferyl-beta-D-glucuronide (MUG)
National Environmental Laboratory Accreditation Conference
Nanometer
National Primary Drinking Water Regulations
Nephelometric  turbidity unit
Ongoing precision and recovery
Ongoing proficiency testing
Performance-based measurement system
Proficiency testing
Public water system
Quality assurance
Quality assurance plan
Quality control
Relative standard deviation
Safe Drinking Water Act
Standard operating procedure
Too numerous to count
Ultraviolet
                                                                              February 2006

-------
                                                     SECTION  1:  INTRODUCTION
The Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR or LT2 Rule; Reference 5.1)
requires public water systems (PWSs) that use surface water or groundwater under the direct influence of
surface water to monitor their source water (influent water prior to treatment plant) for Cryptosporidium,
E. coli, and turbidity for a limited period [40 CFR § 141.701 (a) and (c-h)]. In support of the monitoring
requirements specified by the rule, three documents have been developed to provide guidance to the
affected PWSs and the laboratories that support them:

    •  Source Water Monitoring Guidance Manual for Public Water Systems for the Long Term 2
       Enhanced Surface Water Treatment Rule (LT2 Rule). This guidance manual for PWSs affected by
       the rule provides information on laboratory contracting, sample collection procedures, and data
       evaluation and interpretation. This guidance manual also provides information on grandfathering
       requirements for Cryptosporidium and E. coli data.

    •  Microbial Laboratory Guidance Manual for the Long Term 2 Enhanced Surface Water Treatment
       Rule (LT2 Rule) (this document). The goal of this manual is to provide Cryptosporidium and E.
       coli laboratories analyzing samples in support of the LT2 Rule with guidance and detailed
       procedures for all aspects of microbial analyses under the rule to maximize data quality and
       consistency.

    •  Users' Manual for the LT2/Stage 2 Data Collection and  Tracking System (DCTS). This manual
       provides PWSs and laboratories with instructions on the  entry, review, and approval of electronic
       data using the LT2/Stage 2 DCTS, and for generating reports using the system.

All of these manuals, as well as responses to frequently asked questions and an online microscopy module
to assist analysts with identification of Cryptosporidium oocysts, are available at
http://vvww.cpa.gov/safcvvatcr/disinfcction/lt2/compliancc.

This guidance document is provided to help implement the LT2 Rule. This guidance document does not,
however, substitute for the LT2 Rule or  the analytical methods approved for use under the rule. The
material presented is intended solely for guidance and does not alter any regulatory or analytical method
requirements not altered by the LT2 Rule itself.

Sections 1 and 2 of the microbial laboratory LT2 manual provide LT2 background information and
guidance on issues that apply to both Cryptosporidium and E. coli laboratories. Section 3 provides
guidance specific to Cryptosporidium analyses for the LT2 Rule, and Section 4 provides guidance for E.
coli analyses performed in support of the LT2 Rule.
                                                                                 February 2006

-------
                                                                            Section 1: Introduction
1.1    Background

The LT2 Rule is a National Primary Drinking Water Regulation that requires monitoring, reporting, and
public notification for all PWSs that use surface water sources. The rule requires additional treatment
techniques for some systems, based on Cryptosporidium monitoring results and the treatment currently
provided (40 CFR § 141.711 — 141.712). The LT2 Rule was developed to improve control of microbial
pathogens in drinking water, including specifically the protozoan Cryptosporidium, and to address risk
trade-offs between control of microbial pathogens and control of disinfection byproducts.


1.2    LT2 Rule Microbial Monitoring  Requirements

Filtered PWSs serving a population of at least 10,000 people, and some filtered wholesale PWSs serving
populations less than 10,000 people, are required to collect and analyze source water samples for
Cryptosporidium, E. coli and turbidity for a minimum of 2 years [40 CFR § 141.701(a)(l)]. Unfiltered
PWSs serving a population of at least 10,000 people, and some unfiltered wholesale PWSs serving
populations less than 10,000 people, are required to collect and analyze source water samples for
Cryptosporidium for a minimum of 2 years [40  CFR § 141.701(a)(2)]. Small, filtered systems (PWSs that
serve fewer than 10,000 people) are required to monitor their source water for E. coli for a minimum of 1
year. A subset of small filtered systems would then be required to collect and analyze source water
samples for Cryptosporidium over a 1 or 2-year period if they exceed E. coli trigger levels [40 CFR §
141.701(a)(3-4)]. Small, unfiltered systems are  required to monitor their source water for
Cryptosporidium instead of E. coli. [40 CFR  141.701(a)(6)].

Monitoring requirements for each system size, and the schedule for each stage of monitoring, are
described in Table 1-1. Detailed guidance for sample collection during the LT2 Rule, and procedures for
sample collection, documentation, and shipment, are provided in the Source Water Monitoring Guidance
Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2
Rule) (at http://www.epa.gov/safcwatcr/disinfcclion/ll2/compliancc).

Cryptosporidium samples must be analyzed by a laboratory approved for analysis under the Laboratory
Quality Assurance Evaluation Program  for the Analysis of Cryptosporidium in Water (Section 3.2 of this
manual, below) or a laboratory approved for Cryptosporidium analysis by an equivalent State laboratory
certification program. However, at the time of publication, there were no equivalent State programs.
Samples must be analyzed using EPA Method 1622/1623 [40 CFR § 141.704(a) and part 141.705]
(References 5.2 and 5.3).

E. coli samples must be analyzed by certified laboratories using methods approved under the LT2 Rule
for surface water monitoring [40 CFR § 141.704(b)] (See Section 4 of this  manual for details). Under the
LT2 Rule, turbidity samples must be analyzed by a party approved by the State (usually a certified
operator or a professional engineer) using methods approved in 141.74(a)(Reference 5.4)1)[40 CFR
§141.704(c)].
                                                                                 February 2006

-------
                                                                                  Section 1: Introduction
Table 1-1.      Summary of LT2 Rule Monitoring Requirements (Initial Round of Monitoring)
SCHEDULE
SCHEDULE 1:
Large systems
serving > 100,000 *
SCHEDULE 2:
Large systems
serving >50,000
and <100,000 *
SCHEDULE 3:
Large systems
serving >1 0,000
and<50,000*
SCHEDULE 4:
Small filtered
systems (serving
fewer than
10,000")
Small unfiltered
systems (serving
fewer than 10,000)
and
§ Small filtered
systems exceeding
E. coli trigger
levels d or that
elect to proceed
directly to
Cryptosporidium
monitoring or that
fail to conduct E.
coli monitoring
Monitoring
begins
October 1,
2006
April 1, 2007
April 1, 2008
October 1,
2008
April 1,20 10
Monitoring
duration
2 years a
2 years a
2 years a
1 year a'd
Monitoring parameters and sample frequency
requirements
Cryptosporidium
Minimum
1 sample/month b
Minimum
1 sample/month b
Minimum
1 sample/month "
See next row ^
1 year b, 2 Cryptosporidium
samples per month,
or
2 years8, 1 Cryptosporidium
sample per month
E. coli
Minimum
1 sample/month °
Minimum
1 sample/month c
Minimum
1 sample/month °
Every two weeks
N/A
Turbidity
Minimum
1 sample/month °
Minimum
1 sample/month c
Minimum
1 sample/month °
N/A
N/A
* Wholesale systems must comply with the requirements based on the population of the largest system in
the combined distribution system [40 CFR § 141.700(b)(1)]
  PWSs may be eligible to use (grandfather) data collected prior to the applicable monitoring start date if certain
  requirements are met [40 CFR § 141.707(a)(1)]
b PWSs monitoring for Cryptosporidium may collect more than one sample per month if sampling is evenly spaced
  over the monitoring period [40 CFR § 141.701(a)(7)]
c Unfiltered systems serving > 10,000 are not required to perform E. coli monitoring or turbidity but to conduct source
  water monitoring that includes only Cryptosporidium sampling [40 CFR § 141.701(a)(2)J
d Filtered systems serving fewer than 10,000 people must monitor for Cryptosporidium only if their E. coli annual
  mean concentrations is greater than 10 £. co///100 mL for systems using lakes/reservoirs or is greater than 50 E.
  colinOO mL for systems using flowing streams [40 CFR § 141.701 (a)(4)]
e Small systems collecting one sample per month for 2 years are still required, where applicable, to meet the
  treatment technique implementation deadlines in 40 CFR § 141.713 (c). The same treatment compliance dates
  apply to the PWS regardless of which Cryptosporidium sampling frequency is used (i.e., selecting the 2 year
  Cryptosporidium sampling frequency does not extend Cryptosporidium treatment compliance deadlines).
N/A = Not applicable. No monitoring required.
                                                                                        February 2006

-------
                                                                           Section 1: Introduction
1.3    Use of Cryptosporidium Data

Two types of Cryptosporidium data are collected under the LT2 Rule: Cryplosporidium occurrence data
from the analysis of field samples, and method performance data from the analysis of matrix spike (MS)
samples. The use of occurrence data from field samples is discussed in Section 1.3.1; the use of method
performance data from MS samples is discussed in Section 1.3.2.
1.3.1
Cryptosporidium Monitoring Sample Data
The concentration of Cryptosporidium oocysts in source water samples analyzed during the LT2 Rule will
be used to calculate a mean Cryptosporidium concentration for a PWS and classify the PWSs into a
treatment requirements "bin" (40 CFR § 141.710). These bin classifications are provided in Table 1-2.
The treatment bin classification established for each PWS will be used to determine whether additional
treatment is needed. PWSs in Bin 1 are not required to implement additional treatment. PWSs in Bins 2 -
4 will be required to implement increasing levels of treatment and source water protection to address their
higher risk for high Cryptosporidium source water concentrations.
Table 1-2.
Bin Classifications for Filtered Systems
Average Sample Concentration
Cryptosporidium <0.075 oocyst/L or no monitoring
requirement
0.075 oocysts/L Cryptosporidium < 1 .0 oocysts/L
1 .0 oocysts/L Cryptosporidium < 3.0 oocysts/L
Cryptosporidium, 3.0 oocysts/L
PWSs that serve fewer than 10,000 people and NOT
required to monitor for Cryptosporidium
Bin classification
Bin 1
Bin 2
Bin 3
Bin 4
Bin 1
The method used to average individual sample concentrations to determine a PWS's bin classification
depends on the number of samples collected and the length of the sampling period.
For a filtered PWS monitoring for Cryptosporidium, bin classification would be based on the following
calculations of bin concentration:

    •   For PWSs that collect at least 48 samples during a two year monitoring period, the
       Cryptosporidium bin concentration is equal to the arithmetic mean of all sample concentrations
       [40 CFR § 141.710(b)(l)]

    •   For PWSs that collect at least 24 samples, but not more than 47 samples, during a two year
       monitoring period, the Cryptosporidium bin concentration is equal to the highest arithmetic mean
       (average) of all sample concentrations in any 12 consecutive months in the monitoring period [40
       CFR§ 141.710(b)(2)]

    •   For PWSs serving fewer than 10,000 people and monitoring Cryptosporidium for only one year
       (i.e., collect 24 samples in 12 months), the bin concentration is based on the arithmetic mean of
       all sample concentrations [40 CFR § 141.710(b)(3)]
                                                                                 February 2006

-------
                                                                           Section 1: Introduction
    •  For systems with plants operating only part of the year that monitor fewer than 12 months per
       year, the bin concentration is based on the highest arithmetic mean of all sample concentrations
       during any year of Cryptosporidium monitoring [40 CFR § 141.710(b)(4)]

    •  For  filtered PWSs in which sampling frequency varies, systems must first calculate a
       monthly average concentration for each month of monitoring. Systems must then use
       these monthly average concentrations, rather than individual sample concentrations, in
       the calculation of bin concentration as described in the four examples above [40 CFR §
       141.710(b)(5)]

Unfiltered systems must calculate the arithmetic mean of all Cryptosporidium samples concentrations [40
CFR § 141.712(a)(l)]. For unfiltered systems in which sampling frequency varies, systems must first
calculate  a monthly average for each month of monitoring. Systems must then use these monthly average
concentrations, rather than individual sample concentrations, in  the calculation of the mean
Cryptosporidium level [40 CFR § 141.712(a)(3)]

In all cases, the bin concentration is calculated using individual  sample concentrations. These
concentrations are calculated as "number of oocysts detected / volume (in L) analyzed." Individual
sample concentrations are not calculated as "oocysts detected / 10 L," nor are bin concentrations
calculated as the "sum of the oocysts detected / the sum of the volumes analyzed." As a result, each
sample has an equal weight on the final bin concentration. In cases where no oocysts are detected, the
number of oocysts used to calculate the sample concentration is "0." There should be no adjustment of
the number of oocysts detected based on recovery or oocyst viability.

1.3.2         Cryptosporidium  Matrix Spike Data
During LT2  Rule Cryptosporidium monitoring, PWSs are required to analyze, at a minimum, one MS
sample for every 20 field samples from their source water [Section 9.1.8 of Method 1622/1623]. For all
PWSs, the first MS sample should be collected and analyzed during the first sampling event under the
monitoring program per Section 9.1.8 in EPA Method 1622.1623. Details on MS sample requirements are
provided  in Section 3.3.12.

Based on this requirement, the following PWS categories must analyze at least two MS samples during
LT2 Rule monitoring:

    •  Large PWSs that perform monthly monitoring for two years (resulting in 24 samples)
    •  Small PWSs that are triggered  into Cryptosporidium monitoring and collect semi-monthly
       samples for one year or monthly samples for two years (resulting in 24 samples)

For large PWSs that perform semi-monthly or more frequent monitoring for two years (resulting in 48 or
more samples), a minimum of three MS samples should be collected and analyzed. If a PWS monitors
more frequently or collects more than 60 samples, a minimum of four MS samples should be analyzed.

Although MS sample results will not be used to adjust Cryptosporidium recoveries at any individual
source water, the results will be used collectively to assess overall recovery and variability for EPA
Method 1622/1623 in source water.

                                             5                                  February 2006

-------
                                                                            Section I: Introduction
1.4    Use of E. coli and Turbidity Data

E. coli and turbidity data are being collected by PWSs on Schedules 1 -3  during LT2 Rule monitoring to
confirm or refine the levels of E. coli that would indicate high Cryptosporidium concentrations in a source
water.

Small, filtered systems are permitted to monitor for E. coli, rather than more expensive Cryptosporidium
analyses. Only those small filtered systems with E. coli levels that exceed the trigger level are required to
monitor for Cryptosporidium to determine bin placement [40 CFR § 141.701  (a)(4)]. Based on the data
from the Information Collection Rule (ICR) and Information Collection Rule Supplemental Survey
(1CRSS), the following E. coli trigger levels were set:

    •   A mean of 50 E. colil\00 mL for flowing stream-type source waters

    •   A mean of 10 E. coli/]00 mL for reservoir/lake source waters

Small filtered PWSs that are monitoring for E. coli must collect at least one sample twice per month.
Individual sample concentrations are then averaged to determine a mean E. coli level. A PWS's mean E.
coli level is compared to the E. coli trigger levels above to determine if the PWS is required to monitor
for Cryptosporidium.

The indicator data from large PWS monitoring will be reviewed and, if appropriate,  guidance on alternate
indicator trigger values will be issued to States prior to when small PWSs begin monitoring. States are
allowed to approve alternative approaches to indicator monitoring for small PWSs [40 CFR §
                                                                                 February 2006

-------
                                          SECTION 2: GENERAL MICROBIAL
                                      LABORATORY QUALITY ASSURANCE
All laboratories analyzing Cryptosporidium and E. coli samples for the LT2 Rule should adhere to
defined quality assurance (QA) procedures to ensure that analytical data generated under the rule are
scientifically valid and are of known and acceptable quality. Detailed quality control (QC) requirements
and recommendations specific to Cryptosporidium and E. coli analyses are discussed in Sections 3 and 4
of this manual, respectively. Two QA issues that apply to both analyses—quality assurance plans and
sample temperature monitoring—are discussed below, in Sections 2.1 and 2.2.
2.1    Quality Assurance Plans

As specified in both the Lab QA Program for Cryptosporidium laboratories
(http://ww\v. cpa.gov/safewatcr/disinfection/lt2/. Section 3.2 of this manual, Reference 5.5) and the
Laboratory Certification Manual (Chapter III, page III-4, Reference 5.6) for E. coli laboratories, each
laboratory should operate a formal QA program and document the scope of this program through a QA
plan.

The laboratory's QA plan should be a stand-alone document. However, some information can be
incorporated into the document by reference, including laboratory standard operating procedures (SOPs),
analytical methods, and quality control (QC) and calibration notebooks. Laboratories currently certified
for coliform analysis under the drinking water laboratory certification program may use their current QA
plan; however, this plan  should be updated to address the specific requirements for LT2 Rule monitoring.
Topics that should be addressed in the QA plan are outlined below. Details on LT2 Cryptosporidium
requirements are provided in Section 3 of this manual; details on LT2 E. coli analyses are provided in
Section 4.

For Cryptosporidium laboratories, this QA plan should be available for review during a laboratory's on-
site audit, as part of the EPA's Cryptosporidium Laboratory Quality Assurance Evaluation Program
(Section 3.2 of this manual). For E. coli laboratories, this QA plan should be available for review during
recertification audits as part of the National Primary Drinking Water Regulations. If the laboratory also
performs turbidity testing, then the QA plan should address turbidity requirements.

The following items should be addressed in each QA plan:

1.  Laboratory organization and responsibility
   •   Include a chart showing the laboratory organization and line authority, including QA Managers
   •   List the key individuals who are responsible for ensuring the production of valid measurements
       and the routine assessment of QC measurements
   •   Specify who is responsible for internal audits and reviews of the implementation of the QA plan
       and its requirements

2.  Personnel
   •   List each analyst's academic background and water analysis experience
   •   List each analyst's training on the method
   •   Describe training available to keep personnel up to date on methods and regulations

                                            7                                  February 2006

-------
                                               Section 2: General Microbial Laboratory Quality Assurance
3.  Facilities
    •   Arrangement and size of laboratory
    •   Bench space
    •   Storage space
    •   Lighting
    •   Air system
    •   Lab reagent water system
    •   Waste disposal system
    •   Safety considerations. The laboratory should address biosafety in the laboratory when handling or
        processing Cryptosporidium samples and organism controls. Guidance on laboratory biosafety for
        Cryptosporidium is provided in Appendix A of this guidance manual.

4.  Field sampling procedures (with SOP used by laboratory or sent to PWS clients)
    •   Describe how samples are collected, including sample containers, sample storage, transport times,
        and  sample temperature
    •   Describe sample identification and information recording system

5.  Laboratory sample handling procedures
    •   Describe sample storage conditions
    •   Describe the laboratory's sample tracking system; specify procedures used to maintain the
        integrity of all samples, i.e., logging, tracking samples from receipt by laboratory through
        analysis to disposal
    •   Describe sample acceptance criteria

6.      Equipment
    •   Specifications for each piece of equipment used for Cryptosporidium, E. coli analyses, and/or
        turbidity
    •   Calibration procedures, frequency, standards for each piece of equipment used for
        Cryptosporidium, E. coli analyses, and/or turbidity
    •   Quality control records for each piece of equipment used for Cryptosporidium, E. coli analyses,
        and/or turbidity
    •   Preventative maintenance  and schedules,  documentation for each piece of equipment used for
        Cryptosporidium, E. coli analyses, and/or turbidity

7.  Supplies
    •   Laboratory glassware and  plastic ware acceptance conditions
    •   Chemicals, reagents, dyes and culture media acceptance conditions
    •   Chemicals, reagents, dyes, and culture media storage conditions
    •   Filters acceptance conditions
    •   Description of the laboratory's reagent tracking system

8.  Laboratory practices (may reference SOP)
    •   Preparation of reagent-grade water
    •   Glassware washing and preparation
    •   Sterilization procedures


                                              8                                    February 2006

-------
                                               Section 2: General Microbial Laboratory Quality Assurance
9.  Analytical procedures
    •   Describe all reference methods used
    •   State that the analytical methods described in this manual will be followed
    •   Identify available SOPs

10. Quality control (QC) checks
    •   Confirmation/ verification procedures, frequency
    •   Sterility controls
    •   Replicate analyses; frequency
    •   QC samples, source; frequency
    •   Positive and negative controls, proficiency testing (PT) samples, source; frequency
    •   Spiked field samples
    •   Between-analyst deviation

11. Data reduction, verification, validation, and reporting
    •   Data reduction (conversion of raw data to Cryptosporidium oocysts/L and/or E. co///100 mL)
    •   Procedures to ensure the accuracy of data transcription and calculations
    •   Validation (ensuring that QC steps associated with a field result are acceptable)
    •   Reporting, including procedures and format for reporting data to utilities/EPA

12. Corrective actions
    •   Define the laboratory response to unacceptable results from PT or QC samples and from internal
        QC checks
    •   Identify persons with responsibility to take corrective action
    •   Describe how the actions taken and the effectiveness of the actions taken will be documented

13. Recordkeeping
    •   Describe how records are to be maintained (e.g. electronically, hard copy, etc.)
    •   Describe length of time records are to be kept (see archive requirements, Sections 3.8 and 4.8 of
        this manual)
    •   State where records are to be stored

The laboratory QA plan should be concise, but responsive to the above listed items. Additional guidance
on developing QA plans is available in "Guidance on Quality Assurance Project Plans (QAPP) (G-5),"
(EPA/240/R-02/009, December 2002), which is available as a download from
http://www.epa.uov/quality/qa_docs.ritrnl#noncparqt.

However, the goals of a lab QA plan in general are different from the goals of the Guidance on QAPP,
and not all of the issues that should be addressed for laboratory QA during the LT2 Rule are covered by
this guidance (i.e., laboratory sample handling and record keeping). However, some of the concepts
presented in the QAPP guidance that typically are not included in laboratory QA plans may aid the
laboratory in updating their QA plan to address specific LT2 requirements.
                                                                                   February 2006

-------
                                             Section 2: General Microbial Laboratory Quality Assurance
2.2   Sample Temperature Monitoring

Ctyptosporidium oocysts present in a sample can degrade and E. coli present in a sample can grow or die
off, biasing analytical results. Cryptosporidium and E. coli samples for LT2 Rule monitoring are stored
and maintained between 1 °C and 10°C to reduce biological activity. This is specified in Section 8.0 of
the December 2005 versions of EPA Method 1622/1623 for Cryptosporidium samples and at 40 CFR §
704(b)(3) and Chapter V, Section 6.3, of the Laboratory Certification Manual (Reference 5.6) for E. coli
samples.

Samples for all analyses should remain above freezing at all times. This is a requirement in Section 8.0 of
the December 2005 versions of EPA Method 1622/1623.  Although not a significant concern for 10 L
water samples, this is a greater concern for Cryptosporidium filters and 120 or 250 mL E. coli samples
that are shipped off-site with coolant materials, such as wet ice, blue ice, or gel packs. The sample
collection protocols discussed in the Source Water Monitoring Guidance Manual for Public Water
Systems for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule) provide  sample
packing procedures for E.  coli and Cryptosporidium samples. Utility personnel should follow these
procedures to ensure that samples remain at acceptable temperatures during shipment.

It is recommended that utilities practice the collection, packing, and shipping protocols prior to
monitoring to ensure acceptable temperatures upon receipt at the laboratory.

Because Cryptosporidium samples collected for the LT2 Rule must meet the QC criteria in the methods
[40 CFR  § 704 (a)], and because these QC criteria include receipt of samples at <20°C and not  frozen,
laboratories must reject  LT2 Cryptosporidium  samples that are received at >20°C or frozen unless  the
sample was collected the same day  it was received. This is discussed further in Section 3.3.11 in this
manual. In these cases, the PWS must re-collect and re-ship the sample.

E. coli samples that are received at >10°C or frozen, or E. coli samples that the laboratory has determined
exceeded 10°C or froze during shipment, must be rejected. After receipt, E. coli samples must be stored
at the laboratory between 0°C and 10°C, and not frozen, until processed [40 CFR § 141.704(b)(3)].

Several options are available to measure sample temperature upon receipt at the laboratory and, in some
cases, during shipment:

   •   Temperature sample. One option, for Cryptosporidium filtered samples (not for 10 L bulk
       samples) and E. coli 120 and 250 mL samples, is for the PWS to fill a small, inexpensive sample
       bottle with water and pack this "temperature sample" next to the field sample. The  temperature of
       this extra sample volume is measured upon receipt to estimate the temperature of the field
       sample. Temperature sample bottles are not appropriate for use with 10 L bulk samples  because
       of the potential effect that the difference in sample volume may have in temperature equilibration
       in the sample cooler. Example product: Cote Parmer cat. no. C-06252-20 or equivalent.

   •   Temperature vial. A similar option is to use a thermometer that is securely housed in a liquid-
       filled vial. Temperature vials are not appropriate for use with 10 L bulk samples for the reasons
       stated above. Unlike temperature samples, the laboratory does not need to perform an additional
       step to monitor the temperature of the vial upon receipt, but instead just reads the thermometer.
       Example product:  Eagle-Picher Sentry Temperature Vial 3TR-40CS-F or 3TR-40CS or
       equivalent.
                                             10                                  February 2006

-------
                                             Section 2: General Microbial Laboratory Quality Assurance
    •   iButton. Another option for measuring the sample temperature during shipment and upon receipt
       is a Thermocron® iButton. An iButton is a small, waterproof device that contains a computer
       chip to record temperature at different time intervals. The information is then downloaded from
       the iButton onto a computer. The iButton should be placed in a temperature sample in the cooler,
       rather than placed directly in the cooler, where it may be affected by close contact with the
       coolant. Again, this option is not appropriate for use with 10 L bulk samples. Example product:
       Thermocron® iButtons or equivalent.

       Stick-on temperature strips. Another option is for the laboratory to apply a stick-on temperature
       strip to the outside of the sample container upon receipt at the laboratory. This option does not
       measure temperature as precisely as the other options, but provides an indication of sample
       temperature to verify that the sample temperature is acceptable. This option is appropriate for use
       with both 10 L bulk samples and field-filtered samples, but not for use with E. coli samples.
       Example product: Cole Farmer cat. no. C-90316-00.

       Infrared thermometers. Another option is for the laboratory to measure the temperature of the
       surface of the sample container or filter using an infrared thermometer.  The thermometer is
       pointed at the sample, and measures the temperature without coming in contact with the sample
       volume. This option is appropriate for use with both 10 L bulk samples and field-filtered samples.
       Example product: Cole Farmer cat. no. EW-35625-10.

As with other laboratory equipment, all temperature measurement devices should be calibrated routinely
to ensure accurate measurements. See the EPA Lab Certification Manual (Reference 5.6) for more
information.

All temperature readings should be documented and archived as required by the analytical methods.
                                              11                                   February 2006

-------
This page intentionally left blank
             12                                   February 2006

-------
                      SECTION 3: GUIDANCE FOR CRYPTOSPORIDIUM
                                                                   LABORATORIES
Cryptosporidium analyses conducted in support of the LT2 Rule must be performed using EPA Method
1622 or EPA Method 1623 [40 CFR § 141.704(a)]. Guidance on the use of these methods during the LT2
Rule is provided in this section of the manual.
3.1    LT2 Rule Cryptosporidium Sample Analysis Requirements

LT2 Rule requirements of particular significance are summarized in Sections 3.1.1 through 3.1.6, below,
and discussed in more detail along with guidance in the remainder of Section 3.

3.1.1         Approved Laboratories

Systems must have Cryptosporidium samples analyzed by a laboratory that has passed a quality assurance
(QA) evaluation under EPA's Laboratory Quality Assurance Evaluation Program for Analysis of
Cryptosporidium in Water (Reference 5.5) or a laboratory that has been approved for Cryptosporidium
analysis by an equivalent State laboratory approval program [40 CFR § 141.705(a)]. However,  at the
time of publication of this guidance document there were no equivalent State programs for approval of
Cryptosporidium laboratories. Details on the elements of the Lab QA Program QA evaluation are
provided in Section 3.2. It is suggested that each approved laboratory establish and maintain a
relationship with another approved laboratory and develop a protocol to provide back-up analyses to
clients if needed during LT2. A list of approved laboratories is posted at
http://www.cpa.gov/safcwatcr/disinfcction/lt2/.

3.1.2         Revised Cryptosporidium Method

The LT2 Rule requires that samples collected under the rule be analyzed using the December 2005
version of Methods 1622/1623. EPA proposed the use of the April 2001 versions of EPA Methods
1622/1623 in the LT2 Rule. However EPA requested and received comments on the use of updated
versions to consolidate several method-related changes EPA believes are necessary to address LT2 Rule
monitoring requirements. Methods 1622/1623 were revised and the December 2005 version of these
methods (References 5.2 and 5.3) are included as Appendix B and Appendix C of this guidance manual.
These changes include the following:

   •   Increased flexibility in matrix spike (MS) and initial precision and recovery (IPR) requirements.
       The requirement that the laboratory must analyze an MS  sample on the first sampling event for a
       new PWS has been changed to a recommendation; the revised method allows the IPR test to be
       performed across four different days, rather than restrict analyses to 1 day.

   •   Clarification of some method procedures, including the spiking suspension vortexing procedure;
       the buffer volumes used during immunomagnetic separation (IMS); requiring (rather than
       recommending) that laboratories purchase HC1 and NaOH standards at the normality specified in
       the method; and the use, or not, of methanol during slide staining in Section 14.2 of Method
       1622/1623  is per manufacturer's instructions.
                                           13                                February 2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
    •   Addition of recommendations for minimizing carry-over of debris onto microscope slides after
        IMS and information on microscope cleaning. Clarification of the actions to take in the event of
        QC failures.
    •   A change in the sample receipt temperature requirements to "<20°C, and not frozen," and
        additional guidance on sample storage (between 1 °C and 10°C) and shipping procedures based
        on time of day of collection. The revision includes suggested options for monitoring sample
        temperature during shipment and/or upon receipt at the laboratory.
    •   Addition of the requirement for examination using differential interference contrast (DIC)
        microscopy to the analyst verification procedure.

    •   Addition of an approved method modification using the Pall Gelman Envirochek™ HV filter.
        This approval is based on an interlaboratory validation study demonstrating that three
        laboratories, each analyzing reagent water and a different source water, met all method
        acceptance criteria for Cryptosporidium (but not Giardict; however, individual  laboratories are
        permitted to demonstrate acceptable performance for Giardia in their laboratory).

    •   Incorporation of detailed procedures for concentrating samples using an IDEXX Filta-Max®
        foam filter. (A method modification using this filter was already approved by EPA in the April
        2001  version of the methods.)
    •   Addition of BTF EasySeed™ irradiated oocysts and cysts as acceptable materials for spiking
        routine QC samples. EPA approved the use of EasySeed™ based on side-by-side comparison
        tests of method recoveries using EasySeed™ and live, untreated organisms.

    •   Removal of the Whatman Nuclepore CrypTest™ cartridge filter. Although a method modification
        using this filter was approved by EPA in the April 2001 versions of the methods, the filter is no
        longer available from the manufacturer, and so is no longer an option for sample filtration.
    •   Addition of BTF EasyStain™ monoclonal antibody stain as an acceptable reagent for staining in
        Methods 1622/1623.  The product was validated through an interlaboratory  validation study using
        the Pall Envirochek™ HV filter.
    •   Addition of portable continuous-flow centrifugation (PCFC) as a filtration/concentration
        technique for the detection of Cryptosporidium in Methods 1622/1623. The product met all
        method acceptance criteria for Cryptosporidium using 50 L source water samples (but not
        Giardia; however, individual laboratories are permitted to demonstrate acceptable performance
        for Giardia in their laboratory).


3.1.3          Minimum Sample Volume  Analysis Requirements
Under LT2 Rule Cryptosporidium sample volume requirements [40 CFR § 141.704(a)(l)], PWSs are
required to analyze, at a minimum, either

    •   10 L of sample, or
    •   2 mL of packed pellet volume, or

    •   As much volume as two filters can accommodate before clogging (this condition  applies only to
        filters that have been approved by EPA  for nationwide use with EPA Method 1622/1623—the
        Pall Gelman Envirochek™ and Envirochek™ HV filters, or the IDEXX Filta-Max® foam filter)

                                             14                                  February 2006

-------
                                              Section 3: Guidance for Cryptosporidium Laboratories
The LT2 Rule sample volume analysis requirement of 10 L (rather than 10.0 or 10.00 L) accommodates
the potential for imprecisely filled sample containers or filters. Therefore, sample volumes of 9.5 L and
higher would meet the LT Rule requirements. Sample volumes should be recorded to the nearest 0.25 L
when using a graduated carboy instead of a flow meter to measure volume.

Systems may analyze larger volumes than  10 L, and larger volumes analyzed should increase analytical
sensitivity, provided method performance is acceptable. EPA prefers systems to analyze similar sample
volumes throughout the monitoring period. However, data sets including different sample volumes will be
accepted, provided the system analyzes the minimum sample volume requirements noted above.

Matrix spike samples must be collected from the same location as the field sample and the volume
analyzed must be within 10 percent of the volume analyzed for the field sample (Section 9.5 of Method
1622/1623). It is suggested that the same volume of sample be collected for both the field and the matrix
spike sample to ensure the volumes analyzed are within 10 percent. Additional guidance on matrix spike
samples is provided in Section 3.3.12 of this manual.

Additional guidance on sample volume and sample collection issues is provided in the Source Water
Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water
Treatment Rule (LT2 Rule), available for download from http://www.cpa.gov/safewatcr/disinfcction/lt2/

3.1.4          Spiking Suspensions Requirements for Spiked  Quality Control
               Samples
Flow cytometer-counted spiking suspensions must be used for ongoing precision and recovery (OPR) and
matrix spike (MS) samples [40 CFR § 141.704(a)(3)]. The use of flow cytometer-counted spiking
suspensions is a recommendation in EPA Method 1622/1623, and is a requirement in the LT2 Rule.
Spiking suspensions are discussed in more detail in Section 3.3.1, below.

3.1.5          Acceptable Sample Results
Cryptosporidium sample results reported under the LT2 Rule must be generated at an approved laboratory
and meet the quality control (QC) requirements specified in EPA Method 1622/1623. These requirements
include, but are not limited to, sample temperature requirements, minimum frequencies for ongoing
precision and recovery (OPR), method blank, and matrix spike samples;  acceptable OPR and method
blank results; holding time requirements; and staining control frequency  and results. A checklist for these
requirements is provided as Appendix D. Guidance on implementing Cryptosporidium method QC
requirements is provided in Section 3.3, below.

3.1.6          Cryptosporidium Oocyst Counts to Report
Sample examination using EPA Method 1622/1623 includes an immunofluorescence assay using
fluorescein isothiocyanate (FITC) as the primary antibody stain, 4',6-diamidino-2-phenylindole (DAPI)
staining to detect nuclei, and differential interference contrast microscopy (DIG) to detect internal
structures. Hoffman Modulation Contrast (HMC) optics may be a suitable alternative to DIC, provided
that the laboratory demonstrates acceptable analyst performance and acceptable microscope capability to
the technical auditor during the on-site laboratory audit conducted prior to laboratory approval through
the Lab QA Program. Cryptosporidium oocysts to be reported using Section  15 of EPA Method
1622/1623 are defined as the following:

    •  Those determined by brilliant apple green fluorescence under UV light, size (4 to 6 /^m), and
       shape (round to oval)


                                             15                                 February 2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
    •   Excluding any atypical organisms detected by FITC, DAPI, and DIG (e.g., those possessing
        spikes, stalks, appendages, pores, one or two large nuclei filling the cell, red fluorescing
        chloroplasts, crystals, spores, etc.)

The oocyst counts for a sample, based on the above definition and appropriate magnification and
examination procedures per Method 1622/1623, and the sample volume analyzed, will be used to
calculate the oocyst concentration for each sample during the LT2 Rule.

In a field sample, all organisms that meet the above definition must be counted and assigned to the
appropriate categories.  This reporting requirement cannot be met unless all Cryptosporidium-like
organisms in a field sample are examined first under the FITC filter, then under the DAPI filter, and
finally using DIG. If no organisms meet the above definition, the lab should report zero oocysts. The
number listed in the summary row, "Total FA number", on the Cryptosporidium examination results form
and the volume examined (L) should be the minimum information reported to the utility (see Section 3.7
of this manual for details regarding the LT2/Stagc 2 Data Collection and Tracking System).
3.2    Laboratory Quality Assurance Evaluation  Program

The objectives of the Lab QA Program are to evaluate laboratories' competency to reliably measure for
the occurrence of Cryptosporidium in surface water using EPA Method 1622/1623. Each laboratory
participating in the program will be required to complete the following steps to be qualified through this
program:

    •   Complete an application (including a self-evaluation and initial demonstration of capability)
    •   Perform initial proficiency testing (IPT)
    •   Participate in an on-site evaluation
    •   Perform ongoing proficiency testing (OPT) every four months

Information on the Laboratory QA Program is available at http://www.epa.gov/safewater/disinfection/lt2/
and is summarized below, in Sections 3.2.1 - 3.2.7.

3.2.1         Application
Applications for the program (Appendix E) are available on the website,
http://wwvv.cpa.gov/safewater/disinfcction/lt2/, and may also be requested from the following address:

        Cryptosporidium Laboratory Quality Assurance Coordinator
        c/o CSC Water Programs
        6101 Stevenson Avenue
        Alexandria, VA 22304

EPA reviews each application to verify that the laboratory has submitted the following information:

    •    A completed self-evaluation checklist
    •    Resumes of laboratory personnel
    •    Standard operating procedures for each method version
                                             16                                  February 2006

-------
                                                Section 3: Guidance for Cryptosporidium Laboratories
    •   Initial demonstration of capability (IDC) data, which consist of the following:
           Acceptable initial precision and recovery (IPR) test results
           Acceptable method blank result analyzed with IPR test
           Acceptable matrix spike/matrix spike duplicate (MS/MSD) results with results from the
           unspiked matrix sample collected and analyzed at the same time
    •   Table of contents from the laboratory's quality assurance plan
    •   Documentation of personnel training and list of samples analyzed and duration of time using the
        method
    •   Example of client data reporting form
    •   A statistical summary of percent recoveries for all OPR and MS samples analyzed over the past
        six months.

Completed applications should be submitted to Cryptosporidium Laboratory Quality Assurance
Coordinator, c/o CSC Water Programs, at the address listed above.
3.2.2
Personnel Qualifications and Training
As part of approval, EPA will determine whether laboratory personnel are qualified to analyze Cryptosporidium
samples for LT2 Rule monitoring. Suggested personnel qualifications for the Lab QA Program are provided in
Table 3-1. Each laboratory should have at least one principal analyst.
Table 3-1.
Suggested Laboratory QA Program Personnel Qualifications
Position
Principal Analyst
Analyst
Technician
Education
BS/BA in
Microbiology or
closely related field
2 years college in
Microbiology or
equivalent
No minimum
requirement
Experience with
Crypto and IFA
Microscopy
1 year continuous
6 months continuous
No minimum
required
Experience Using
Method 1622/1623
6 months
3 months
3 months performing
specific parts of
procedures
Number of
Samples Analyzed
Using Method
1622/1623
100 (50 if approved
as an analyst during
Information
Collection Rule
[ICR])
50 (25 if approved
as an analyst during
ICR)
50 (25 if approved
as an analyst during
ICR)
During the on-site evaluation (Section 3.2.4 of this manual), EPA will review laboratory records to verify
that the personnel performing EPA Method 1622/1623 analyses are qualified to do the analyses required
under LT2. For new staff that is added after the on-site evaluation, the laboratory should send a letter to
EPA providing the following information on the new staff member:

    •  Resume including education
    •  Number of samples analyzed using EPA Method 1622/1623

    •  Number of months of experience
    •  Verification that analyst training followed the laboratory's training SOP
                                              17
                                                                   February 2006

-------
                                                Section 3: Guidance forCryptosporidium Laboratories
In addition, the following steps should be completed by new personnel as part of their training prior to
analyzing samples for LT2 (specify in the laboratory's training SOP):

    •   Review laboratory SOPs for analysis of samples using Method 1622/1623

    •   Observation of an experienced analyst performing the method

    •   Performance of the method while being observed by an experienced analyst

    •   Acceptable  performance of a set of IPR samples using blind spikes

    •   Analysis of as many MS/MSD samples as possible

    •   Repeated study of microscopy module
It is also helpful for trainees to examine and characterize oocysts that are live and inactivated to note the
differences between the two states and variations which may occur in the staining and morphological
quality of the oocysts. Comparing the quality of the organisms between live and inactivated oocysts may
help define and troubleshoot any problems that may occur during processing and eliminate the possibility
that the staining or morphological differences are due to the inactivation techniques used for the
organisms.

After the initial training is successfully completed,  a trainee should analyze as many Ongoing Precision
and Recovery (OPR) samples as possible. In addition, the trainee should analyze non LT2 samples using
the same method to  gain experience. To optimize the experience gained in the required 3 month period
and 50 samples analyzed, the trainee should complete the analysis of 20 MS/MSD sets from a variety of
source waters. At a minimum, it is recommended that samples analyzed to gain experience include
different matrices that may demonstrate possible interferences with processing and/or examination.

After the initial training is successfully completed,  a trainee may assist with LT2 samples with an
experienced analyst (i.e., both trainee and analyst may sign the laboratory bench sheet and slide
examination form) to gain experience with a variety of source  waters. It should be noted that any such
assistance with processing should be supervised closely enough to proactively eliminate processing
mistakes and preserve the analysis. The trainee may re-examine a slide after complete examination by an
experienced analyst. The experienced analyst's microscopy results should be used for reporting LT2
results. When a trainee successfully completes the required experience and number of samples,
documentation supporting the completion should be sent to the EPA and the trainee's status will be
changed to analyst or technician. The troubleshooting in Section 9.7.5 of Method 1622/1623 may be used
during analyst training also. Proficiency of skill in photomicrography and development of an analyst
photo library is encouraged.

3.2.3         Initial Proficiency Testing
After the laboratory's application has been reviewed and accepted, EPA will send the laboratory a set of
eight initial proficiency testing (IPT) samples, which consist of a suspension of Cryptosporidium oocysts
in a concentrated matrix. Laboratories will resuspend these spikes in reagent water to produce simulated
source water samples, and analyze the samples using the version of EPA Method 1622/1623 that the
laboratory plans to use for routine Cryptosporidium analyses.

Laboratory IPT data will be evaluated against the mean recovery and precision (as relative standard
deviation [RSD]) criteria that EPA has established for IPT samples. If a laboratory fails the IPT criteria
twice, EPA recommends that the laboratory

    •    Receive additional training in performing the method (discussed further in Section 3.2.6 of this
        manual)

                                              18                                   February 2006

-------
                                                Section 3: Guidance forCryptosporidium Laboratories
    •  Repeat their IPR analyses until acceptable and submit the results to EPA

If the laboratory does not pass the third IPT following additional training, they may consult with the EPA
regarding the level and type of training undertaken. After satisfying these requirements, the laboratory
may re-apply for IPT samples.

3.2.4         On-Site Evaluation

Each laboratory that passes the IPT is eligible to participate in an on-site evaluation next, which consists
of two concurrently performed assessments: a data and QA evaluation and a technical evaluation.

3.2.4.1        Data and QA Evaluation
During the data and QA evaluation, laboratory documentation will be evaluated to verify compliance with
QA program requirements. The evaluation will cover the following:

    •  Equipment and personnel records

    •  Data recording procedures, based on field sample data and quality control sample data

    •  Quality control test frequency and acceptability

    •  Quality assurance plans

    •  Standard operating procedures

To ensure consistency and thoroughness for all audits, the data auditor uses a detailed checklist
(Appendix F) to evaluate specific factors under each of these categories.

To prepare for the on-site evaluation, the laboratory can  use the checklists provided with the program
application to perform a self-audit.

3.2.4.2       Technical Evaluation
During the technical evaluation, laboratory sample processing and analysis using EPA Method  1622/1623
will be evaluated. The laboratory will be assessed on its  capabilities including the following:

    •  Sample processing and analyses

    •  Microscopy

To ensure consistency and thoroughness for all audits, the technical auditor uses a detailed checklist
(Appendix F) to evaluate specific factors under each of these categories.

3.2.5         Approval and Ongoing Proficiency Testing

Laboratories will be approved after they have submitted an acceptable application, passed the IPT and
passed the on-site evaluation, as summarized in steps 1-3 in Figure 3-1. Laboratories that are approved
will also receive a set of three ongoing proficiency testing (OPT) samples approximately every  four
months that should be analyzed in the same manner as the IPT samples. EPA will evaluate the precision
and recovery data for OPT samples to determine if the laboratory continues to be qualified under the
Laboratory QA Program. Laboratories must successfully evaluate the OPT samples to maintain EPA
approval  for the LT2.
                                              19                                  February 2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
If a laboratory fails to meet the precision or recovery criteria for a set of OPT samples, the laboratory will
be shipped a second set of samples. If the laboratory's next set of OPT data arc acceptable, no further
action is required.

If a laboratory fails the next set of OPT samples (two sets of OPTs in a row), it is recommended that the
laboratory receive additional training and examine laboratory control charts. If the laboratory continues to
fail OPT samples, EPA will reevaluate the laboratory's approval for this program.

3.2.6          Additional Training

Additional training can be received at another approved laboratory or through training opportunities
supported by the EPA.  Laboratories also can consult universities, vendors, websites, and microscope
service companies for possible training opportunities. EPA has produced an online microscopy module
detailing the characterization ofGiardict and Cryptosporidium. Viewing this module from the LT2
website, http://www.epa.gov/safewater/disinfection/lt2/, is open to all analysts as a supplement to one-on-
one microscopy training. Practice samples may be performed including method blanks, OPR samples,
MS samples, and non-LT2 samples. It is recommended that samples analyzed to gain additional practice
include  different matrices which may demonstrate possible interferences with processing and/or
examination.

3.2.7          Notifying Utilities of Change in Laboratory Status

Two actions are necessary if a laboratory is disapproved:

    •   The laboratory should notify clients

    •   EPA will remove the laboratory from the approved list
        (http://www.epa.fiov/saftcwater/disinfection/lt2/index.htmn

If a laboratory receives notice that they have been "disapproved," the laboratory will no longer be able to
analyze samples under LT2 until they have been re-approved [141.705(a)]. The laboratory should
immediately notify their clients of their status change and cease analyzing samples for LT2. As suggested
in Section 3.1.1  of this manual, laboratories should have a protocol in place to divert samples to another
approved laboratory in the event back-up analyses are needed.

All samples being processed by the laboratory at the time of the disapproval are considered acceptable,
provided all QC and holding time requirements (EPA Methods 1622 and 1623) are met.  Analysis of
these samples should be completed by the laboratory. However, no new LT2 sample analyses may be
initiated unless/until the laboratory is re-approved.
3.3    Cryptosporidium Method Quality Control

During the LT2 Rule, Cryptosporidium samples must meet the quality control (QC) requirements listed in
EPA Methods 1622/1623. The requirements discussed in this guidance manual are based on the
December 2005 versions of EPA Methods 1622/1623 (References 5.2 and 5.3). Section 3.1 in this
manual included QC for samples, Section 3.2 included QC for laboratories, and this section covers QC for
the method.

Sections 3.3.1 through 3.3.13 provide guidance on the implementation of the QC requirements specified
in the December 2005 version of EPA Method 1622/1623. Routine QC requirements that must be verified
internally by the laboratory before reporting LT2 Rule monitoring results are summarized in Table 3-2.
QC guidance for method modifications and use of multiple method variations are covered in 3.3.14. This

                                             20                                  February 2006

-------
                                                        Section 3: Guidance for Cryptosporidium Laboratories
guidance is provided to help implement the QC requirements in the methods and does not substitute for,
or alter, the method requirements.
Figure 3-1.     Process for Receiving and Monitoring Laboratory Approval
        Step 1 :

         Submit
        Program
       Application
        Analyze
    Initial Proficiency
    Testing Samples
         (IPTs)
                        1 Receive training in deficient areas
                         Repeat IPR analysis until acceptable
                                            -Yes	
        Step 3.

        On-Site
       Evaluation
                      On-site evaluation
                        of laboratory
 Receive audit
report from EPA
   Respond to any
deficiencies cited in EPA
    audit report
                                                       Provide additional
                                                      information to EPA if
                                                      responses are deemec
                                                         unacceptable
                                                                                 1 Receive training in deficient areas
                                                                                 2 Evaluate laboratory control charts
        Step 4.

    Analyze Ongoing
       Proficiency
    Testing Samples
         (OPTs)
                                                      21
                                         February 2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
 Table 3-2.
Summary of Routine QC Requirements
QC sample
or
procedure
IDC
IPR
Method
Blank

OPR


MS

Positive
staining
control

Negative
staining
control
Verification
of Analyst
Performance
Matrix
Reagent
water
and
source
water
Reagent
water
Reagent
water

Reagent
water


Source
water

none

none
Reagent
Water
Number
of
samples
8
4
1

1


2

1

1
N/A
Frequency
Once
Initial use of
method
Each IPR and
OPR set
At least each
week samples
are processed
or every 20
samples,
whichever is
more frequent
For each source
water - initial
sampling and
every 20
samples
Process each
time samples
are stained;
examine each
microscope
session
Each time
samples are
stained
Monthly
Purpose
To demonstrate control over the
analytical system; consists of IPR set,
Method blank ,and MS/MSD as a
requirement of Lab QA Program
To establish initial control over the
analytical system and demonstrate
acceptable method performance
(recovery and precision)
To demonstrate the absence of
contamination throughout the
analytical process

To demonstrate ongoing control of
the analytical system and verify
continuing method performance
(recovery and precision)

To determine the effect of the matrix
on (oo)cyst recoveries; must be
accompanied by an unspiked field
sample collected at the same time as
the MS sample

To demonstrate ongoing control of
the staining process and performance
of reagents and microscope

To demonstrate the absence of
contamination through staining
process
Refine consistency of organism
characterizations between analysts
Control
Charts
No

Required


Recommended


No
3.3.1
Cryptosporidium Spiking Materials
During LT2 Cryptosporidium monitoring, laboratories must analyze samples spiked with
Cryptosporidium oocysts to assess ongoing laboratory and method performance in accordance with
method QC requirements. These ongoing spiked sample analyses include initial precision and recovery
samples (IPRs), ongoing precision and recovery samples (OPRs), matrix spike samples, and positive
staining controls (Sections 3.3.2, 3.3.4, 3.3.12, and 3.3.8 of this manual, respectively). Flow cytometer-
counted spiking suspensions must be used for the IPR, OPR and matrix spike samples [40 CFR §
141.704(a)(3)], and those suspensions must be used within the noted expiration (Section 8.3 of Method
1622/1623). The laboratory should spike samples according to the procedures provided in Section 11.4 of
EPA Method 1622/1623 or according to the procedures provided by the spiking suspension vendor.
                                             22
                                                                 February 2006

-------
                                                Section 3: Guidance for Cryptosporidium Laboratories
The commercial staining kits contain positive control organisms which may be used for routine positive
staining control slides. These organisms are typically treated by various inactivation techniques and need
to be evaluated for appropriate FITC fluorescence, DAPI-stained nuclei, and internal morphology. If an
analyst notes that the appropriate features are not present, e.g., the majority of oocysts are DAPI negative
or exhibit weak nuclei staining, it is suggested that a different vendor of positive control organism be
utilized.  Laboratories are encouraged to use the online microscopy module for comparison
(http://www.epa.gov/safcwatcr/disiiifcction/lt2/). Laboratories may also obtain positive staining control
organisms from the vendors listed below and in Method 1622/1623 Section 7.10 which may be live or
inactivated.

Sources of flow cytometer-counted Cryptosporidium spiking suspensions for use with routine, spiked
Cryptosporidium QC samples include the following:

1.     Wisconsin State Laboratory of Hygiene
               Flow Cytometry Unit
               http://www.slh.wisc.edu/
               2601 Agriculture Drive
               Madison, WI 53718
               Phone: (608)224-6260
               Fax:   (608)224-6213

       The Wisconsin State Laboratory of Hygiene prepares and distributes live Cryptosporidium
       parvum oocysts and Giardia intestinalis cysts that have not been treated to reduce viability.

2.     BioTechnology Frontiers (BTF)
               http://www.bttbio.coni
               Unit 1, 35-41 Waterloo Road
               North Ryde NSW 2113
               Australia
               Phone:+61 288779150
               Fax: +61  2 8877 9101
               Email: contactfgjblfbio.com

       BTF prepares and distributes Cryptosporidium parvum oocysts and  Giardia intestinalis cysts that
       have been irradiated to inactivate the organisms. Note: Irradiated, flow cytometer-counted
       spiking suspensions may be used for routine laboratory QC samples, including initial precision
       and recovery (IPR) samples, ongoing precision and recovery (OPR) samples, and matrix spike
       (MS) samples.  In accordance with EPA Method 1622/1623, irradiated organisms may not be  used
       for interlaboratory validation studies performed to seek nationwide approval of modified versions
       of the methods.

3.3.2         Initial Precision and Recovery Test
The initial precision and recovery (IPR) test required by EPA Method 1622/1623 consists of four reagent
water samples spiked with -100 to 500 oocysts and is used to demonstrate acceptable performance with
the method. Section 9 of EPA Method 1622/1623 also requires the IPR to be performed for each  method
modification (additional guidance on QC when using multiple method variations is provided in Section
3.3.13 of this manual).

The results of the four analyses are used to calculate the mean percent recovery and the relative standard
deviation (RSD) of the recoveries for Cryptosporidium (Section 3.6.3 of this manual). For EPA Method


                                              23                                  February  2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
1622/1623, the mean Cryptosporidium recovery should be from 24 percent to 100 percent and the RSD of
the four recoveries should be less than or equal to 55 percent. Characterization of the first three
Cryptosporidium oocysts and three Giardia cysts must be reported on the slide examination form for each
IPR sample following Section 9.4.5 in EPA Method 1622/1623.

3.3.3          Method Blank Test
The method blank test required in Section 9.6 of EPA Method 1622/1623 consists of analysis of an
unspiked reagent water sample to demonstrate freedom from contamination. The method requires that one
method blank sample must be analyzed each week or every 20 field and matrix spike samples, whichever
is more frequent (Section 9.1.7 in Method 1622/1623). A week is defined as any 168 hour (7 day) period
that begins with the processing of the OPR. If more than one method variation will be used for filtration
and/or another technique which exposes samples to different apparatus and/or reagents, a separate method
blank is required for each variation.

Method blank samples should be analyzed before any field samples in a batch are processed to verify
acceptable performance. If one or more Cryptosporidium oocysts (as defined in Section 13 of Method
1622/1623) are found in a blank, the method blank is unacceptable and analysis of additional  samples is
halted until the source of contamination is eliminated and a blank shows no evidence of contamination.
Troubleshooting the problem and repeating the method blank to bring the analytical system under control
is required before proceeding with sample analysis. If the repeated method blank is acceptable and field
samples can be processed within holding times, no replacement samples are necessary.
Note: If oocysts are detected in the method blank, analysis of additional samples is halted until
the source of contamination is eliminated.
3.3.4         Ongoing Precision and Recovery Test
The ongoing precision and recovery (OPR) in Section 9.7 of EPA Method 1622/1623 entails analysis of a
reagent water sample spiked with -100 to 500 oocysts to demonstrate ongoing acceptable performance.
One OPR sample should be analyzed each week or every 20 field and MS samples, whichever is more
frequent (Section 9.1.7 of Method 1622/1623). A week is defined as any 168 hour (7 day) period that
begins with the processing of the OPR (Section 9.1.7 of Method 1622/1623). If more than one method
variation will be used for filtration and/or another technique, a separate OPR may be required for each
variation.

OPR samples should be analyzed before any field samples in a batch are processed to verify acceptable
performance. OPR Cryptosporidium recovery should be from 11  percent to 100 percent to be  considered
acceptable (Section 9.7.3 of Method 1622/1623). Characterization of the first three Cryptosporidium
oocysts and three Giardia cysts must be reported on  the slide examination form as per Section 9.7.1.2 of
EPA Method 1622/1623. If the OPR Cryptosporidium recovery is not acceptable, no samples may be
processed. Troubleshooting the problem and repeating the OPR to bring the analytical system under
control is required before proceeding with sample analysis.  If the repeated OPR is acceptable  and field
samples can be processed within holding times, no replacement samples are necessary.
Note: Ongoing precision and recovery results should be 11 percent to 100 percent.
                                             24                                 February 2006

-------
                                                Section 3: Guidance forCryptosporidium Laboratories
3.3.5
Ongoing Precision and Recovery Control Charts
As noted in Section 9.7.6 of the December 2005 version of EPA Method 1622/1623, laboratories should
maintain a quality control (QC) chart of OPR recoveries, graphically displaying the results of continuing
performance. The control chart should be developed using the most recent 20 to 30 test results.

The control chart is developed by plotting percent recovery of each OPR sample over time (Figure 3.2).
Based on the mean of the recoveries (x ) on the chart, the upper and lower control limits should be
established as follows
    •  Upper control limit = x + 2 standard deviations
    •  Lower control limit = x - 2 standard deviations
After each 5 to 10 new recovery measurements, new control limits should be recalculated using the most
recent 20 to 30 data points. Control charts can be used to track the laboratory's performance and
determine if any trends in recovery are occurring. Control charts can also be used to compare
performance of different method variations, different analysts, and/or other changes implemented by the
laboratory. If recovery measurements fall outside the control limits, laboratories should take corrective
action, investigating potential causes of the outlying result. The troubleshooting guidance for OPR
failures provided in Section 9.7.5 of EPA Method 1622/1623 is also useful for investigating the cause of
acceptable, but outlying, OPR measurements identified through the use of control charts.

Figure 3-2.     Ongoing Precision and Recovery Control Chart Example
   100%
   90%
                                     Recovery
                                     Mean + 2 Standard Deviations
                                     Mean - 2 Standard Deviations
    01/04/2001
               02/01/2001
            03/01/2001
03/29/2001
04/26/2001
                                                             05/24/2001
06/21/2001
07/19/2001
                                              25
                                                                    February 2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
3.3.6          Quality Control Batches
All LT2 Cryptosporidium samples must be associated with an acceptable OPR and method blank sample
as stated in Section 9 of the EPA Method 1622/1623. LT2 samples arc associated with QC samples
through a "QC batch." A QC batch consists of an OPR and method blank and a maximum of 20 field and
MS samples that are eluted, concentrated, and purified in the same week as the OPR and method blank
samples using the same reagents (e.g., eluting solution). A week is defined as any 168 hour (7-day) period
that begins with the processing of the OPR. If more than 20 field and MS samples are processed in a
week, the OPR and method blank samples are associated with the field and MS samples which are eluted,
concentrated, and purified using the same reagents as the OPR and MB and processed prior to the next
OPR/MB. The next QC batch may begin concurrently with the processing of the last of the 20 samples
associated with the previous OPR/MB. A field sample and its associated MS sample should be analyzed
in the same "QC batch". QC samples do not need to be analyzed necessarily during weeks in which no
field samples are analyzed unless analytical practice would be beneficial for the laboratory.

3.3.7          Holding Time Requirements
During Cryptosporidium analyses for the LT2 Rule, sample processing should be completed as soon as
possible by the laboratory. The laboratory should complete sample filtration (if sample is received in
bulk), elution, concentration, purification, and staining the day the sample is received whenever possible.
However, the laboratory is permitted to split up the sample processing steps if processing a sample

completely in one day  is not possible. If this is necessary, sample processing can be halted after filtration,
application of the purified sample onto the slide, or staining.
The following holding times must be met for samples analyzed by EPA Method 1622/1623 during the
LT2 Rule:

    •   Sample collection and filtration. Sample elution must be initiated within 96 hours of sample
       collection whether shipped to the laboratory as a bulk  sample or filtered in the field.

    •   Sample elution, concentration, and purification. The laboratory must complete the elution,
       concentration,  purification, and application of the sample to the slide in one work day. It is
       critical that these steps be completed in one work day to minimize the time that any target
       organisms present in the sample sit in eluate or concentrated matrix. This process ends with the
       application of the purified sample on the slide for drying. Follow the stain manufacturer's
       instructions regarding the drying procedure to use.  Drying options include: on the lab bench, on
       the lab bench with air gently moving around the slides, on a slide warmer between 35 °C and
       42°C, in a humid chamber or incubator between 35°C and 42°C, or in the refrigerator. The slides
       must be completely dried before staining and stored to maintain the dried state until stained.
       Storage of dried slides consists of storing on the lab benchtop (protected), in a desiccator box in
       the refrigerator, or in a desiccator box on the lab bench (if the  lab is humid).

    •   Staining. The sample must be stained within 72 hours of application of the purified sample to the
       slide including drying time.

    •   Examination.  Although immunofluorescence assay (FA) and 4',6-diamidino-2-phenylindole
       (DAPI) and differential interference contrast  (DIC) microscopy examination and confirmation  is
       ideally performed immediately after staining is complete, laboratories have up to 168 hours (7
       days) from completion of sample staining to complete the examination and confirmation of
       samples. However, if fading/diffusion of FITC or DAPI staining is noticed, the laboratory should
       reduce this holding time. In addition, the laboratory may adjust the concentration of the DAPI
       staining solution so that fading/diffusion does not occur per Section 14.6 in Method 1622/1623.

                                             26                                 February 2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
       The laboratory also may evaluate the use of another mounting medium (alternatives are provided
       in Section 3.8.2 of this manual, below).
LT2 Rule requirement:
               Each sample must meet the QC criteria for the methods. Per EPA
               Method 1622/1623, samples must be processed or examined
               within each of the holding times specified in Section 8.2 of the
               method.
A breakdown of the holding times for each set of steps is provided in Table 3-3

Table 3-3.      Method 1622/1623 Holding Times (adapted from Table 1 EPA Method 1622/1623)
Sample Processing Step
Collection
Filtration
Elution
Concentration
Purification
Application to slide
Staining
Examination
Maximum Allowable Time between Breaks
(samples should be processed as soon as possible)
Up to 96 hours are permitted between sample collection and
initiation of elution
These steps must be completed in 1 working day
Up to 72 hours are permitted from application of the purified
sample to the slide to staining
Up to 168 hours (7 days) are permitted between sample staining
and examination
3.3.8
Staining Controls
Positive staining controls are used to verify that the FITC and DAPI stains are fluorescing appropriately.
Positive staining controls are prepared by applying 200 to 400 intact oocysts to a slide and staining the
slide with the same reagents and staining procedure used to stain field samples. The analyst examines
several fields of view to verify that the stain is fluorescing at the appropriate intensity and uniformity.
Each analyst must characterize a minimum of 3 Cryptosporidium oocysts on the positive staining control
slide before examining field sample slides per Section 15.2.1.1 in Method 1622/1623. Control slides and
sample slides should be read on the same day. If sample slides from the same staining batch are read over
multiple days, the control slide should be rechecked each day before examination of the sample slides. If
the laboratory has a large batch of slides that will be examined over several days and is concerned that a
single positive control may fade due to multiple examinations, the laboratory should prepare multiple
control slides at the same time with the batch of field slides and alternate between the positive controls
when performing the positive control check.

Negative staining controls are used to verify that no oocysts or interfering particulates are present.
Negative staining controls are prepared by staining and examining a slide with phosphate buffered saline
solution.

The analyst should indicate on each Cryptosporidium slide examination form whether the positive
staining control and negative staining control were acceptable.
                                              27
                                                                   February 2006

-------
                                                Section 3: Guidance for Cryptosporidium Laboratories
 LT2 Rule requirement:      Each sample must meet the QC criteria for the methods. Per EPA
                              Method 1622/1623, positive and negative staining controls must
                              be acceptable (Section 15.2.1).
 3.3.9         Examination Preparation

 To help the analyst identify the target analyte during field sample slide examination, each analyst must
 characterize a minimum of three Cryptosporidium oocysts on the positive staining control slide before
 examining field sample slides. This characterization must be performed by each analyst during each

 microscope examination session. FITC examination must be conducted at a minimum of 200X total
 magnification, DAPI examination must be conducted at a minimum of 400X, and DIG examination and
 size measurements must be conducted at a minimum of 1000X.

 Size, shape, and DIG and DAPI characteristics of the three Cryptosporidium oocysts must be recorded by
 the analyst in a microscope log (Section 15.2.1.1 of Method 1622/1623).

 3.3.10        Verification of Analyst Performance

 Analyst verifications are ongoing comparisons of slide counts and characterizations used to assess and
 maintain consistency in slide examination among analysts. The goal is to encourage comparison and
 discussion among analysts to continually refine their microscopy  skills. At least monthly when
 microscopic examinations are being performed, the laboratory shall prepare at least one slide containing
 40 to 200 oocysts. More than 50 percent of the oocysts must be DAPI positive and undamaged under
 DIG. Another option is to order prepared slides from Wisconsin State Laboratory of Hygiene, Flow
 Cytometry Unit (http://www.slh.wisc.edu/, (608) 224-6260).

 For laboratories with multiple analysts, each analyst shall determine the DAPI category (DAPI negative,
 DAPI positive intense internal blue staining, and DAPI positive with number of nuclei) and the DIG
 category (empty, containing amorphous structures, or internal structure characterization) of the same 10
 selected oocysts. It is recommended that the DAPI and DIG categorization of the selected oocysts occur
 with all analysts at the same time, i.e. each analyst determines the category independently, then the
 differences in the DAPI and DIG categorizations among analysts are discussed and resolved, and these
 resolutions documented. This round-robin approach with all analysts may encourage further discussion
 among analysts and lead to more consistent characterizations. Alternatively, organism coordinates may be
 recorded for each analyst to locate and categorize the organisms at different times. Differences among
 analysts must still be discussed and resolved.

 Laboratories should be aware that both  FITC and especially DAPI fluorescence may fade during the
process of analyst verification.  Therefore, DAPI comparisons should be performed first and the UV light
shutter should remain closed except for a few seconds during observation by the analyst. Repeat
comparisons with new FITC organisms, again taking care to keep the shutter closed whenever the oculars
are not being used.

Each analyst shall also determine the total number of oocysts by FITC fluorescence at 20X magnification
for the entire slide. It is recommended that this count be performed last or on a separate slide than that
used for the DAPI and DIG characterization so that fading will not influence counts. The total number of
oocysts enumerated by each analyst must be within ±10 percent of each other. If the number is not within
this range, the analysts must identify the source of any variability  between analysts' examination criteria,
prepare a new slide, and repeat the performance verification.

                                             28                                 February 2006

-------
                                              Section 3: Guidance for Cryptosporidium Laboratories
Laboratories with only one analyst should maintain a protozoa library and compare the results of slide
examinations to photographs of oocysts and cysts and interfering organisms to verify that examination
results are consistent with these references. These laboratories may also perform repetitive counts of a
single verification slide for FITC and DAPI. These laboratories are encouraged to coordinate with other
laboratories to share slides and compare counts.

Analyst verification serves as an on-going and consistent training venue for new and experienced
analysts. In addition to the monthly verification of analyst performance, other training options exist at
universities, microscope service companies, and existing laboratories. EPA has produced an online
microscopy module detailing the characterization ofGictrdia and Cryptosporidium. Viewing this module

from the LT2 website, http://www.epa.gov/safewater/disinfection/lt2/, is open to all analysts as a
supplement to one-on-one microscopy training.

3.3.11        Acceptance Criteria for Receipt of Field Samples
Cryptosporidium samples for LT2 Rule monitoring should be stored between 1 °C and 10°C to reduce
biological activity and so they  cannot freeze. This is specified in Section 8.0 of the December 2005
versions of EPA Method 1622/1623. Because Cryptosporidium samples collected for the LT2 Rule must
meet the QC criteria in the methods, and because these QC criteria include receipt of samples at <20°C
and not frozen, laboratories must reject LT2 Cryptosporidium samples that are received at >20°C or
frozen. In these cases, the PWS must re-collect and re-ship the sample.

Several options available to measure sample temperature upon receipt at the laboratory and, in some
cases, during shipment, are provided in Section 2.2 of this manual.
LT2 Rule requirement:     Each sample must meet the QC criteria for the methods. Per EPA
                            Method 1622/1623, samples not received on the day of collection
                            must be received at the laboratory at <20°C and not frozen
                            (Section 8.1 of the method).
3.3.12        Matrix Spike Samples
The matrix spike (MS) in EPA Method 1622/1623 (Section 9.5.1 of the December 2005 version) entails
analysis of an extra bulk water sample spiked with -100 to 500 oocysts in the laboratory to determine the
effect of the source water matrix on the method's oocyst recovery. The laboratory should analyze an MS
sample when samples are first received from a PWS location for which the laboratory has never before
analyzed samples to identify potential method performance issues with the matrix.
LT2 Rule requirement:     Each sample must meet EPA Method 1622/1623 requirements [40
                            CFR § 141.704(a)], which include the following: (1) The MS and
                            field sample must be collected from the same sampling location by
                            splitting the sample stream or collecting the samples sequentially;
                            (2) The volume of the MS sample analyzed must be within 10
                            percent of the volume of the field sample analyze); and  (3) The
                            MS and field sample must be analyzed by the same procedure
                            (Section 9.5.1 of Method 1622/1623).	
                                            29                                 February 2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
3.3.12.1       Matrix Spike Frequency
For all PWSs, the first MS sample should be collected and analyzed during the first sampling event under
the monitoring program per EPA Method 1622/1623 (Section 9.1 .8 of Method 1622/1623). If it is not
possible to analyze an MS sample for the first sampling event, the first MS sample should be analyzed as
soon as possible to identify potential method performance issues with the matrix. The laboratory and
PWS should evaluate the MS recoveries, as well as other attributes of sample processing and
examination, and work together to determine whether sample filtration and processing procedures are
working acceptably or need to be re-evaluated. Matrix spike samples may be analyzed more frequently
than one every 20 field samples to better characterize method performance in the matrix.

Based on this requirement, the following PWS categories must analyze at least two MS samples during
LT2 Rule monitoring:
    •   Large PWSs that perform monthly monitoring for two years (resulting in 24 samples)
    •   Small PWSs that are triggered into Cryptosporidium monitoring and collect semi-monthly
       samples for one year or monthly samples for two years (resulting in 24 samples)

For large PWSs that perform semi-monthly or more frequent monitoring for two years (resulting in 48 or
more samples),  a minimum of three MS samples should be collected and analyzed. If a PWS monitors
more frequently or collects more than 60 samples, a minimum of four MS samples must be analyzed.

3.3.12.2       Matrix Spike Samples Associated with Field-Filtered Samples
Matrix spike samples must be collected as bulk samples and spiked in the laboratory prior to filtration.
The volume of the MS sample must be within 10 percent  of the volume of the associated field sample.
PWSs that field-filter 10 L samples may field filter the monitoring sample, but must collect and ship the
10 L MS sample in bulk to the laboratory for spiking, filtering, and analysis.

For PWSs that field-filter >10 L samples, all but 10 L of the MS sample may be filtered in the field. The
remaining 10 L of source water for MS analysis may be collected in bulk and shipped to the laboratory.
The laboratory will then spike the 10 L bulk sample and pump it through the filter containing the balance
of the sample already filtered in the field. The associated monitoring sample must be collected as usual
[40CFR§ 14
3.3.12.3       Matrix Spike Control Charts
As with the OPR samples and described in Section 9.5.1.4 of the December 2005 version of EPA
Methods 1622/1623, laboratories should assess precision of MS recoveries. This can be accomplished by
maintaining a control chart that graphically displays the results of continuing performance. It is suggested
that the precision assessment be maintained across all MS samples as well as stratified by source. The
control chart across all MS samples should be developed when at least 5 MS samples have been
completed. It is the laboratory's decision as to the maximum number of samples used to determine
precision assessments. For individual sources completing only two MS samples, precision estimates
should be calculated using percent difference instead of standard deviation. If more MS samples are
completed per source, the laboratory should generate precision assessments using the mean and standard
deviation.
                                             30                                  February 2006

-------
                                               Section 3: Guidance forCryptosporidium Laboratories
The control chart is developed by plotting percent recovery of each matrix spike sample versus time. Based on the
mean of the recoveries (x/on the chart, the upper and lower control limits should be established as follows:
    •   Upper control limit = x + 2 standard deviations
    •   Lower control limit = x - 2 standard deviations

An example of a control chart (using OPR data, not MS data) is provided in Figure 3-2. Control charts
can be used to track the laboratory's performance and determine if any trends in recovery are occurring.
Control charts can also be used to compare performance of different method variations, different analysts,
and other changes implemented by the laboratory along with performance in different matrices. If
recovery measurements fall outside the control limits, laboratories may take corrective action,
investigating potential causes of the outlying result.

3.3.13         QC Guidance for Method Modifications and Use of Multiple Method
               Variations
EPA Methods  1622/1623 are performance-based methods and, therefore, allow method modifications if a
laboratory can meet applicable QC criteria (EPA Method 1622/1623 [Section 9.1.2]). Table 3-4 presents
examples of changes to EPA Method 1622/1623 that EPA considers to be "routine," "occasional," and
"substantive," and what QC steps are necessary to demonstrate acceptability before implementing these
changes.
                                              31                                  February 2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
Table 3-4.      Recommended QC for Different Types of Changes to EPA Method 1622/1623
"Routine" Changes
Using new lots of the same method component, such as
• new lots of filters
. IMS kits
• stains
• other reagents including reagent water
Changing to new equipment that meets existing specifications in the
method (e.g., pumps or centrifuges)
"Occasional" Changes
Using different equipment, reagents or procedures for which specifications
are not included in the method, such as
lab shaker
IMS magnets
Leighton tubes
slides
mounting medium
centrifuge speeds
slide drying procedures
staining procedure using the same brand of reagents
vortex speeds during IMS
microscope
"Substantive" Changes
Changing to a different filter
Changing flow rate (e.g., using a flow rate of 4 L/min with the Envirochek™
HV version of Method 1623, rather than the 2 L/min flow rate specified in
the method)
Changing sample volumes (e.g., processing 50 L samples rather than 10 L
samples)
Changing to a different Filta-Max® concentration or elution procedure:
• Standard wash station and concentrator tube
• Stomacher and concentrator tube
• Stomacher and centrifugation
Incorporating the use of multiple filter membranes in the Filta-Max®
concentrator tube
Changing to a different antibody staining kit
Incorporating additional rinses and transfers to reduce carryover from IMS
to the slide
Changing vendors for spike organisms
Changing laboratory space or location

Verification of reagent
acceptability, per routine
OPRs/method blanks, is
recommended.

Initial precision and recovery,
method blank, matrix spike/matrix
spike duplicate, and unspiked
field sample are recommended.




New demonstration of acceptable
performance is required through
initial precision and recovery, and
method blank. In addition, matrix
spike/matrix spike duplicate, and
unspiked field sample is strongly
recommended.




3.3.13.1       Making a Substantive Change to the Method
Substantive changes to Method 1622/1623 as described in Table 3-4 essentially comprise a new variation
on the method. A method variation is the complete set of sample processing components (including the
filter, IMS, and stain) and sample processing procedures (including filtration, concentration, purification,
and staining) used to process a water sample for examination. Per EPA Method 1622/1623 (Section
9.1.2), if a laboratory intends to switch completely from one method variation to another, then the
laboratory should demonstrate acceptable QC as outlined in Table 3-4 using the new method variation
before implementing this procedure for the analysis of field samples. If the laboratory demonstrates
                                             32
February 2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
acceptable initial laboratory performance and implements the new method variation and discontinues use
of the old method variation, then the laboratory must demonstrate acceptable ongoing laboratory
performance (through the OPR, method blank, and OPT tests) using the new variation.

Guidance on requirements for initial and ongoing demonstrations of acceptable laboratory performance
for different method variations is provided in Sections 3.3.14.2 of this manual. The examples of
substantive changes given in Table 3-4 have been shown to be effective in multiple laboratories with
different source water matrices. Guidance to demonstrate acceptability of substantive changes that are
novel is discussed in Section 3.3.13.3 of this manual.

3.3.13.2       Using Multiple Method Variations
Per EPA Method 1622/1623 (Section 9.1.2), if a laboratory intends to use multiple method variations
(that differ through a substantive change) concurrently, then the laboratory demonstrates acceptable initial
laboratory performance (through the IDC test per the Lab QA Program) using each method variation
before implementing this procedure for the analysis of LT2 Rule samples. In addition, the laboratory
demonstrates acceptable ongoing laboratory performance (through the OPR, method blank, and OPT
tests) for each substantive method change for all but the following method variations:

   •  Antibody staining kits. If a laboratory alternates among more than one antibody staining kit, the
       laboratory performs positive and negative staining controls for each antibody kit for each batch of
       slides for which the kit is used and should alternate between the kits for ongoing demonstrations
       of acceptable laboratory performance. MS samples should be processed using the same method
       variation as the associated field sample, regardless of the method variation used to demonstrate
       ongoing acceptable laboratory performance.

   •  Additional rinses and transfers. If the laboratory uses additional rinses and transfers for some
       samples to reduce carryover from IMS onto the slide, the laboratory should use this procedure
       (which may reduce recoveries) to demonstrate acceptable ongoing laboratory performance on the
       same percentage of OPR samples as percentage of field samples analyzed with this variation. MS
       samples should be processed using the same method variation as the associated field sample,
       regardless of the method variation used to demonstrate ongoing acceptable  laboratory
       performance.

   •  Multiple membranes for Filta-Max® concentration. If the laboratory uses multiple membrane
       filters in the Filta-Max® particle concentrator for some samples, the laboratory should use
       multiple memebrane filters to demonstrate acceptable ongoing laboratory performance on at least
       the  same percentage of OPR samples as the percentage of field samples analyzed with multiple
       membranes. MS samples should be processed using the same method variation (and same
       number of membranes) as the associated field sample, regardless of the method variation used to
       demonstrate ongoing acceptable laboratory performance.
    •  Multiple sample volumes. See discussion in Section 3.3.14.

3.3.13.3       Substantive Change Acceptability through Alternate Test Procedure (ATP)
               or Tier 2 validation
Novel changes with new kinds of equipment or reagents cannot be evaluated using the QC tests and
criteria described above because experiments with more than one water matrix or a study designed to
compare the new procedure with an established test procedure are necessary for maintaining the same
level of data quality. EPA's Microbiological ATP Protocol (Reference 5.7) describes a process for

                                             33                                  February 2006

-------
                                              Section 3: Guidance for Cryptosporidium Laboratories
conducting QC acceptance criteria-based studies and for conducting side-by-side comparisons to
demonstrate comparability with an EPA-approved method. For example, the ATP protocol would be
useful for evaluating new elution systems or new staining or immunomagnetic separation techniques.

The Tier 2 validation is used if nationwide approval of a modification is sought (Section 9.1.2.1.2 of
Method 1622/1623). A Tier 2 validation includes analysis of an IPR set, method blank, MS/MSD, and
field sample at a minimum of 3 laboratories and compares the results to the methods' QC criteria.

3.3.14       Guidance on QC for Different Sample Volumes

A laboratory with multiple PWS clients representing a range of sample volumes is not responsible for
performing QC tests at all of the volumes. However, if the laboratory does analyze both 10 L and 50 L
sample volumes for clients—or any volumes in between—then the laboratory should demonstrate
acceptable performance in a manner representative of the sample volumes they process. Guidance on
initial and ongoing demonstrations of acceptable laboratory performance is provided below, in Sections
3.3.14.1 through 3.3.14.3.

3.3.14.1      Initial Precision and  Recovery Tests for Different Sample Volumes
A laboratory with multiple PWS clients representing a range of sample volumes should successfully
perform the IPR and method blank test, as well as successfully analyze matrix spike/matrix spike
duplicate (MS/MSD) and initial proficiency testing (IPT) samples (for the Lab QA Program's initial
demonstration of capability [IDC]), at the largest (most challenging) volume. The laboratory should
demonstrate acceptable performance for these tests using spikes of no greater than 500 oocysts.

3.3.14.2      Ongoing Precision and Recovery Tests and Method Blank Tests for
              Different Sample Volumes
A laboratory demonstrates ongoing acceptable performance at both extremes of the volume spectrum by
performing OPRs and method blanks at a volume consistent with the highest sample volume submitted by
clients (e.g. 50 L) as well as OPRs and method blanks at a volume consistent with the lowest sample
volume submitted by clients (but not less than 10 L). Labs analyzing multiple sample volumes, should
demonstrate acceptable performance in a manner representative of the sample volumes they process i.e. at
the  same percentage as volumes in field samples submitted to the laboratory. Frequency of OPRs with
different volumes should be consistent with the frequency of samples with different volumes, e.g. if half
of the samples received have 50 L volumes and half have  10 L volumes, then half of the OPRs should be
performed with 50 L and half with 10 L. Laboratories should work with their PWS clients to attempt to
schedule clients with different sample volume sizes for different periods during the week, so the field
samples can be batched with QC samples of comparable volume.

3.3.14.3      Ongoing Proficiency Tests for Different Sample Volumes
Laboratories with multiple PWS clients representing a range of sample volumes are not required to
analyze OPT samples at each sample volume. The laboratory should notify EPA of the sample volume
most representative  of the LT2 samples processed and perform the OPT test using this volume.
3.4   Sample Collection Procedures
Several options are available to the PWS for collecting untreated surface water samples for
Cryptosporidium analysis, including the following.
    •   On-site filtration of water samples from pressurized or unpressurized sources using the Pall
       Gelman Envirochek™ or Envirochek™ HV capsule filter.


                                            34                                 February 2006

-------
                                                Section 3: Guidance for Cryptosporidium Laboratories
    •  On-site filtration of water samples from pressurized or unpressurized sources using the IDEXX
       Filta-Max® foam filter.

    •  Collection of bulk water samples for shipment to the laboratory for filtration and analysis.

Detailed procedures for each of these options, as well as packing and shipping the samples from the PWS
to the Cryptosporidium analysis laboratory, are provided as appendices in the Source Water Monitoring
Guidance Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water Treatment
Rule (LT2 Rule) (Reference 5.15).

As noted in the PWS guidance manual, EPA recommends that the laboratory and PWS conduct at least
one practice sampling and analysis event prior to starting official LT2 monitoring. Based on previous
experiences in the Information Collection Rule (ICR) and ICR Supplemental Surveys, unanticipated
problems are often encountered during the first sampling event, but are addressed in subsequent events.
Rather than risking sampling problems during official LT2 monitoring, the PWS and laboratory can
identify and resolve any problems by conducting the practice sampling and analysis.

The contract laboratory is often involved with PWSs regarding sampling issues including scheduling,
multiple sources, location, and replacement samples.  These issues are detailed in the Source Water
Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water
Treatment Rule (LT2 Rule)', however, an overview is below.

    •  Scheduling: PWSs are encouraged to work with the contract laboratory to establish a schedule
       that will comply with LT2 Rule requirements and is mutually acceptable to the PWS and the
       laboratory.

    •  Sampling Location:  PWSs are required to collect source water samples for the LT2 Rule from
       the plant intake prior to chemical treatment, unless approved by the State to collect the source
       water sample after chemical treatment [40 CFR §  141.703(b)(2)]. Systems that recycle filter
       backwash water must collect source water samples prior to the point of filter backwash water
       addition [40 CFR § 141.703(c)]. All Cryptosporidium, E. coli, and turbidity source water samples
       collected under LT2  Rule requirements should be collected from the same appropriate sampling
       location.

    •  Multiple Sources:  The use of multiple sources during monitoring must be consistent with routine
       operational practice [40 CFR § 141.703(e)].  If there is a tap prior to treatment where sources are
    combined, the sample must be collected there. If not, the PWS must pursue one of the following
    options:
           collect manually from each  source prior to treatment and composite into one sample in the
           same proportion  as used by  the plant at the time of collection
           collect manually from each  source prior to treatment, analyze separately, and calculate a
           weighted average of the analysis results.

    •  Replacement Samples:  Certain  situations may dictate the need for a replacement sample, i.e. the
       PWS is unable to report a valid  Cryptosporidium analytical result for a scheduled sampling date.
       The following list details some of the possible situations:
           sample not collected during required time frame due to extreme conditions or situations that
           may pose a danger to the sampler
           sample is lost or contaminated

                                              35                                  February 2006

-------
                                                Section 3: Guidance for Cryptosporidium Laboratories
           laboratory exceeds analytical method holding time
           sample receipt temperature fails criteria
           volume requirements not met (field and/or MS sample)
           QC samples fail acceptance criteria
           problems encountered during processing
           failure of an approved laboratory to analyze the sample
        The PWS must submit an explanation for the delayed sampling date to the EPA/State concurrent
        with the shipment of the replacement sample to the laboratory. The system must collect a
        replacement sample as close to the required date as feasible but within 21 days of being notified
        by the laboratory that a result cannot be reported for that date [40 CFR § 141.702(b)(2)].

3.5    Recordkeeping

An effective record keeping system provides information on sample collection and preservation,
analytical methods, raw data, calculations, reported results, and a record of persons responsible for
sampling and analyses. For EPA Methods 1622/1623, original data, including microscope examination
counts and notes, must be recorded. The data may be recorded on bench sheets (Appendix G) and slide
examination forms (Appendix H).

Data should be recorded in ink and a single  line drawn through any change with an initialed, dated
correction entered next to it. Data files may also be microfiche or electronic. Electronic data should be
backed up by a protected tape or disk or hard copy. Under the LT2 Rule, monitoring data (both initial and
second round of monitoring) must be kept until 3 years after bin classification for filtered systems or
determination of mean Cryptosporidium levels for unfiltered systems [40 CFR § 141.722(a)]. Although it
is the PWS's responsibility to meet LT2 Rule data storage requirements for compliance monitoring
samples, the PWS may contract this work to the laboratory.

As laboratories perform Cryptosporidium analyses during the LT2 Rule, the following data recording
practices should be followed:

    •   Record sample identification information, including  sample collection and receipt dates and
        conditions
    •   Record all raw data (primary measurements) used to calculate final concentrations of oocysts/L
        for each sample
    •   Record the date and time of each method step associated with a holding time to verify that all
        method holding times have been met
    •   Record the name of the analyst performing each method step to verify that only qualified
        technicians and analysts are performing the method

The minimum data elements that should be recorded for Cryptosporidium samples during the LT2 are
discussed in detail below. These data elements are critical to ensuring that final sample concentrations can
be verified using primary data and are necessary to demonstrate that all method-specified holding times
were met. Standardized bench sheets and Cryptosporidium examination results forms are available for
download as Appendices G and H on the LT2 website
http://www.epa.gov/safewater/disinfcction/lt2/comp]iance_m]jiianua].htrnl.
                                              36                                  February 2006

-------
                                                Section 3: Guidance for Cryptosporidium Laboratories
Sampling records provided by the PWS with the sample should include the following information, at a
minimum:

    •   Public water system name and ID number*

    •   Facility name and number*

    •   Sample Collection Point Name and ID
    •   Date and start/stop times of collection*

    •   Sampler's name and phone number (or alternate contact for laboratory if problems are
        encountered)
    •   Source water temperature and turbidity

    •   Volume  filtered information (if the sample was filtered in the field)

    •   Whether the filter clogged (if the sample was filtered in the field)

    •   Analyses requested (e.g. routine field sample analysis or field sample + MS analysis)

*       Note: These three elements are used to identify the LT2 sample for tracking sample collection,
        analysis, reporting, and use.

Detailed guidance on sample collection data recording, as well as forms and sample collection and
shipping procedures can be found in the Source Water Monitoring Guidance Manual for Public Water
Systems for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule). This manual is
available for download from http://www.cpa.gov/safcwatcr/disintcction/lt2/compliance.htnil.

Upon receipt of the sample at the laboratory, laboratory personnel should record, at a minimum, the
information  in Table 3-5. Laboratories should immediately notify utilities of any deficiencies requiring a
resample.

Table 3-5.     Sample Receipt Data Elements to Record in the Laboratory	
 Public water system name and ID
 Facility name and ID
 Sample collection point name and ID
 Turbidity at the collection point taken immediately after sample collection
 Date and time of sample collection (start and stop times if field filtered)
 Date and time of sample receipt by laboratory
 Volume filtered (if sample is filtered in the field)
 Name of laboratory person receiving the sample
 Temperature of sample upon receipt
 Any deficiencies (deficiencies may include but are not limited to: exceeded sample holding time, transport
 temperature exceeded 20°C, or sample leaked during transport)
Laboratories analyzing samples for Cryptosporidium using EPA Method 1622/1623 in support of the LT2
Rule should record the primary elements required to calculate the final concentrations and percent
recoveries for matrix spike (MS), ongoing precision and recovery (OPR), and proficiency test samples.
These primary data elements are provided in Table 3-6, and should be recorded on the EPA Method
1622/1623 bench sheet (Appendix G) and slide examination form (Appendix H).

                                              37                                   February 2006

-------
                                                 Section 3: Guidance for Cryptosporidium Laboratories
Table 3-6.     Primary Data Elements to Record for Calculations
 Estimated number of oocysts spiked (MS and OPR samples), based on information provided by the flow-cytometry
 laboratory with the spiking suspension
 Sample volume spiked, in L (MS, OPR and proficiency test samples)
 Sample volume filtered, to nearest 1/4 L
 Number of filters used (if the filter clogged)
 Pellet volume after concentration, to the nearest 0.1 ml
 Total volume of resuspended concentrate, in mL
 Volume of the resuspended concentrate transferred to IMS, in ml_
 Number of subsamples analyzed
 Total number of oocysts detected in the sample
To determine that all method QC requirements were met and that the samples were analyzed by qualified
personnel according to the requirements of the Laboratory QA Program for the Analysis of
Cryptosporidium the laboratory should record the elements in Appendices G and H.  These appendices
include, but are not limited to, the key QC data elements in Table 3-7.

Table 3-7.     Key QC Data Elements to Record	
 Elution date and time (must be within 96 hours of sample collection)
 Slide preparation date and time (must be completed in same working day as elution)
 Sample staining date and time (must be completed within 72 hours of slide preparation)
 Sample examination date and time (must be completed within 168 hours (7 days) of sample staining)
 Person (PWS or Lab employee) performing filtration
 Analyst performing elution
 Analyst performing IMS
 Analyst performing sample staining
 Analyst performing sample examination
 Results of the positive and negative staining controls
The laboratory should also record any additional information that will support the results obtained or
allow problems with sample results and laboratory performance to be identified. This additional
information includes the following:
        Information on the version of EPA Method 1622/1623 used to perform the analysis including
        filter type, elution procedure, concentration procedure, IMS system used, detection kit used, and
        source of oocysts for spiking suspensions

        Lot numbers of reagents and materials used during the analysis, including the filter, elution
        buffer, IMS system, .detection kit, and spiking suspension

        FITC, DAPI, and DIG information of all oocysts detected in the field samples using the slide
        examination form
                                               38                                   February 2006

-------
                                              Section 3: Guidance for Cryptosporidium Laboratories
This information should be recorded on the EPA Method 1622/1623 bench sheet (Appendix G) and
Cryptosporidium slide examination form (Appendix H), as appropriate.

Size, shape, and DIG and DAPI characteristics of the three Cryptosporidium oocysts on the positive
staining control slide (Section 15.2.1.1 of Method 1622/1623) must be recorded by the analyst on a
microscope log.

3.6   Calculations for EPA Methods 1622/1623

During LT2 Rule monitoring, field sample results will be reported using the LT2/Stage 2 Data Collection
and Tracking System (DCTS) described in Section 3.7 of this manual. The DCTS will reduce the data
elements entered by the laboratory to yield final sample results in oocysts/L. The DCTS will also verify
that LT2 Rule Cryptosporidium sample volume analysis requirements were met and calculate MS
recoveries.  Sections 3.6.1 through 3.6.3 are guidance for laboratories to perform the same type of
calculations as the DCTS for oocyst concentrations, matrix spike recoveries, and OPR recoveries. The
laboratory may choose to report the total oocysts and volume analyzed, along with the oocysts/L, and
other primary data elements listed in Table 3-6 to the PWS.  The PWS may then use that report during the
DCTS data review process discussed in Section 3.7.2 of this manual. For QC samples, the laboratory
calculates recoveries for OPR and MS samples to report to their PWS and to maintain QC control charts
as discussed in Sections 3.3.5 and 3.3.12.3 also in this manual.

3.6.1         Calculating Oocyst Concentrations
To calculate the concentration of Cryptosporidium in a sample, reported as oocysts/L, the following
information is needed:

    •   Number of oocysts detected in the sample (recorded as a primary measurement from the slide
       examination form)

    •   Volume analyzed

Using these two data elements, the final concentration should be calculated as:

                                              oocysts detected  in the sample
                  final concentration (oocysts/L) =
                                                    volume analyzed (L)
If 100% of the sample volume filtered is examined, then the volume analyzed equals the volume filtered.
This applies whether one filter or more than one filter was used; if more than one filter was used, and all
of the volume filtered through the multiple filters is processed through the remainder of the method, then
the volume examined is simply the sum of the volumes filtered through each of the filters used.

If <100% of the volume filtered was processed through the remainder of the method, then additional
calculations are needed to determine the volume analyzed. This is discussed in Section 3.6.1.1 through
3.6.1.3 of this manual.

3.6.1.1       Determining  Volume Analyzed when Less than 100% of Sample Was
              Examined
When <100% of the sample filtered is processed through the remainder of the method and examined
(such as when the volume filtered yields >2 mL of packed pellet volume after centrifugation), then the
volume analyzed should be determined using the following equations to determine the percentage of the
sample that was examined.

                                             39                                 February 2006

-------
                                               Section 3: Guidance forCryptosporidium Laboratories
   r^r,~^* Qx,om;,-,^ -   total volume of resuspended concentrate transferred to IMS (ml) (see Section 3.6.1 .2
   perceni         —
                  —    rn«_,tu -i -H^TIM^T^
     (expressed as a     of Method 1622/1623)
        decimal)                           total volume of resuspended concentrate (ml)

  volume analyzed (L) = percent examined * sample volume filtered (L)

3.6.1.2        Determining the Volume of Resuspended Concentrate to Use for Packed
               Pellets > 0.5 mL
Packed pellets with a volume >0.5 mL must be divided into subsamples. You should use the formula
below to determine the total volume of resuspension required in the centrifuge tube before separating  the
concentrate into two or more subsamples and transferring to IMS.

                                                  pellet volume (mL) after centrifugation
  total volume of resuspended concentrate (mL) required =  -  x 5 mL
                                                                   0.5 mL


3.6.1.3        Example Calculation
Example.       A 10 L field sample was filtered and processed, producing a packed pellet volume of 2.7
               mL. The laboratory transferred 20 mL of the total resuspended concentrate (27 mL) to
               IMS and examination (because a minimum of 2 mL of pellet is required for analysis if 10
               L cannot be examined or 2 filters did not clog [see Section 3.1 .3 of this manual]). The
               laboratory detected 20 oocysts during examination. The following calculations were
               performed to determine the volume analyzed and final concentration.

                                                             2.7 mL
          total volume  of resuspended concentrate (mL) required =                   * 5 mL = 27 mL
                                                             0.5 mL
                                                             20 mL
                                        percent examined -                    = 0.74 (74%)
                                                             27 mL
                                      volume analyzed (L) =    0.74*10L         =  7.4 L
                                                            20 oocysts
                              final concentration (oocysts/L) =                   = 2.7 oocysts/L
                                                              7.4 L
3.6.2          Matrix Spike Recovery Calculations
To determine the percent recovery for a matrix spike (MS) sample the following information is needed:
    •   The number of oocysts counted in the MS sample
    •   The estimated number of oocysts spiked into the MS sample
    •   The number of oocysts counted in the unspiked field sample (to correct for background
       concentration)
                                             40                                  February 2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
                     oocysts counted in MS sample - oocysts counted in unspiked field sample
 percent recovery =                                                                    x 100%
                                      oocysts spiked into MS sample

This calculation assumes that the same sample volume was examined for both the field and MS samples.
If the sample volumes examined are different, you should calculate the number of oocysts per L for both
the field and MS samples before calculating percent recovery. If both a matrix spike (MS) and a matrix
spike duplicate (MSD) are analyzed, then the mean recovery and relative percent difference should be
calculated and compared to the acceptance criteria in Tables  3 and 4 of the December 2005 version of
EPA Method 1623. The percent recovery for each sample should be calculated as described above to
determine the mean recovery.
To calculate the mean percent recovery, you should calculate the percent recovery for each sample, as
described above, and then use the following formula:

                                percent recovery of MS sample + percent recovery of MSD sample
         mean percent recovery =
To calculate the relative percent difference (RPD), the absolute value (without sign) of the difference
between the number of oocysts counted in the MS and MSD should be divided by the mean of the oocysts
counted in both samples to yield a percentage of the difference.  This calculation assumes that the same
volume is analyzed for both the MS and MSD. You should calculate the number counted per L before
calculating the RPD if volumes analyzed are different.
                           oocysts counted in MS - oocysts counted in MSD|
                RPD =  	 x 100%
                         ((oocysts counted in MS + oocysts counted in MSD)/2)

Example.       The laboratory prepared both the MS and MSD by spiking two 10 L samples with 100
               oocysts each. The laboratory detected 45 oocysts in the MS sample and 50 oocysts in the
               MSD. In the 10 L unspiked field sample only 2 oocysts were detected. To determine the
               percent recovery for each sample and the mean recovery and relative percent difference
               of the MS and MSD, the following  calculations were performed. For both the MS and
               MSD, as well as the unspiked field  sample, the entire 10 L sample was filtered and 100%
               of the sample was examined.
                                          45 oocysts - 2 oocysts
                    MS percent recovery = 	 x 100% = 43%
                                              100 oocysts
                                          50 oocysts - 2 oocysts
                  MSD percent recovery =  	 x 100% = 48%
                                              100 oocysts
                                              41                                   February 2006

-------
                                                Section 3: Guidance for Cryptosporidium Laboratories
                                           43% + 48%
                          mean recovery =                 = 45,5%
                                  145 oocysts - 50 oocysts [
                      RPD = 	  * 100% = 10.5%
                               ((45 oocysts + 50 oocysts) / 2)
3.6.3         OPR Sample Calculations
The percent recovery of an OPR sample should be calculated using the following formula:
                                             oocysts detected
                          percent recovery =                     * 100%
                                              oocysts spiked
Example:      The laboratory prepared the OPR sample by spiking 50 L with 150 oocysts. The entire
               sample was filtered and examined. The laboratory detected 76 oocysts.

                                            76 oocysts
                        percent recovery =                  x 100% = 50.7%
                                            150 oocysts

OPR recoveries are compared to the limits for ongoing recovery in Tables 3 and 4 of the December 2005
version of EPA Method 1623. These recoveries are tracked over time using control charts to assess
precision, as discussed in Section 3.3.5, above.

3.7   Electronic Data Reporting

During the LT2 Rule, laboratories will report Cryptosporidium data to their PWS clients electronically
through EPA's LT2/Stage2 Data Collection and Tracking System (DCTS). The DCTS is a web-based
application that allows laboratory users to enter or upload data, then electronically "release" the data to
the PWS  for review, approval, and submission to EPA and the State. Although ownership of the data
resides with the PWS throughout this process, the DCTS increases the ease and efficiency of the data
entry and transfer process from one party to another by transferring the ability to access the data from the
laboratory to the PWS to EPA and the State, and ensuring that data cannot be viewed or changed by
unauthorized parties. A summary of the data entry, review, and transfer process through the DCTS is
provided  in Table 3-8, below.

The data  reporting process is described in more detail below, in Sections 3.7.1 through 3.7.3, and is also
described in detail in the Users' Manual for the LT2/Stage 2 Data Collection and Tracking  System
(DCTS). The DCTS users' manual also provides detailed information on the PWS user registration
process. Information on the DCTS and a downloadable users' manual are available at
http://www.epa.gov/satcwatcr/disii.ifcction/lt2/.
                                             42                                  February 2006

-------
                                                  Section 3: Guidance for Cryptosporidium Laboratories
Table 3-8.     LT2/Stage 2 Data Collection and Tracking System Data Entry, Review, and Transfer
 Laboratory actions

 •  Laboratory posts analytical results to the DCTS

 •  DCTS reduces data and checks data for completeness and compliance with LT2 Rule requirements
 •  Laboratory Principal Analyst confirms that data meets quality control requirements

 •  Laboratory "releases" results electronically to the PWS for review
                                                                                                CD
    Laboratory user cannot edit data after it is released to the PWS
                                                                                                CD
                                                                                                (/]
                                                                                                s
                                                                                                O
                                                                                                CD
 PWS actions
                                                                                                CD
    PWS reviews electronic data through the DCTS
                                                                                                o
    PWS cannot edit data - only review data and either return to laboratory to resolve errors or submit to
EPA
                                                                                                §
                                                                                               T3
                                                                                               <
PWS "releases" data back to the laboratory if it has questions
If no questions, PWS submits data to EPA as "approved" or "contested" (indicating that samples have
been correctly analyzed, but that the PWS contends that they are not valid for use in LT2 binning)

If the PWS does not review the sample result by the deadline for submitting it to EPA (no later than 10
days after the end of the first month following the month when the sample was collected) the sample
result status in the DCTS is automatically changed to "approved" to prevent a monitoring violation report
from generating.
 EPA and State actions

 •   EPA and State users cannot edit data - only review data
 •   EPA and State review data through the DCTS and approve results where appropriate

 •   Contested results

         If EPA/the State rejects the PWS explanation for the contested sample, the sample is marked
         "EPA approved" in the DCTS

         If EPA/the State accepts the PWS explanation for the contested sample, the sample is invalidated
         and the PWS must resample
3.7.1          Data Entry/Upload

The analyst or another laboratory staff member enters a subset of the data recorded at the bench (Section
3.5 of this manual) into the DCTS, either by entering the data using web forms or by uploading data in
XML format (see the DCTS users' manual). In accordance with 40 CFR § 141.706(e)(l), this information
includes the following:

    •   PWS ID

    •   Facility ID

    •   Sample collection date

    •   Sample type (field or MS)

    •   Sample volume filtered (L), to nearest % L
                                                43                                    February 2006

-------
                                                Section 3: Guidance for Cryptosporidium Laboratories
    •  Was 100% of filtered volume examined?
    •  Number of oocysts counted
    •  For samples in which less than 10 L is filtered or less than 100% of the sample volume is
       examined, the laboratory also must enter or upload the number of filters used and the packed
       pellet volume
    •  For samples in which less than 100% of sample volume is examined, the laboratory also must
       report the volume of resuspended concentrate and volume of this resuspension processed through
       immunomagnetic separation
    •  For matrix spike samples, the laboratory also must report the sample volume spiked and
       estimated number of oocysts spiked; these data are not required for field samples

By entering Cryptosporidium data into the system, the laboratory acknowledges that the following QC
requirements were met including: all holding times, sample condition on receipt, results of associated
method blank, OPR, and positive and negative staining controls. The DCTS allows for replacement
samples to be entered and marked as replacement samples. See the DCTS users' manual for guidance.

After the information has been entered or uploaded into the data system, the system will reduce the data
to yield final sample results, in oocysts/L, verify that LT2 Rule Cryptosporidium sample volume analysis
requirements were met for samples in which less than 10 L were analyzed, and calculate MS recoveries.

The laboratory's Principal Analyst under the Lab QA Program is generally responsible for  verifying the
quality and accuracy of all sample results in the laboratory. If inaccuracies or other problems are
identified, the Principal Analyst discusses the sample information with the analyst or data entry staff and
resolves the issues before the data are released for PWS review.

If no inaccuracies or other issues are identified, the laboratory approves the reported data for "release" to
the PWS for review (EPA does not receive the data at this point). When the data are approved, the rights
to the data are transferred electronically by the system to the PWS, and the data can no longer be changed
by the laboratory.

3.7.2          PWS Data Review
After the laboratory has released Cryptosporidium data electronically to the PWS using the DCTS, the
PWS will review the results. The PWS user cannot  edit the data, but if the PWS has an issue with the
sample result, such as if the PWS believes that the sample collection point ID or collection  date is
incorrect, the PWS can release the results back to the  laboratory for issue resolution. In addition to noting
the reason in the DCTS for the return of the data to the laboratory, the PWS may also contact the
laboratory verbally to discuss the issue.

If the PWS determines that the data are accurate, the PWS releases the results to EPA (and  the State, if
applicable) as "approved" results. If the PWS determines that the data are accurate, but believes that the
data are not valid for LT2 binning purposes, the PWS can release the results to EPA and the State as
"contested." Contested samples are those that have been correctly analyzed, but that the PWS contends
are not valid for use in LT2 binning,  and have been submitted to EPA for evaluation.
                                              44                                   February 2006

-------
                                               Section 3: Guidance for Cryptosporidium Laboratories
3.7.3          EPA/State Review
After the PWS has released the results as approved or contested, they are available to EPA and State users
to review through the DCTS. EPA and State users cannot edit the data. EPA or State users approve
results where appropriate. Pursuant to 141.702(a)(2), resampling must occur whenever EPA or the State
rejects results or indicates agreement with a PWS action to contest a result.
3.8   Data Archiving

The PWS is required to keep all original, hardcopy monitoring results associated with LT2 sample
analyses (both initial and second round of monitoring) for 3 years after bin classification for filtered
systems or determination of mean Cryptosporidium level for unfiltered systems [40 CFR § 141.722(a)].
Although it is the PWS's responsibility to meet LT2 Rule data storage requirements for compliance
monitoring samples, including MS samples, the PWS may contract this work to the laboratory.

3.8.1          Hardcopy Data
The following data should be archived:

    •  Bench sheets and slide examination forms for all LT2 monitoring samples, including both field
       samples and MS samples
    •  Bench sheets and slide examination forms for all OPR samples and method blank samples, and
       records of the compliance monitoring samples associated with each OPR sample and blank
       sample

    •  Spike enumeration information received from Cryptosporidium spiking suspension vendors
    •  Bench sheets and slide examination forms for all OPT samples

As part of the Lab QA Program, the laboratory also should maintain the same documentation for their IPR
and IPT data for each method variation used for LT2 samples.

3.8.2         Slides
Although not required, laboratories also may want to archive slides and/or take photographs of slides to
maintain for clients. Slides  should be stored in the dark between 1 ° and  10°C and not frozen, and in the
appropriate type of chamber for the mounting medium used.
As an alternate to the DABCO/glycerol mounting medium, currently specified in EPA Method 1622 and
1623, laboratories may wish to evaluate the use of the elvanol mounting medium, which hardens, and
may be useful for archiving slides. EPA recommends quality control assessment before changing the
mounting medium currently used by the laboratory including initial precision and recovery, method
blank, matrix spike/matrix spike duplicate, and unspiked field samples.  Reagents for the mounting
medium include the following:
    •  8.0 g elvanol (polyvinyl alcohol (PVA))—MP Biomedical cat. no. 151937, Solon, Ohio, or
       equivalent

    •  48.0 g (40 mL) glycerol

    •  10% NaN3 (sodium azide)
    •  DABCO—Sigma-Aldrich, cat no. D-2522, or equivalent
                                             45                                  February 2006

-------
                                              Section 3: Guidance forCryptosporidium Laboratories
    •   Tris buffer—Dissolve 1.2 g Tris (Fisher cat. no. BP152) in 95 mL reagent water, adjust pH to 8.5
       with 1 N HC1

To prepare the medium, you should use the following procedure:

    •   Add 48.0 g (40 mL) glycerol to 8.0 g elvanol and stir.

    •   Add 49.0 mL of reagent water and 1.0 mL 10% NaN3 and stir. Let stand 4 hours at room
       temperature.
    •   Add DABCO in Tris buffer (4.75  g of DABCO in 100 mL Tris buffer, adjusted to pH 8.5 with
       cone. HC1) and stir.
    •   Place mixture in a boiling water bath until the mixture becomes homogenous.

    •   Centrifuge mixture at 2000XG for 10 minutes. Centrifugation of entire mixture in one tube is
       preferable.
    •   Dispense 3- to 5 mL aliquots of the mixture into tubes and store at 0°C to 10°C.

If the mounting medium sets up in the tube during storage, re-heat in boiling water bath or microwave for
a short time to restore mixture to liquid state.

Make sure to test any new reagent first on QC samples to verify that the mounting medium performs
properly before using the medium on any monitoring samples.

Commercially prepared mounting media for archiving slides are also available. For example, Waterborne
Inc. produces an archiving mounting medium (product no. M102) that is fade-retardant and self-sealing.
Vector Laboratories produces both an anti-fade mounting medium and an archiving anti-fade mounting
medium (Vectashield® Mounting Medium and HardSet™ Mounting Medium, product no. H-1000 and
H-1400).
3.9   Equipment, Supplies, Reagents, and Standards
See Sections 6 and 7 of EPA Method 1622/1623 for details on the materials needed to perform the
Cryptosporidium analyses specified in the methods.
                                            46                                February 2006

-------
                SECTION 4: GUIDANCE FOR E. COLI LABORATORIES
The Long-Term 2 Enhanced Surface Water Treatment (LT2) rule requires that large filtered PWSs (those
serving >10,000) perform E. coli and turbidity analyses on source water samples [40 CFR § 141.701(a)].
The Cryptosporidium, E. coli, and turbidity data will be analyzed to confirm or refine the relationship
between Cryptosporidium and E. coli levels in source waters and refine the previously established trigger
levels for E. coli. To reduce costs, small filtered PWSs (those serving <10,000) will use E. coli
monitoring to determine the need for implementing more expensive Cryptosporidium monitoring or
improved treatment.

Turbidity measurement requirements and recommendations are addressed in the Source Water
Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced Surface Water
Treatment Rule (LT2 Rule) (http://www.epa.gov/safewater/disinfection/lt2/). Laboratories performing E.
coli analyses under LT2 must be certified under the Drinking Water Laboratory Certification program
(described below) to analyze  drinking water compliance samples. The procedures required of a
laboratory to become certified are described in the Manual for the Certification of Laboratories Analyzing
Drinking Water, 5th Edition (Reference 5.6). This manual can also be found online at
http://www.cpa.uov/safcwater/labccrt/labindcx.hlml.

LT2 compliance monitoring differs from monitoring currently  being conducted under the Surface Water
Treatment Rule (SWTR) and Total Coliform Rule (TCR). It is important for both PWSs and laboratories
to note the differences in monitoring requirements so that there is no confusion surrounding the samples
that are taken and analyzed for these three regulations.

   •   LT2  requires samples to be analyzed for E.coli while the SWTR requires  that samples be
       analyzed for fecal coliforms.

   •   LT2  requires all E. coli analyses to be quantitative [40 CFR 141.704(b)],  while the TCR requires
       only presence/absence analyses.

   •   LT2  allows the use of mTEC medium (Standard Methods 9213 or EPA Method 1103.1) and m-
       ColiBlue 24® to be used for the detection of E. coli in source water samples. These methods are
       not approved for use  in analysis of samples under the SWTR.

   •   The holding time for LT2 samples is 30 hours, with a possible  extension to 48 hours for samples
       analyzed by the Colilert reagent version of Standard Method 9223B if approved by the State [40
       CFR 141.704(b)]. This extended holding time is for use only with LT2 samples; SWTR samples
       are still required to be analyzed within the 8 hour holding time.

Compliance monitoring for LT2 is in addition to, and does not replace, the monitoring mandated under
SWTR and TCR
                                            47                                 February 2006

-------
                                                         Section 4: Guidance for E. coli Laboratories
4.1    Laboratory Certification Program

Since 1978, the U.S. Environmental Protection Agency has implemented a certification program for
laboratories performing drinking water analyses for compliance with regulations issued pursuant to the
Safe Drinking Water Act and subsequent National Primary Drinking Water Regulations (NPDWR). All
laboratories analyzing drinking water compliance samples must be certified for the analyses they perform
[40 CFR 141.28]. The laboratory certification process and detailed specifications for certification are
described in the Manual for the Certification of Laboratories Analyzing Drinking Water, referred to as
"laboratory certification manual", 5th Edition (Reference 5.6). This manual can be found online at:
http://www.epa.gov/safewater/labcert/labindex.htm1.  Laboratories performing E. coli analyses for LT2
monitoring must be certified by EPA, the National Environmental Laboratory Accreditation Conference
(NELAC), or the State to conduct total or fecal coliform analysis in drinking water under 40 CFR 141.74
using the same E.coli technique that they are certified to use for coliform analysis in drinking water [40
CFR § 141.705(b)]. Approved E. coli methods and references are provided in Table 4-1 [40 CFR §
141.704(b)].

EPA notes that this approach deviates from the approach typically used in its Laboratory Certification
program in that the latter program is based on certification for the specific method (not simply the same
technique) being used in compliance monitoring. EPA strongly encourages all laboratories using these E.
coli methods to seek certification for those methods as soon as is practical.

Table 4-1.      Approved E. coli Methods for LT2 Rule
E. coli Methods Approved for
LT2 Rule
Standard Methods
9221B.1/9221F (LTB-EC-MUG)
Standard Methods 9223B
(Colilert®/Colilert-18®)
Standard Methods 9222B/9222G1
(mEndo/LES-Endo-NA-MUG)
Standard Methods 9222D/9222G
(mFC-NA-MUG)
Standard Methods 921 3D/
EPA Method 1103.1 (mTEC)
EPA Method 1603 Modified
mTEC
EPA Method 1604 Ml medium1
m-ColiBlue24® Broth1
Method Format
multiple tube fermentation/
most probable number
multiple tube/multiple well
membrane filtration, two step
membrane filtration, two step
membrane filtration, one step
membrane filtration, one step
membrane filtration, one step
membrane filtration, one step
Method Citation
Standard Methods for the
Examination of Water and
Wastewater (Reference 5.8)2
Standard Methods for the
Examination of Water and
Wastewater (Reference 5.8)2;
IDEXX Laboratories, Inc.
(Reference 5.9)
Standard Methods for the
Examination of Water and
Wastewater (Reference 5.8)2
Standard Methods for the
Examination of Water and
Wastewater (Reference 5.8)2
EPA Method 1103.1 (Reference
5.1 0); Standard Methods for the
Examination of Water and
Wastewater (Reference 5.8)2
EPA Method 1603 (Reference
5.11)
EPA Method 1604 (Reference
5.12)
Hach Company (Reference 5.13)
 If high levels of non-E. coli total coliforms interfere with the ability to accurately enumerate E. coli despite additional
dilutions, an alternate method should be used (i.e., SM 9222D/9222G, SM 9213D/EPA Method 1103.1, EPA Method
1603, SM 9221B.1/9221F, and SM 9223B)
218ih, 19th, or 20th Editions of Standard Methods for the Examination of Water and Wastewater may be used.
                                              48
February 2006

-------
                                                       Section 4: Guidance for E. coli Laboratories
4.2   Summary of LT2 Rule E. coli Methods

The methods approved under the LT2 Rule were developed by EPA, voluntary consensus standards
bodies (VCSB) (i.e., American Public Health Association (APHA), American Water Works Association
(AWWA), and Water Environment Foundation (WEF) who jointly publish Standard Methods for the
Examination of Water and Waste-water, referred to as "Standard Methods'"), and commercial vendors with
methods submitted to the EPA Office of Water Alternate Test Procedure (ATP) process. For several
procedures, an EPA Method, VCSB method, and/or a commercially available method (submitted to the
ATP program) are approved.

Laboratories should obtain a copy of the methods approved for LT2 monitoring prior to seeking
certification for these methods.  The method summaries provided below only offer a brief overview of the
methods and are not meant to provide details of the methods. Copies of analytical methods may be
obtained from the citations listed in Table 4-1. Copies of analytical methods published by EPA are
available for a nominal cost through the National Technical Information Service (NTIS); U.S. Department
of Commerce; 5285 Port Royal Road; Springfield, VA 22161.  Copies of the EPA methods may also be
downloaded from the EPA Office of Research and Development; National Exposure Research Laboratory
(NERL)-Cincinnati Microbiology home page at www.epa.gov/microbes/.  All other methods must be
obtained from the publisher.  Publishers for all methods are included in Table 4-1; addresses for
publishers can be found in Section 5.

E. coli sample analyses performed under the LT2 Rule must be quantitative; presence/absence E. coli
results are unacceptable. The analytical methods approved under LT2 are for enumeration of E. coli in
ambient water, and the results are reported as number of E. coli per 100 mL water. Public Water Systems
monitoring for E. coli under the LT2 Rule should collect and analyze at least 100 mL of sample to ensure
sufficient volume for sample analysis. In order to obtain quantitative results for the source water samples
for LT2 monitoring, the laboratory will need to select the appropriate configuration of tubes/filtrations
and dilutions/volumes to account for the quality, character, consistency, and anticipated E.  coli density of
the water sample.  See Section 4.5 below for guidance on sample volume and dilutions for LT2 sample
analyses.

To assess the comparability of results obtained with individual methods, it is  suggested that side-by-side
tests be conducted across seasons of the year with water samples routinely tested in accordance with the
most current Standard Methods for  the Examination of Water and Wastewater or EPA alternate test
procedure (ATP) guidelines.  The methods summarized below are approved for the analysis of E. coli
samples under the LT2 Rule [40 CFR § 141.704(b)].

4.2.1         Most Probable  Number (MPN) Methods

4.2.1.1       Standard Methods 9223B: Colilert® and Colilert-18®
Colilert® and Colilert-18® tests are cnromogenic/fluorogenic enzyme substrate tests for the simultaneous
determination of total coliforms and E. coli in water. All tests must be conducted in a format that
provides quantitative results, such as the multiple tube or multiple well formats, e.g., Quanti-tray® (51
well analysis) and Quanti-tray® 2000 (97 well analysis). Using multiple-tube procedures,  laboratories
will need to employ an appropriate tube and dilution configuration of the sample as needed. After the
appropriate sample dilutions/volumes are added, the tubes or trays are incubated for 18 hours when using
Colilert-18® or 24 hours when using Colilert®. Each tube or well is then compared to the reference color
"comparator" available from the manufacturer. A yellow color greater or equal to the comparator
indicates the presence of total coliforms in the sample. The tube or well is then checked for fluorescence
under long-wavelength UV light. A yellow well with fluorescence greater than or equal to the

                                             49                                 February 2006

-------
                                                        Section 4: Guidance forE. coli Laboratories
comparator is positive for E. coli. The most probable number (MPN) value is determined by the number
of positive tubes or wells using MPN tables provided by the manufacturer. E. coli densities are then
calculated and reported as MPN/100 mL (discussed in Section 4.6.1 of this manual below).

4.2.1.2        Standard Methods 9221B.1/9221F: LTB-EC-MUG
The multiple-tube fermentation (MTF) method uses multiple tubes and serial dilutions/volumes in a two-
step procedure to determine E. coli densities in water. In the first step a series of tubes containing lauryl
tryptose broth (LTB) are inoculated with undiluted sample and/or dilutions/volumes of the sample and
mixed. After incubation, tubes are examined for growth (turbidity) and gas, which constitute a positive
presumptive test for coliforms, which include E. coli. Note: Lactose broth may be used in lieu of LTB, if
at least 25 parallel tests are conducted between this broth and LTB using the water samples normally
tested, and this comparison demonstrates that the false-positive rate and false-negative rate for total
coliform using lactose broth is less than 10 percent.

Growth from all presumptive tubes (showing any amount of gas, growth or activity within 48+3 hours of
incubation) is transferred to tubes containing EC-MUG broth for E. coli enumeration.  After incubation,
tubes that exhibit turbidity and fluorescence are considered to be positive for E. coli.  The density of E.
coli is determined from the number of positive tubes using the MPN table provided in SM 9221C. E. coli
densities are reported as MPN/100 mL (See Section 4.6.3 of this manual for guidance  on determining the
MPN). Note: there is no requirement to run the completed phase on 10 percent of all total coliform-
positive tubes on a seasonal basis.

4.2.2         Membrane Filtration  (MF) Methods

Note: When the MF method has not been used previously to test ambient water with high turbidity, large
number of non-coliform bacteria, or samples that may contain organisms stressed by chlorine, a parallel
test should be conducted with a multiple-tube technique to demonstrate applicability and comparability of
results.

4.2.2.1         Standard Methods 9222B/9222G:  mEndo/LES-Endo—NA-MUG and
               Standard Methods 9222D/9222G:  mFC — NA-MUG

These membrane filter methods are two-step MF procedures that provide a direct count of E. coli  in
water. First, a sample is filtered through a 0.45 um, white gridded membrane filter. The filter is then
placed on a pad saturated with mEndo broth or a plate containing mEndo or LES-Endo agar and
incubated. Pink to red colonies with a metallic (golden-green) sheen on mEndo (or LES-Endo) are total
coliforms. If initial determination of fecal coliforms is desired, mFC media can be substituted for
mEndo/LES-Endo. Blue colonies on the filter are fecal coliforms.

In the second step of these methods, the filter is transferred to nutrient agar containing MUG (NA-MUG)
medium and incubated. Sheen colonies from mEndo/LES-Endo or blue colonies from mFC that fluoresce
under a long-wavelength UV light after incubation on NA-MUG are positive for E. coli.  E. coli densities
are reported as CFU/100 mL. Guidance on  determining the number of E.coli/\OQ mL is discussed below
in Section 4.6.2.

If high levels of total coliforms interfere with the ability to accurately enumerate E. coli despite additional
dilutions, an alternate method (e.g., SM 9213D, EPA Method 1603 or 9223B) should be used.
                                             50                                  February 2006

-------
                                                       Section 4: Guidance forE. coli Laboratories
4.2.2.2       Standard Methods 9213D/EPA Method 1103.1: mTEC
The mTEC agar method is a two-step MF procedure that provides a direct count of E. coli in water. In this
method, a water sample is filtered through a 0.45 um white gridded membrane filter, the filter is placed
on mTEC agar (a selective primary isolation medium), and the plate is incubated at 35 °C ± 0.5 °C for 2
hours to resuscitate injured or stressed bacteria and then at 44.5°C ± 0.2 °C for 22-24 hours in a water
bath.  Following incubation, the filter is transferred to a pad saturated with urea substrate medium.  After
15 minutes, all yellow, yellow-brown, or yellow-green colonies are counted as E. coli. E. coli densities
are reported as CPU/100 mL.

4.2.2.3       EPA Method 1603: Modified mTEC

The modified mTEC agar method is a single-step MF procedure that provides a direct count of E. coli in
water. This is a modification of the standard mTEC media (SM 9213D). In this method, a water sample is
filtered through a 0.45 um white gridded membrane filter, the filter is placed on modified mTEC agar,
incubated at 35 °C ± 0.5°C for 2 hours to resuscitate injured or stressed bacteria, and then incubated for
22-24 hours in a 44.5°C ± 0.2 °C  water bath. Following incubation, all red or magenta colonies are
counted as E.  coli. E. coli concentrations are reported as CFU/100 mL.

4.2.2.4       EPA Method 1604: Ml Medium
The MI medium method is a single-step MF procedure used to simultaneously enumerate total coliforms
and E. coli in water. In this method, a water sample is filtered through a 0.45 um white  gridded membrane
filter, the filter is placed on an MI medium and incubated. Following incubation, all blue colonies  under
ambient light  are counted as E. coli. These colonies can be fluorescent or non-fluorescent under long-
wavelength ultraviolet light. E. coli concentrations are reported as CFU/100 mL.

If high levels  of total coliforms interfere with the ability to accurately enumerate E. coli despite additional
dilutions, an alternate method (e.g., SM 9213D, EPA Method 1603 or 9223B) should be used.

4.2.2.5       m-ColiBlue24® Broth
This broth method is a single-step MF test for enumerating total coliforms and E. coli in water. In this
method, a water sample is filtered through a 0.45 um white gridded membrane filter. The filter is placed
on a plate containing an absorbent pad saturated with m-ColiBlue24® broth and incubated.  Following
incubation, all blue colonies are counted as E.  coli. E. coli concentrations are reported as CFU/100 mL.

If high levels  of total coliforms interfere with the ability to accurately enumerate E. coli despite additional
dilutions, an alternate method (e.g., SM 9213D, EPA Method 1603) should be used.
4.3   Quality Assurance/Quality Control for E. coli Analyses

The Manual for the Certification of Laboratories Analyzing Drinking Water (Reference 5.6) describes
criteria and procedures that should be considered when a laboratory undergoes the certification process.
This manual contains elements that are required by federal regulations as well as criteria that are
recommended by EPA for laboratory certification. Additional QA/QC specifications beyond those found
in the laboratory certification manual may be found in Standard Methods, Section 9020 (Reference 5.8).
                                             51                                  February 2006

-------
                                                       Section 4: Guidance fort., coli Laboratories
Each laboratory should have a Quality Assurance plan describing the QA program and QC activities
necessary to meet the laboratory's specific needs. The QA Plan for LT2 monitoring must meet the
specifications of the laboratory's certifying authority and the method used. The QA plan should address
the following issues: personnel policies, equipment and instrument specifications, specifications for
supplies, analytical methods and QC measures, standard operating procedures (SOPs), documentation

specifications, performance evaluation samples, internal and external lab audits, and corrective actions.
For more suggestions on what items should be included in a QA plan, see the Laboratory Certification
Manual, Chapters.

Chapter V, Critical Elements of Microbiology, of the Laboratory Certification Manual details both
required and recommended criteria for a laboratory that is analyzing water samples for microbes.  E. coli
sample results reported under the LT2 Rule should meet the quality control (QC) specifications set forth
in the Laboratory Certification Manual and the methods listed in Table 4-1 above.

4.3.1          Quality Control Specifications Applicable to LT2 Samples

4.3.1.1         Holding Time  and Temperature Requirements for Field  Samples
Source water samples are dynamic environments and, depending on sample constituents and
environmental conditions, E. coli present in a sample can grow or die off, biasing  analytical results.
Samples that are not analyzed immediately after sample collection during LT2 Rule monitoring must be
chilled to reduce biological activity, and preserve the state of source water samples between collection
and analysis. Samples for E. coli analyses must be maintained between 0°C and 10°C if they are shipped
[40 CFR 141.704(b)(3)]. Samples should not be allowed to freeze.
For best results, samples should be analyzed as soon as possible after collection.  Due to the need by some
utilities to ship samples overnight to an off-site laboratory for analysis, the holding time for LT2 E. coli
samples is 30 hours. If the State determines on a case-by-case basis that analyzing an E. coli sample
within 30 hours is not feasible, the State may authorize the holding of an E. coli sample for up to 48 hours
between collection  and initiation of analysis.  E. coli samples held between 30 to 48 hours must be
analyzed by  the Colilert® reagent version of Standard Method 9223B [40 CFR §  141.704(b)(2)]. The E.
coli holding time established for source water monitoring under the LT2 Rule does not apply to E.coli
sample holding time requirements that have been established under other programs and regulations.

Given the importance of maintaining sample temperatures for E.  coli, laboratories should establish
acceptance criteria  for receipt of E. coli samples transported to their laboratory. Several options are
available to measure sample temperature upon receipt at the laboratory and, in some cases, during
shipment; these are provided in Section 2.2 of this manual.

4.3.2          Quality Control Specifications for E. coli Methods

In addition to the overall QC specifications set forth in the Laboratory Certification Manual and in the
analytical methods, laboratories analyzing samples for LT2 Rule E. coli analyses must meet method-
specific incubation  time and temperature requirements.

4.3.2.1         Incubation Time/Temperature Specifications for MPN Methods

The required incubation times and temperatures for MPN methods are provided in Table 4-2.
                                             52                                  February 2006

-------
                                                      Section 4: Guidance for E. coli Laboratories
Table 4-2.     Incubation Time and Temperature Specifications for MPN Methods
Method
Standard Methods 9223B
Standard Methods 9221 B/9221 F
Media
Colilert®
Coliert-18®
LIB
EC-MUG
Incubation Time/Temperature
24 - 28 hours at 35°C ± 0.5°C
18 - 22 hours at 35°C ± 0.5°C
24 ± 2 hours at 35°C ± 0.5CC and
48±3hoursat35°C±0.5cC
24 + 2 hours at 44.5°C ± 0.2°C
4.3.2.2        Incubation Time and Temperature Specifications for MF Methods
The required incubation times and temperatures for MF methods are provided in Table 4-3.

Table 4-3.     Incubation Time and Temperature Specifications for Membrane Filter Methods
Method
Standard Methods 9222B/9222G
Standard Methods 9222D/9222G
Standard Methods 9213D/EPA
Method 1103.1
EPA 1603
EPA 1604
m-ColiBlue24® Broth
Media
mENDONA-MUG
Les-ENDO-NA-MUG
mFONA-MUG
mTEC agar
Modified mTEC
Ml agar
m-ColiBlue24® Broth
Incubation Time/Temperature
22 - 24 hours at 35°C ± 0.5°C-
4hoursat35°C±0.5°C
22 - 24 hours at 35°C ± 0.5°C-
4hoursat35°C±0.5°C
22 - 26 hours at 44.5°C ± 0.2°C •
4 hours at 35°C±0.5°C
2 hours at 35°C + 0.5°C -
22 - 24 hours at 44.5°C ± 0.2°C
2hoursat35°C + 0.5°C -
22 - 24 hours at 44.5°C ± 0.2°C
24±2 hours at 35°C±0.5°C
24 hours at 35°C ± 0.5°C
4.4   Sample Collection Procedures

PWSs are required to collect source water samples for the LT2 Rule from the plant intake prior to
chemical treatment, unless approved by the State to collect the source water sample after chemical
treatment [40 CFR § 141.703(b)(2)]. Systems that recycle filter backwash water must collect source water
samples prior to the point of filter backwash water addition [40 CFR § 141.703(c)]. All Cryptosporidium,
E. coli, and turbidity source water samples collected under LT2 Rule requirements must be collected from
the same appropriate sampling location. Detailed guidance on sample collection procedures for E. coli
samples for on-site analysis and for shipment to an off-site laboratory for analysis are provided in the
Source Water Monitoring Guidance Manual for Public Water Systems for the Long Term 2 Enhanced
Surface Water Treatment Rule (LT2 Rule). This manual is available for download from
http://www.epa.gov/safewalcr/disinFcction/lt2/.
                                            53
February 2006

-------
                                                       Section 4: Guidance for E. coli Laboratories
4.5    Sample Volume and Dilution Guidance

Because E. coli analyses will be performed on source waters, rather than finished drinking waters,
multiple sample volumes/dilutions may be necessary to assess high E. coli levels. Initially all laboratories
should consider analyzing four sample volumes (100, 10, 1.0, and 0.1 mL) for all methods except MTF.
For MTF methods, the PWS should initially consider analyzing five sample volumes of 10, 1.0, 0.1, 0.01,
and 0.001  mL. Sample volumes may need to be adjusted based on confounding conditions (e.g., high
turbidity, heavy rainfall, etc.).

If the PWS has historical data demonstrating that E. coli levels are consistently low, they may drop the
smaller sample volumes. If there has been a substantial rainfall in the 24 hours prior to sample collection
causing runoff, sample volumes may need to be adjusted in order to obtain valid results and avoid data
that are above the analytical range of the method. This could also be a concern if the PWS eliminates the
100 mL sample volume due to historical data indicating that values tend to be very high and the E. coli
levels are significantly lower than anticipated, requiring the PWS to report data that are below the
analytical  range of the method. EPA recommends bracketing the target sample volume (i.e., analyzing a
sample volume above and below the sample volume expected to yield useable data) to account for
potential variability.

4.5.1         Sample Volume and Dilution Guidance  for Multiple-Well Methods
The analysis of water samples under LT2 using multiple-well methods generally requires the use of four
aliquots of samples (100, 10, 1.0, and 0.1 mL). As mentioned above, sample volumes may need to be
adjusted based on confounding conditions (e.g., high turbidity, heavy rainfall, etc.). Because a 100 mL
volume is  necessary to fill all of the wells, it will be necessary to add the aliquots of the sample to sterile
reagent water blanks.  The total volume after the sample aliquot is added to the reagent water blank should
be approximately 100 mL (i.e., a 90 mL blank should be used for the 10 mL sample volume, a 99- mL
reagent water blank should be used for the 1.0 mL aliquot).  Colilert® or Colilert|8® reagent should be
added to the sterile reagent water blanks prior to adding sample. If the sample is added to the reagent
water before the Colilert® or Colilerti8® reagent, bacterial cells may lyse. (Reference 5.14)

4.5.2         Sample Volume and Dilution Guidance for Multiple-Tube Methods
For the analysis of water samples by a multiple-tube technique (e.g., Colilert® in a multiple-tube format, '
SM 9222B LTB/EC-MUG) under LT2, laboratories should consider the use of a 20-tube most probable
number format (i.e., 5 tubes at each of the four dilutions, 10.0, 1.0, 0.1, and 0.01 mL). Initially, it is
recommended that five sample volumes (i.e., 5 tubes for each sample volume, 10.0, 1.0, 0.1, 0.01, and
0.001 mL) are analyzed. Additional guidance on selection of dilutions for multiple-tube methods is
available in Standard Methods 9221 (Reference 5.8).

4.5.3         Sample Volume and Dilution Guidance for Membrane Filtration
For the analysis of water samples under LT2, it is recommended that four different aliquots of sample
(100, 10, 1.0,  and 0.1 mL) be analyzed when using membrane filtration methods.  Alternate sample
volumes may be used if necessary. To ensure adequate distribution of bacteria during filtration, dispense
aliquots of the sample, with the exception of the 100 mL volume, into sterile buffered water blanks (at
least 30 mL, depending on the sample volume analyzed). Alternately, 10 mL of sterile buffer may be
added to the filter apparatus prior to adding the sample. The buffered water minimizes clumping of the
bacteria on the filter surface.
                                            54                                 February 2006

-------
                                                       Section 4: Guidance for E. coli Laboratories
4.6   E. coli Data Recording and Calculations

Laboratories performing E. coli analyses during the LT2 Rule must follow any data recording practices
required by the certification authority granting the certification to the laboratory, and should follow any
recommended data recording practices outlined in the Laboratory Certification Manual, Chapter V,
Section 8.

Some of the recommended information to be recorded for each E. coli sample is as follows. Data for each
sample should be recorded on a sample collection form used in the field and maintained in the same file
as the E. coli laboratory data or on the laboratory data form. Note that some of these data will be entered
into the LT2/Stage 2 Data Collection and Tracking System (DCTS):

    •   PWS ID
    •   Facility ID
    •   Sample Identification (if any)
    •   Sample collection point ID
    •   Sample collection date and time
    •   Sample type
    •   Analytical method number
    •   Method type
    •   Name of sampler
    •   Turbidity
    •   Source water type

The laboratory should record all of the primary measurements associated with each analysis, as they are
needed to calculate the final concentration ofE. coli per 100 mL. Primary measurements for membrane
filtration  methods will include the volumes filtered and the plate counts for each volume filtered. The
multiple-well and multiple-tube formats will include the volumes or dilutions of samples analyzed and the
number of positive wells or tubes per each volume analyzed. Method-specific data to record for each of
the individual method types are discussed in Sections 4.6.1 — 4.6.3 of this manual.

The suggested data elements shown below are important as they allow the laboratory to ensure that final
sample results can be verified using primary data and to demonstrate that sample analyses were
performed within method-specified holding times.  Please note that not all of the data that may be
recorded  will be reported in the DCTS. E. coli data reporting under the LT2 Rule is discussed  in Section
4.7 of this manual.

The final E. coli concentration for field samples will be reported as CPU/100 mL or MPN/100 mL
depending on the method used for analysis. If no E. coli are detected in the sample, the detection limit
based on  the volume of sample analyzed may be reported (e.g., <1  CPU /100 mL or <1.8 MPN/100 mL)
or a zero  for purposes of the DCTS.

In addition, this section also provides standardized procedures for determining E. coli concentration for
LT2 samples for the various analytical techniques that are approved for use under the rule. Because these
analyses will be performed on source waters rather than finished drinking waters, and multiple dilutions
may be necessary to assess higher E. coli levels, it is recommended that laboratories consistently use the
same analytical procedures for determining E. coli sample concentrations to reduce variability.
                                             55                                  February 2006

-------
                                                        Section 4: Guidance for E. coli Laboratories
4.6.1          Multiple-Well Data
In addition to the data elements contained in the bullcted list in Section 4.6 of this manual, laboratories
using Colilert® and Colilert-18® methods for E. coli sample analyses in support of the LT2 Rule should
record the data elements specific to multiple-well techniques. Data elements to be recorded for the 97-
well format (Quanti-Tray 2000®) are noted in Table 4-4; data to be recorded for the 51-well format
(Quanti-Tray®) are noted in Table 4-5. These elements include the primary measurements needed to
calculate the E. coli concentration in the sample as well as all method-required incubation and read times
needed to verify that the sample analyses were conducted under analytical control.

Calculations for determining the E. coli concentration using the Quanti-Tray 2000® (97-well) and
Quanti-Tray® (51-well) formats are provided after each table.


4.6.1.1         Data Elements for Quanti-Tray 2000® (97-well) Analyses
The recommended  data elements to record for Quanti-Tray 2000® (97-well) analyses are provided in
Table 4-4.

Table 4-4.      Recommended Minimum Record for Quanti-tray 2000® Colilert® and Colilert-18®
	Analyses	
Primary Measurements
ml_ of sample added to tray (does not include reagent water volume)
Large wells positive: Total coliform positive and UV fluorescence
Small wells positive: Total coliform positive and UV fluorescence
Holding Time / Incubation Time Information
Incubation start: date/time
Incubation end: date/time
Additional incubation start: date/time
Additional incubation end: date/time
4.6.1.2        Determining E. coli Concentration Using Colilert® and Colilert-18® Quanti-
               Tray 2000® Data

A.     Select appropriate dilution to yield countable results. If multiple dilutions are used, the tray
       exhibiting positive wells in the 40 percent and 80 percent range (39 to 78 total positive large and
       small wells) should be used to determine MPN value.
Note:          The analytical result can be automatically calculated using the LT2/Stage 2 Data
               Collection and Tracking System. See Section 4.7 of this manual for additional
               information.
                                              56                                  February 2006

-------
                                                        Section 4: Guidance for E. coli Laboratories
B.      Determine MPN. Use the MPN tables provided by the vendor. To determine the MPN using
        these tables, locate the number at the intersection of large positive wells and small positive wells
        from the appropriate dilution, identify the corresponding MPN/100 mL in the table provided by
        the vendor. Large well values are located in the left column; small well values are located in the
        top row. For example, if a 100 mL sample was analyzed, and there were 29 large positive wells
        and 5 small positive wells, the corresponding MPN would be 49.6 MPN/100 mL.

C.      Adjust for dilution factor. Because the MPN/100 mL values in the table are based on 100 mL
        samples, the MPN value should be adjusted if less than 100 mL of sample volume was analyzed.
        Use the following calculation to adjust the MPN to account for the dilution:

                                                  100
      Analytical result =   MPN value x 	
                                           mL of sample analyzed

Example:
               Volume analyzed = 10 mL of sample (in 90 mL of dilution water)
               Large wells positive = 39
               Small wells positive = 5
               The MPN value calculated based on the intersection of 39 and 5 in the table.

               MPN = 81.3
                                   100
         Analytical result =   81.3 x  	   = 813 E. coli MPN/100 mL
                                    10
4.6.1.3        Data Elements for Quanti-Tray® (51 -well) Analyses
The recommended data elements to record for Quanti-Tray® (51 -well) analyses are provided in Table 4-
5.

Table 4-5.      Recommended Data to Record for Quanti-Tray® Colilert® and Colilert-18®
	Analyses	
Primary Measurements
mL of sample added to tray (does not include reagent water volume)
 Number of wells positive: Total coliform positive and UV fluorescence
Holding Time / incubation Time information
 Incubation start: date/time
 Incubation end: date/time
Additional incubation start: date/time
Additional incubation end: date/time
                                              57                                  February 2006

-------
                                                       Section 4: Guidance for E. coli Laboratories
 4.6.1.4        Determining E. coli Concentration Using Colilert® and Colilert-18® (51-well)
               Data
 A.      Select appropriate dilution. If multiple dilutions are used, the tray exhibiting 80 percent positive
        wells (41 positive wells) should be used to determine MPN value.
Note:          The analytical result can be automatically calculated using the LT2/Stage 2 Data
               Collection and Tracking System (DOTS). See Section 4.6 of this manual for additional
               information.
B.      Determine MPN. Using the number of positive wells from the appropriate dilution, identify the
        corresponding MPN/IOO mL using the MPN table provided by the vendor. For example, if a 100
        mL sample was analyzed, and there were 41 positive wells, the corresponding MPN would be
        83.1 MPN/lOOmL.

C.      Adjust for dilution factor. Because the MPN/IOO mL values in the table are based on 100 mL
        samples, the MPN value should be adjusted if less than 100 mL of sample volume was analyzed.
        Use the following calculation to adjust the MPN to account for the dilution:

                                    100
        MPN value x    	       =  E. coli MPN/100 mL
                            mL of sample analyzed

Example:

              Volume analyzed (mL) =10 mL (in 90 mL of dilution water)
              Number of positive wells = 41
              MPN = 83.1

              The analytical result is calculated as follows:

                                   100
         Analytical result =   83.1 x 	   = 831 £. coli MPN/100 mL
                                   10

4.6.2         Membrane Filtration Data

In addition to the general sample data contained in the bulleted list in Section 4.6 of this manual,
laboratories using membrane filtration methods for E. coli sample analyses in support of the LT2 Rule
should record the data elements specific to this technique. These are noted in Table 4-6, and include the
primary measurements needed to calculate the E. coli concentration in the sample, as well as all method-
required incubation and read times needed to verify that the sample analyses were conducted under
analytical control.

4.6.2.1        Data Elements for Membrane Filtration Analyses

The recommended data elements to record for membrane filtration analyses are provided in Table 4-6.
                                             58                                 February 2006

-------
                                                        Section 4: Guidance for E. coli Laboratories
 Table 4-6.     Recommended Data Elements for Record for Membrane Filtration Analyses
Primary Measurements
Filter 1 volume (mL) (e.g., 100 mL)
CPU on Filter 1
Filter 2 volume (mL) (e.g., 10 mL)
CFU on Filter 2
Filter 3 volume (mL) (e.g., 1.0 mL)
CFU on Filter 3
Filter 4 volume (mL) (e.g., 0.1 mL)
CFU on Filter 4
Holding Time / Incubation Time Information
Primary isolation medium (e.g., mENDO, mFC) incubation start: date/time
Primary isolation medium (e.g., mENDO, mFC) incubation end: date/time
Secondary isolation medium (e.g, NA-MUG) incubation start: date/time
Secondary isolation medium (e.g, NA-MUG) incubation end: date/time
4.6.2.2        Determining E. coli Concentrations Using Membrane Filter Data
A.     E. coli counts should be determined from the volume(s) filtered that yielded 20 to 80
       E. coli colonies (20-60 for mFC-NA-MUG), and not more than 200 total colonies per plate.
       (Guidance for samples that do not yield countable plates is provided in Sections E and F below)
       (References 5.8 and 5.10-5.12)
Note:         The analytical result can be automatically calculated using the LT2/Stage 2 Data
               Collection and Tracking System (DCTS). See Section 4.7 of this manual for additional
               information.
B.  If there are greater than 200 colonies per membrane, even for the lowest dilution, the result is
    recorded as "too numerous to count" (TNTC). These results should not be reported for LT2
    monitoring, as they cannot be used for the required data analyses. During the next sampling event,
    analyze an additional, lower dilution volume (the highest dilution volume may be omitted) unless
    conditions were unusual (e.g., heavy rains, flooding, etc.) during the sampling event yielding TNTC
    for all dilutions.

C.  If colonies are not sufficiently distinct for accurate counting, the result is recorded as "confluent
    growth" (CNFG). To prevent CNFG from occurring, smaller sample aliquots should be filtered. For
    example, if sample volumes of 100, 10, 1 and 0.1 mL are analyzed and even the 0.1 mL plate results
    in CNFG, then potentially 0.01 mL should be analyzed during the next sampling event. For sample
    volumes less than 1 mL, serial dilutions should be used, and 1  mL volumes of the dilutions should be
    filtered.  The 100 mL volume can be eliminated. Note: If growth is due to high levels of total coliforms
    but low E. coli then another method should be chosen for analyses that does not rely on total coliform
    determination prior to or simultaneously with E. coli determination.
                                              59                                  February 2006

-------
                                                      Section 4: Guidance for E. coli Laboratories
Note:         Results that are TNTC or CNFG are not appropriate for LT2 microbial data
              analysis, and cannot be entered into the L T2/Stage 2 Data Collection and
              Tracking System (DOTS). These results should not be reported.
D.     Using the E. coli counts from the appropriate dilution, E. coli CPU/100 mL should be calculated
       based on the following equation:

                                100
           E. coli CFU x   	   = £. coli CFU/100 mL
                           mL sample filtered

       Example 1:

              Filter 1 volume = 100 mL                    CFU = TNTC
              Filter 2 volume = 10 mL                     CFU = 40
              Filter 3 volume = 1.0 mL                    CFU = 9
              Filter 4 volume = 0.1 mL                    CFU = 0
Using the guidance on countable colonies in Step A, the count from the 10 mL plate will be used to
calculate the E. coli concentration for the sample:

                         100
        40 E. coli CFU x  	 = 400 E. coli CFU/100 mL
                         10 mL
E.     If no E. coli colonies are present, the detection limit (i.e., 1 CFU per volume filtered)  is
       calculated and reported per 100 mL (see example below).

       Example 2:

              Filter 1 volume (mL) = 100 mL        CFU = 0
              Filter 2 volume (mL) = 10 mL         CFU = 0
              Filter 3 volume (mL) = 1.0 mL         CFU = 0

           Detection            100mL
                      	   = E. coli CFU/100 mL
             limit =       Largest volume filtered


         100 mL
         100mL
                  = <1 £. co///100mL
                                            60                                February 2006

-------
                                                     Section 4: Guidance for E. coli Laboratories
       Example 3:

              Filter 1 volume (mL) =100 mL        CPU = Lab accident, no data available
              Filter 2 volume (mL) = 10 mL         CPU = 0
              Filter 3 volume (mL) = 1.0 mL        CPU = 0

              Calculation of E. coli/'100 mL:

         100mL
                  = <10£. co//CFU/100mL
         10 mL
F.     If there are no filters with E. coli counts in the 20-80 colony range (20-60 for mFC-NA-MUG),
       sum the E. coli counts on all filters, divide by the volume filtered and report as number per 100
       mL.

       Example 4:

              Filter 1 volume (mL) = 50 mL                CPU = 15
              Filter 2 volume (mL) = 25 mL                CPU = 6
              Filter 3 volume (mL) = 10 mL                CPU = 0

              The analytical result is calculated as:

                         100
         (15 + 6+ 0)x 	 = 25£ co/;CFU/100mL
                      (50+25+10)

       Example 5:

              Filter 1 volume (mL) = 50 mL                CPU = 105
              Filter 2 volume (mL) = 25 mL                CPU = 92
              Filter 3 volume (mL) = 10 mL                CPU = 85

              The analytical result is calculated as:

                             100
        (105+ 92 +85) x  	 = 332£ co//CFU/100mL
                         (50 + 25+ 10)

       Example 6:

              Filter 1 volume (mL) = 100 mL               CPU = 82
              Filter 2 volume (mL) = 10 mL                CPU = 18
              Filter 3 volume (mL) = 1.0 mL               CPU = 0

              The analytical result is calculated as:
                             100
         (82 + 18 + 0) x     	 = 90 £ coli CFU/100 mL
                         (100 + 10+1)

                                           61                                 February 2006

-------
                                                        Section 4: Guidance for E. coli Laboratories
        Example 7:

               Filter 1 volume (mL) = 50 mL                 CPU = TNTC
               Filter 2 volume (mL) = 25 mL                 CPU = TNTC
               Filter 3 volume (mL) = 10 mL                 CPU = 83

               The analytical result is calculated as:

                 100
         83 x  	 = 830 E. coli CFU/100 mL
                  10

4.6.3          Multiple-Tube Data
In addition to the general sample data contained in the bulleted list in Section 4.6 of this manual,
laboratories using multiple-tube methods for E. coli sample analyses in support of the LT2 Rule should
record the data elements specific to these techniques. These data elements are noted in Table 4-7 for 15-
tube most probable number methods and Table 4-8 for 15-tube multiple-tube fermentation methods. The
data elements include the primary measurements needed to calculate the E. coli concentration in the
sample, as well as all method-required incubation and read times needed to verify that the sample
analyses were conducted under analytical control.

Calculations for determining the E. coli concentration using multiple tube formats are provided in Section
4.6.3.2.

4.6.3.1         Data Elements for Multiple-Tube Analyses
The recommended data elements to record for 15-tubc most probable number methods are provided in
Table 4-7 and Table 4-8 for 15-tube multiple-tube fermentation methods.

Table 4-7.      Recommended Data Elements to Record for 15-Tube MPN Methods  (Colilert®)
Primary Measurements (Note: not all dilutions listed below may be used)
Number of positive 10.0 mL tubes: Total coliform positive and UV fluorescence
Number of positive 1.0 mL tubes. Total coliform positive and UV fluorescence
Number of positive 0.1 mL tubes: Total coliform positive and UV fluorescence
Number of positive 0.01 mL tubes: Total coliform positive and UV fluorescence
Number of positive 0.001 mL tubes: Total coliform positive and UV fluorescence
Holding Time /Incubation Time Information
Incubation start: date/time
Incubation end: date/time
Additional incubation start: date/time
Additional incubation end: date/time
                                             62                                  February 2006

-------
                                                        Section 4: Guidance for E. coli Laboratories
Table 4-8.      Recommended Data Elements to Record for 15-Tube Fermentation Methods
Primary Measurements (Note: not all dilutions listed below may be used)
Number of positive tubes 10.0 ml
Number of positive tubes 1.0mL
Number of positive tubes 0.1 mL
Numberof positive tubes 0.01 mL
Number of positive tubes 0.001 ml
Number of positive tubes 0.0001 ml
Holding Time /Incubation Time Information
LIB incubation start date/time
LIB 24-hour incubation end date/time
LTB 48-hour incubation read date/time
EC-MUG incubation 24-hour read date/time (from 24-hour LTB)
EC-MUG incubation 24-hour read date/time (from 48-hour LTB)
4.6.3.2        Determination of E. coli Concentrations Using Multiple-Tube Methods1:
The guidance and examples for determining E. coli concentrations using multiple-tube methods are based
on the revision of Standard Methods 9221C included in the 2007 Supplement to the 20?h Edition of
Standard Methods, approved by the Standard Methods Committee in 1999.
Note:         The analytical result can be automatically calculated using the LT2/Stage 2 Data
               Collectionand Tracking  System (DCTS). See Section 4.7 of this manual for
               additional information.
A.     For each sample volume (e.g., 10, 1, 0.1, and 0.01 mL or additional sample volumes as
       necessary), determine the number of positive tubes out of five.

B.     A dilution refers to the volume of original sample that was inoculated into each series of tubes.
       Only three of the dilution series will be used to estimate the MPN. The three selected dilutions are
       called significant dilutions and are selected according to the following criteria. Examples of
       significant dilution selections are provided in Table 4-9, below.

       •   Choose the highest dilution (the most dilute, with the least amount of sample) giving positive
           results in all five tubes inoculated and the two succeeding higher (more  dilute) dilutions.
           (Table 4-9, Example A).

       •   When the lowest dilution (least dilute) tested has less than five tubes with positive results,
           select it and the two next succeeding higher dilutions (Table 4-9, Examples B and C).
        Adapted from 2001 Supplement to the 20"' Edition of Standard Methods 9221 C: Explanation of Bacterial
Density. This supplement is available for download at hUp://www.techstreet.coni/cgi-bin/delail?prodiict id-923645.


                                              63                                  February 2006

-------
                                                          Section 4: Guidance for E. coli Laboratories
C.
•   When a positive result occurs in a dilution higher (more dilute) than the three significant
    dilutions selected according to the rules above, change the selection to the lowest dilution
    (least dilute) that has less than five positive results and the next two higher dilutions (more
    dilute) (Table 4-9, Example D).

•   When the selection rules above have left unselected any higher dilutions (more dilute) with
    positive results, add those higher-dilution positive results to the results for the highest
    selected dilution (Table 4-9, Example E).

•   When there are not enough higher dilutions tested to select three dilutions, then select the
    next lower dilution (Table 4-9, Example F).

MPN values must be adjusted based on the significant dilutions series selected above. Because
the MPN/100 mL values in the table are based on the analysis of 10, 1, and 0.1 mL dilutions, per
method requirements, the MPN value must be adjusted if these are not the significant dilutions
selected. Use the following calculation to adjust the MPN  when the 10, 1, and 0.1 mL dilutions
are not the significant dilutions selected:
                             MPN value
     Analytical result =
                       # of mL in middle dilution
                                        = £. coli MPN/100 mL
Table 4-9.      Examples of Different Combinations of Positive Tubes (Significant Dilution
Results Are in Bo/dand Underlined)
Example
A
B
C
D
E
F
Least dilute
Most dilute
(Lowest)
(Highest)
10
mL
5
4
0
5
5
5
1 mL
5
5
0
4
4
5
0.1
mL
1
1_
1
4
4
5
0.01
mL
0
0
0
1
0
5
0.001
mL
0
0
0
0
1
2
Combination
of positives
5-1-0
4-5-1
0-0-1
4-4-1
4-4-1
5-5-2
MPN Index from
Standard Methods
33
48
1.8
40
40
540
E. coW/100 mL
(after adjustment)
330
48
1.8
400
400
54,000
Example A:
               The significant dilution series for the 5-1-0 combination of positives includes the
               1, 0.1, and 0.01 mL dilutions. Because the 10, 1, and 0.1 mL dilutions were not
               selected, an adjustment is necessary to account for the dilutions selected:
                                            33
                         Analytical result =
                                           = 3305. coli/100 mL
                                           0.1
                                               64
                                                                            February 2006

-------
                                                        Section 4: Guidance for E. coli Laboratories
Example B:


Example C:


Examples D and E:
       Because the 10, 1, and 0.1 mL dilutions are the significant dilutions, no
       adjustment is necessary and the result is 48 E. co//'/100 mL.

       Because the 10, 1, and 0.1 mL dilutions are the significant dilutions, no
       adjustment is necessary and the result is 1.8 E. coli/100 mL.

       The significant dilution series for the 4-4-1 combination of positives includes the
       1, 0.1, and 0.01 mL dilutions. Because the 10, 1, and 0.1 mL dilutions were not
       selected, an adjustment is necessary to account for the dilutions selected:
Example F:
                      Analytical result =
                                         40
                                    = 400E. coli /100mL
                                         0.1
       The significant dilution series for the 5-5-2 combination of positives includes the
       0.1, 0.01 and 0.001 mL dilutions. Because the 10, 1, and 0.1 mL dilutions were
       not selected, an adjustment is necessary to account for the dilutions selected:
                                        540
                      Analytical result =
                                        0.01
                                 = 54,000 E. co///100mL
4.7    Electronic Data Reporting

During the LT2 Rule, laboratories will report E. coli data electronically through EPA's LT2/Stage 2 Data
Collection and Tracking System (DCTS) to the PWS staff responsible for approving and submitting
monitoring results to EPA. The DCTS is a web-based application that allows laboratory users to enter or
upload data, then electronically "release" the data to the appropriate PWS staff for review, approval, and
submission to EPA and the State. Although ownership of the data resides with the PWS throughout this
process, the  DCTS increases the ease and efficiency of the data entry and transfer process from one party
to another by transferring the ability to access the data from the laboratory to the PWS to EPA and the
State, and ensuring that data cannot be viewed or changed by unauthorized parties. A summary of the data
entry, review, and transfer process through the DCTS for both  Cryptosporidium and E. coli samples is
provided in Table 3-8, in Section 3.7.

The data reporting process is summarized below, in Sections 4.7.1 through 4.7.3, and discussed in detail
in the Users' Manual for the LT2/Stage 2 Data Collection and Tracking System (DCTS). The DCTS
users' manual also provides detailed information on the laboratory registration process. Information on
the  DCTS and a downloadable users' manual are available at
http://www.epa.gov/safewater/lt2/index.html.
4.7.1
Data Entry/Upload
The analyst or another laboratory staff member enters a subset of the data recorded at the bench (Section
4.6 of this manual) into the DCTS either by entering the data using web forms or by uploading data in
XML format. This information includes the following:

    •   Sample ID (optional)
    •   PWS ID
                                              65
                                                                   February 2006

-------
                                                         Section 4: Guidance forE. coll Laboratories
    •   Facility ID

    •   Sample collection date

    •   Analytical method number

    •   Method type

    •   Source water type (provided by PWS on sample collection form)

    •   E. colil 100 mL (see note below)

    •   Turbidity result (provided by PWS on sample collection form)

Note: The laboratory may enter the final result for the E. coli sample or may enter the primary
measurements recorded at the bench, and have the DCTS automatically calculate the final E. coli
concentration. Because this information is specific to method type (membrane filtration, multiple-tube,
51-well, and 97-well), the system provides different entry screens for each method type. By entering E.
coli data into the system, the laboratory acknowledges that the following QC requirements were met: all
holding and incubation times and temperatures, sample condition on receipt, all method-specific QC
requirements, and all QA/QC criteria and procedures specified in the Lab Certification Manual.

The laboratory should establish a contact person that is responsible for verifying the quality and accuracy
of all sample results in the laboratory, and should review and approve  the results before they are
submitted to the PWS for review. If inaccuracies or other problems are identified, the official contact
discusses the sample information with the analyst or data entry staff and resolves the issues before the
data are released to the PWS for review.

If no inaccuracies or other issues are identified, the  laboratory's official contact approves the data for
"release" to the PWS for review (EPA does not receive the data at this point). When the data are approved
by the laboratory, the rights to the data are transferred electronically by the system to the PWS, and the
data can no longer be changed by the laboratory.

4.7.2         PWS Data Review

After the laboratory has released E. coli data  electronically to the PWS using the DCTS, the PWS will
review the results.  The PWS user cannot edit the data, but if the PWS has an issue with the sample result,
such as if the PWS believes that the sample collection point or collection date is incorrect, the PWS can
release the results back to the laboratory for issue resolution. In addition to noting the reason in the DCTS
for the return of the data to the laboratory, the PWS should contact the laboratory verbally to discuss the
issue.
If the PWS determines that the data are accurate, the PWS releases the results to EPA (and the State, if
applicable) as "approved" results. If the PWS determines that the data  are accurate,  but believes that the
data are not valid for other reasons, the PWS  can release the results as  "contested."

4.7.3         EPA/State Review

After the PWS has released the results as approved or contested, they are available to EPA and State users
to review through the DCTS. EPA and State users cannot edit the data. EPA or State users approve
results where appropriate. Pursuant to 141.702(b)(2), resampling must occur whenever EPA or the State
rejects results or indicates agreement with a PWS action to contest a result.
                                               66                                  February 2006

-------
                                                        Section 4: Guidance for E. coli Laboratories
4.8   Data Archiving

The PWS is required to keep all original, hardcopy monitoring results associated with LT2 sample
analyses (both initial and second round of monitoring) for 3 years after bin classification for filtered
systems or determination of the mean Cryptosporidium level for unfiltered systems [40 CFR §
141.722(a)J. Although it is the PWS's responsibility to meet LT2 Rule data storage requirements for
compliance monitoring samples, including MS samples, the PWS may contract this work to the
laboratory.
                                             67                                 February 2006

-------
This page intentionally left blank
             68                                   February 2006

-------
                                                     SECTION 5: REFERENCES

5.1    USEPA. 2006. National Primary Drinking Water Regulations: Long Term 2 Enhanced Surface
       Water Treatment Rule. 40 CFR §s 9, 141, and 142.

5.2    USEPA. 2005. Method 1622: Cryptosporidium in Water by Filtration/IMS/FA. U.S.
       Environmental Protection Agency, Office of Water, Washington, D.C. EPA-815-R-05-001.


5.3    USEPA. 2005. Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. U.S.
       Environmental Protection Agency, Office of Water, Washington, D.C. EPA-815-R-05-002.
5.4    USEPA. 1998. National Primary and Secondary Drinking Water Regulations: Analytical
       Methods for Regulated Drinking Water Contaminants. Final Rule, Federal Register: September
       3, 1998 63(171), FR 47097-47114..


5.5    USEPA. 2002. Laboratory Quality Assurance Evaluation Program for Analysis of
       Cryptosporidium Under the Safe Drinking Water Act; Agency Information Collection: Proposed
       Collection; Comment Request. Federal Register: March 4, 2002. 67 (42) FR 9731 - 9734.


5.6    USEPA 2005. Manual for the Certification of Laboratories Analyzing Drinking Water; Criteria
       and Procedures; Quality Assurance. Fifth Edition. EPA 815-R-05-004. Office of Ground Water
       and Drinking Water, U.S. Environmental Protection Agency, 26 Martin Luther King Drive,
       Cincinnati, OH 45268.

5.7    USEPA. 2004. EPA Microbiological Alternate Test  Procedure (ATP) Protocol for Drinking
       Water, Ambient Water, and Wastewater Monitoring Methods Guidance. Washington, DC.  EPA
       821-B-03-004.


5.8    American Public Health Association. 1998. Standard Methods for the Examination of Water and
       Wastewater, 20th Edition. American Public Health Association, Washington, D.C. Standard
       Methods may be ordered from: American Water Works Association Bookstore, 6666 West
       Quincy Avenue,  Denver, CO 80235.

5.9    IDEXX Laboratories, Inc., Description of Colilert®, Colilert-18®, Quanti-Tray®, Quanti-
       Tray®/2000, and Colisure™ methods may be obtained from: IDEXX Laboratories, Inc., One
       IDEXX Drive, Westbrook, Maine 04092.

5.10   USEPA. 2002. Method 1103.1: Escherichia coli in Water by Membrane Filtration Using
       membrane-Thermotolerant&c/jen'c/ji'a coli Agar  (mTEC). U.S. Environmental Protection
       Agency, Office of Water, Washington, D.C. EPA-821-R-02-020.

5.11   USEPA. 2002. Method 1603: Escherichia coli (E.coli) in Water by Membrane Filtration Using
       Modified membrane-Thermotolerant Escherichia  coli agar (Modified mTEC). U.S.
       Environmental Protection Agency, Office of Water,  Washington, D.C. EPA-821-R-02-023.

5.12   USEPA. 2002. Method 1604: Total coliforms and Escherichia coli (E. coli) in Water by
       Membrane Filtration Using a Simultaneous Detection Technique (MI Medium). U.S.
       Environmental Protection Agency, Office of Water,  Washington, D.C. EPA-821-R-02-024.

                                           69                               February 2006

-------
                                                                           Section 5: References
5.13   Hach Company, Inc. m-ColiBlue24 Method is available from: Hach Company, P.O. Box 389,
       Loveland, CO 80539

5.14   Noble, Rachel T., Dorsey, J., Leccaster, M., Mazur, M., McGee, C., Moore, D., Victoria, O.,
       Reid, D., Schiff, K., Vainik P., Weisberg, S. 1999. Southern California Bight  1998 Regional
       Monitoring Program, Vol I: Summer shoreline microbiology. Southern California Coastal Water
       Research Project, Westminster, CA.

5.15   USEPA. 2005. Source Water Monitoring Guidance Manual for Public Water Systems for the Long Term 2
       Enhanced Surface Water Treatment Rule (LT2 Rule). This manual is available for download from
       http://www.cpa.gov/safcwatcr/disinfcction/U2/pwsguidc.htrnl
                                             70                                  February 2006

-------
                    Appendix A

Biosafety Guidelines for Laboratories Analyzing Environmental
              Samples for Cryptosporidium

-------
This page intentionally left blank

-------
                     BIOSAFETY GUIDELINES FOR LABORATORIES
          ANALYZING ENVIRONMENTAL SAMPLES FOR CRYPTOSPORIDIUM
 1.0    Introduction

 Laboratory-related infections with Cryptosporidium can occur if personnel are not properly trained in
 biosafety techniques. In addition to waterborne, fecal-oral, person to person transmission, and animal to
 person, circumstantial evidence suggests that airborne transmission of oocysts may occur1. Adoption of a
 biosafety policy by laboratory management that includes commitment to technician safety, training and
 supervision, as well as, rigid adherence to biosafety guidelines will prevent the occurrence of
 Cryptosporidium infection (cryptosporidiosis) in laboratory personnel.

 Symptoms associated with cryptosporidiosis may include: watery  diarrhea, abdominal cramps, nausea,
 low-grade fever, dehydration, weight loss, and loss of appetite. Symptoms may develop within 2 to 10
 days after infection. There are no antibiotics or drug treatments that will cure cryptosporidiosis. For
 additional information : www.cdc.gov/ncidod/dpd/parasites/crvptosporidiosis/default.htm

 Biosafety Level 2 (BSL 2) practices and facilities are recommended for activities with infective stages of
 Cryptosporidium. A BSL 2 facility is appropriate for agents known to cause disease in humans.


 Biosafety Level 2 practices and facilities include the following requirements:

 •   Laboratory personnel  have specific training in handling pathogenic agents and are directed by
    competent scientists

 •   Access to the laboratory is limited when work is being conducted

 •   Certain procedures in  which infectious aerosols or splashes may be created are conducted in
    biological safety II cabinets

 2.0   Scope and Application

 The biosafety guidelines described in this document are adapted from Laboratory Safety: Principles and
Practices, Second Edition (Reference 8.1) and Biosafety in Microbiological and Biomedical Laboratories
 (BMBL) Fourth Edition (Reference 8.2). A readily available laboratory-specific biosafety manual may
 be developed, and maintained, to address the safety, handling, and laboratory practices described below.
 The manual can be distributed to all employees and available at all times. It can be reviewed annually, or
 as recommended, by the laboratory safety officer. Personnel may read and sign off on the document on a
 regular basis, as determined by the laboratory safety officer.


 3.0   General Safety Practices

 3.1    Basic Laboratory  Safety Recommendations

       3.1.1  Closed-toed shoes worn in the laboratory.

       3.1.2  All work surfaces and floors cleaned regularly and free of clutter.

       3.1.3  All emergency numbers posted in the laboratory.
       'Hojlyng, N., Holten-Andersen, W., and S. Jepsen. 1987. Cryptosporidiosis: a case of airborne
transmission. Lancet. 2:271-272.
       Blagburn, B.L., and W.L. Current.  1983. Accidental infection of a researcher with human
Cryptosporidium. J. Infect. Dis. 142:772-773.

                                             ~\February 2006

-------
LT2 Biosafetv Guidance
        3.1.4  All employees trained in the use and location of all safety/emergency equipment in each
               work area.

        3.1.5  Biological safety II cabinets tested and certified annually.

        3.1.6  All laboratory personnel trained in the proper procedures to clean up biological spills.


4.0    Recommended Microbiological Practices

4.1     Access to the laboratory is limited or restricted at the discretion of the laboratory director or
        laboratory safety officer when experiments are in progress.

4.2     Persons wash their hands after they handle viable materials and animals, after removing gloves,
        and before leaving the laboratory.

4.3     Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in
        the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a
        face shield. Food is stored outside the work area in cabinets or refrigerators designated for this
        purpose only.

4.4     Mouth pipetting recognized as poor practice.

4.5     All procedures are performed carefully to minimize the creation of splashes or aerosols.  Ensure
        that lids are used during all centrifugation and vortexing. Any procedures with open containers
        are performed inside a BSL 2 hood.

4.6     Work surfaces are decontaminated before and after each use and after any spill of viable material.

4.7     All cultures, stocks, and other regulated wastes are decontaminated before disposal by an
        approved decontamination method such as autoclaving.

        4.7.1   Materials to be decontaminated outside of the immediate laboratory are to be placed in a
               durable, leakproof container and closed for transport from the laboratory.

        4.7.2  Materials to be decontaminated off-site from the laboratory are packaged  in accordance
               with applicable local, state, and federal regulations before removal from the facility.


5.0     Recommended Special Practices when Processing Cryptosporidium
        Samples

5.1     In general, persons who are at increased risk of acquiring infection or for whom infection may be
        unusually hazardous are not allowed in the laboratory or animal rooms. For example, persons
        who are immunocompromised may be at risk of acquiring infections.

5.2     The laboratory safety  officer has the final responsibility for assessing each circumstance and
        determining who may enter or work in the laboratory. The laboratory safety officer will report to
        the laboratory director on a regular basis on the status of safety in the laboratory, conduct training
        and maintain outside professional contacts to exchange safety information relevant to laboratory
        operations.

5.3     The laboratory director or laboratory safety officer, establishes policies and procedures, whereby
        only persons who have been advised of the potential hazard and meet specific entry requirements
        are allowed to enter the laboratory or animal rooms.

5.4     When the infectious agent(s) in use in the laboratory require special provisions or special training
        for entry, a hazard warning sign incorporating the universal biohazard symbol is posted on the
        access door to the laboratory work area. The hazard warning sign identifies the infectious agent,
                                              A-2                                February 2006

-------
                                                                           LT2 Biosafety Guidance
        lists the name and telephone number of the laboratory director or other responsible person(s), and
        indicates the special requirement(s) for entering the laboratory.

5.5     Laboratory personnel receive appropriate training on the potential hazards associated with the
        work involved, the necessary precautions to prevent exposures, and the exposure evaluation
        procedures. Personnel receive annual updates, or additional training as necessary for procedural
        or policy changes.

5.6     Materials containing Cryptosporidium are placed in a container that prevents leakage during
        collection, handling, processing, storage, transport, or shipping.

5.7     Laboratory equipment is decontaminated with an appropriate disinfectant before and after the
        equipment is used, and especially after overt spills, splashes, or other contamination by infectious
        materials. Contaminated equipment is decontaminated according to any local, state, or federal
        regulations before it is sent for repair or maintenance or packaged for transport in accordance
        with applicable local, state, or federal regulations before removal from the facility.

5.8     Spills and accidents which result in overt exposures to infectious materials are immediately
        reported to the laboratory director or laboratory safety officer. Medical evaluation, surveillance,
        and treatment are provided as appropriate and written records  are maintained.


6.0     Recommended Safety Equipment (Primary Barriers)

6.1     Properly maintained biological safety 11 cabinets, and other appropriate personal protective
        equipment or physical containment devices are used whenever:

        6.1.1   Procedures with potential for creating infectious aerosols or splashes are conducted.
               These may include centrifuging, vortexing, grinding, blending, vigorous shaking or
               mixing, sonic disruption, or opening containers of infectious materials whose internal
               pressures may be different from ambient pressures.

        6.1.2  High concentrations or large volumes of infectious agents are used. Such materials may
               be centrifuged in the open laboratory if sealed rotor heads or centrifuge safety cups are
               used, and  if these rotors or safety cups are opened only in a biological safety cabinet.

6.2     Face protection (goggles, mask, faceshield, or other splatter guards) is used for anticipated
        splashes or sprays of infectious or other hazardous materials to the face, when the
        microorganisms are manipulated outside the biological safety  cabinet.

6.3     Protective laboratory coats, gowns, smocks, or uniforms designated for lab use are worn while in
        the laboratory. This protective clothing is removed and left in  the laboratory before leaving for
        non-laboratory areas (e.g., cafeteria, library, administrative offices). All protective clothing is
        either disposed of in the laboratory or laundered by the institution; and is not taken home by
        personnel.

6.4     Gloves are worn when handling infected animals and when hands may contact infectious
        materials, contaminated surfaces, or equipment.

        6.4.1   Wearing two pairs of gloves may be appropriate; if a spill or splatter occurs, the hand will
               be protected after the contaminated glove is removed.

        6.4.2  Gloves are disposed of when contaminated, removed when work with infectious
               materials is complete, and are not worn outside the laboratory.

        6.4.3  Disposable gloves are not washed or reused.
                                               A-3                                  February 2006

-------
LT2 Biosafety Guidance
7.0     Recommendations for Laboratory Facilities (Secondary Barriers)
7.1     Each laboratory contains a sink for handwashing.
7.2     The laboratory is designed so that it can be easily cleaned. Rugs in laboratories are not
        appropriate, because proper decontamination following a spill is extremely difficult to achieve.
7.3     Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate
        heat.
7.4     Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are
        accessible for cleaning.
7.5     If the laboratory has windows that open, they are fitted with fly screens.
7.6     A method for decontamination of infectious or regulated laboratory wastes is available (e.g.,
        autoclave, chemical disinfection, incinerator, or other approved decontamination system).
7.7     An eyewash facility is readily available.
7.8     The laboratory facilities  are clean, temperature and  humidity controlled, and have adequate
        lighting at bench tops.
8.0    References
8.1    D. O. Fleming, J. H. Richardson, J. J. Tulis, and D. Vesley. Laboratory Safety: Principles and
       Practices. 1995.  Second Edition. American Society for Microbiology, Washington, D.C.,
       publisher.
8.2    US Department of Health and Human Services. Biosafety in Microbiological and Biomedical
       Laboratories. 1999. Fourth Edition. U.S. Department of Health and Human Services, Centers for
       Disease Control and Prevention, and National Institutes of Health, US Government Printing
       Office, Washington, D.C., publisher.
8.3    Fayer, Ronald, Ed. 1997. Cryptosporidium and Cryptosporidiosis. CRC Press, Inc., Boca Raton.
                                              A-4                                February 2006

-------
                   Appendix B



Method 1622: Cryptosporidium in Water by Filtration/IMS/FA

-------
This page intentionally left blank

-------
&EPA
   United States
   Environmental Protection
   Agency
   Method 1622: Cryptosporidium in Water
   by Filtration/I MS/FA

   December 2005

-------
Office of Water (4607)
EPA815-R-05-001
http://www.epa.gov/microbcs/
December 2005
                                                    Printed on Recycled Paper

-------
                                  Acknowledgments

This method was prepared under the direction of William A. Telliard of the Engineering and Analysis
Division within the U.S. Environmental Protection Agency (U.S. EPA) Office of Water. This document
was prepared by CSC under a U.S. EPA contract, with assistance from its subcontractor, Interface, Inc.

The U.S. EPA Office of Water gratefully acknowledges the contributions of the following persons and
organizations to the development of this method:

Mike Arrowood, Centers for Disease Control and Prevention, Division of Parasitic Diseases (MS-F13),
       4770 Buford Highway, N.E., Atlanta, GA 30341-3724, USA

Phil Berger, Office of Groundwater and Drinking Water, U.S. Environmental Protection Agency, 401 M
       Street,  S.W., Washington, DC 20460, USA

Jennifer Clancy, Clancy Environmental Consultants, Inc., P.O. Box 314, St. Albans, VT 05478, USA

Kevin Connell, CSC, 6101 Stevenson Avenue, Alexandria, VA 22314, USA

Ricardo DeLeon, Metropolitan Water District of Southern California,  700 Moreno Avenue, LaVerne, CA
       91760, USA

Shirley Dzogan, EnviroTest Laboratories, 745 Logan Avenue, Winnipeg, Manitoba R3E 3L5, Canada

Mary Ann Feige (retired), Technical Support Center, Office of Ground Water and Drinking Water, U.S.
       Environmental Protection Agency, 26 W. Martin Luther King Drive, Cincinnati, OH 45268-1320,
       USA

Colin Fricker, Thames Water Utilities, Manor Farm Road, Reading, Berkshire, RG2 OJN, England

Carrie Moulton (Hancock), Technical Support Center, Office of Ground Water and Drinking Water, U.S.
       Environmental Protection Agency, 26 W. Martin Luther King Drive, Cincinnati, OH 45268-1320,
       USA
Stephanie Harris,Manchester Laboratory, U.S. Environmental Protection Agency, Region 10, 7411
       Beach Drive  East, Port Orchard, WA 98366, USA
Dale Rushneck, Interface, Inc., 3194 Worthington Avenue, Fort Collins, CO 80526, USA
Frank Schaefer HI, National Exposure Research Laboratory, U.S. Environmental Protection Agency, 26
       W. Martin Luther King Drive, Cincinnati, OH 45268-1320, USA
Steve Schaub, Health and Ecological Criteria Division (4304), Office of Science and Technology, U.S.
       Environmental Protection Agency, 401 M Street, S.W., Washington, DC 20460, USA

Ajaib Singh, City of Milwaukee Health Department, 841 North Broadway, Milwaukee, WI 53202, USA

Huw Smith, Department of Bacteriology, Scottish Parasite Diagnostic Laboratory, Stobhill NHS Trust,
       Springburn, Glasgow, G21 3UW,  Scotland

Timothy Straub, Lockheed Martin, 7411 Beach Drive East, Port Orchard, WA 98366, USA
William A. Telliard, Office of Science and Technology, U.S. Environmental Protection Agency, 401 M
       Street, S.W.,  Washington, DC 20460, USA

Cryptosporidium cover photo courtesy of the U.S. Centers for Disease Control

-------
                                       Disclaimer

This method has been reviewed by the U.S. EPA Office of Water and approved for publication. Mention
of trade names or commercial products does not constitute endorsement or recommendation for use.
Questions regarding this method or its application should be addressed to:

       Carrie Moulton
       Coordinator, Laboratory Quality Assurance Program for the Analysis of Cryptosporidium
       U.S. Environmental Protection Agency
       Office of Ground Water and Drinking Water
       Technical Support Center, MCI40
       26 West Martin Luther King Drive
       Cincinnati, OH 45268-1320
       (513)569-7919
       (513)569-7191 (fax)
       moulton.carrie@epa.gov

-------
                                       Introduction

To support future regulation of protozoa in drinking water, the Safe Drinking Water Act Amendments of
1996 require the U.S. Environmental Protection Agency (EPA) to evaluate the risk to public health posed
by drinking water contaminants, including waterborne parasites, such as Cryptosporidium. To implement
these requirements, EPA must assess Cryptosporidium occurrence in raw surface waters used as source
waters for drinking water treatment plants. EPA Method 1622 was developed to support this assessment.

Method Development and Validation

EPA initiated an effort in 1996 to identify new and innovative technologies for protozoan monitoring and
analysis. After evaluating potential alternatives to the then-current method through literature searches,
discussions with research and commercial laboratories, and meetings with experts in the field, the
Engineering and Analysis Division within the Office of Science and Technology within EPA's Office of
Water developed draft Method 1622 for Cryptosporidium detection in December 1996. This
Cryptosporidium-only method was validated through an interlaboratory study in August 1998, and was
revised as a final, valid method for detecting Cryptosporidium in water in  January 1999.

The interlaboratory validated versions of Method 1622 (January 1999; EPA-821-R-99-001) and Method
1623 (April 1999; EPA-821-R-99-006) were used to analyze approximately 3,000 field and QC samples
during the Information Collection Rule Supplemental Surveys (ICRSS) between March  1999 and
February 2000. Method 1622 was used to analyze samples from March 1999 to mid-July 1999; Method
1623 was used from mid-July 1999 to February 2000.

Changes in the April 2001 Version of the Method

The method was revised in April 2001, after completion of the ICRSS and multiple meetings with
researchers and experienced laboratory staff to discuss potential method updates. Changes incorporated in
the April 2001 revision of the method (EPA-821-R-01-025) included the following:
•      Nationwide approval of modified versions of the methods using the following components:
       (a)     Whatman Nuclepore CrypTest™ filter
       (b)     IDEXX Filta-Max® filter
       (c)     Waterborne Aqua-Glo™ G/C Direct FL antibody stain
       (d)     Waterborne Crypt-a-Glo™ and Giardi-a-Glo™ antibody stains
•      Clarified sample acceptance criteria
•      Modified capsule filter elution procedure

•      Modified concentrate aspiration procedure

•      Modified IMS acid dissociation procedure

•      Updated QC acceptance criteria for IPR and OPR tests

•      Addition of a troubleshooting section for QC failures

•      Modified holding times

•      Inclusion of flow cytometry-sorted spiking suspensions

-------
Changes in the June 2003 Version of the Method
The method was revised again in June 2003 to support proposal of EPA's Long Term 2 Enhanced Surface
Water Treatment Rule. Changes incorporated into the December 2002 version include:
•      Nationwide approval of a modified version of the methods using the Pall Gelman Envirochek™
       HV filter
•      Removal of Whatman Nuclepore CrypTest™ filter from the methods as a result of discontinuation
       of the product by the manufacturer
       Nationwide approval of the use of BTF EasySeed™ irradiated oocysts  for use in routine quality
       control (QC) samples
•      Minor clarifications and corrections
•      Rejection criteria for sample condition upon receipt
•      Guidance on measuring sample temperatures
       Clarification of QC sample requirements and use of QC sample results
•      Guidance on minimizing carry-over debris onto microscope slides after IMS
Changes in the December 2005 Version of the Method

The method was revised again in 2005 to support promulgation of EPA's Long Term 2 Enhanced Surface
Water Treatment Rule. Changes incorporated into the June 2003 version include:

       Nationwide approval of the use of portable continuous-flow centrifugation as a modified version
       of the method. The product met all method acceptance criteria for Cryptosporidium using 50-L
       source water samples.

•      Addition of BTF EasyStain™ monoclonal antibody stain as an acceptable reagent for staining in
       Methods 1622. The product was validated through an interlaboratory validation study using the
       Pall Envirochek™ HV filter.

•      Clarification of the analyst verification procedure

       Clarification of sample condition criteria upon receipt

Performance-Based Method Concept and Modifications Approved for Nationwide Use

EPA Method 1622 is a performance-based method applicable to the determination of Cryptosporidium in
aqueous matrices. EPA Method 1622  requires filtration, immunomagnetic separation of the oocysts from
the material captured, and enumeration of the target organisms based on the results of
immunofluorescence assay, 4',6-diamidino-2-phenylindole (DAPI) staining results, and differential
interference contrast microscopy.

The interlaboratory validation of EPA Method 1622 conducted by EPA used the Pall Gelman capsule
filtration procedure, Dynal immunomagnetic separation (IMS) procedure, and Meridian sample staining
procedure described in this document. Alternate procedures are allowed, provided that required quality
control tests are performed and all quality control acceptance criteria in this method are met.
                                              IV

-------
Since the interlaboratory validation of EPA Method 1622, interlaboratory validation studies have been
performed to demonstrate the equivalency of modified versions of the method using the following
components:
•       Whatman Nuclepore CryptTest™ filter (no longer available)
        IDEXX Filta-Max® filter
        Pall Gelman Envirochek™ HV filter
•       Portable Continuous-Flow Centrifugation (PCFC)
        Waterborne Aqua-Glo™ G/C Direct FL antibody stain
•       Waterborne Crypt-a-Glo™ and Giardi-a-Glo™ antibody stains
•       BTF EasyStain™ antibody stain
        BTF EasySeed™ irradiated oocysts for use in routine QC samples

The validation studies for the modified versions  of the method met EPA's performance-based
measurement system Tier 2 validation for nationwide use (see Section 9.1.2 for details), and have been
accepted by EPA as equivalent in performance to the original version of the method validated by EPA.
The equipment and reagents used in these modified versions of the method are noted in Sections 6 and 7
of the method.

Because this is a performance-based method, other alternative components not listed in the method may be
available for evaluation and use by the laboratory. Confirming the acceptable performance of a modified
version of the method using alternate components in a single laboratory does not require that an
interlaboratory validation study be conducted. However, method modifications validated only in a single
laboratory have not undergone sufficient testing  to merit inclusion in the method. Only those modified
versions of the method that have been demonstrated as equivalent at multiple laboratories  on multiple
water sources through  a Tier 2 interlaboratory study will be cited in the method.

-------
                                   Table of Contents







 1.0    Scope and Application  	 1




2.0    Summary of Method	 1




3.0    Definitions 	2




4.0    Contamination, Interferences, and Organism Degradation	2




5.0    Safety 	3




6.0    Equipment and Supplies	3




7.0    Reagents and  Standards  	 8




8.0    Sample Collection and Storage	 11




9.0    Quality Control	 13




10.0   Microscope Calibration and Analyst Verification 	21




11.0   Oocyst Suspension Enumeration and Sample Spiking	28




12.0   Sample Filtration and Elution	 36




13.0   Sample Concentration and Separation (Purification)	45




14.0   Sample Staining  	 51




15.0   Examination  	 52




16.0   Analysis of Complex Samples  	 53




17.0   Method Performance	 54




18.0   Pollution Prevention	54




19.0   Waste Management	54




20.0   References	 54




21.0   Tables and Figures 	56




22.0   Glossary of Definitions and Purposes	65
                                              VI

-------
                 Method 1622:  Cryptosporidium in Water
                                by Filtration/IMS/FA

1.0   Scope and Application
•j -j    This method is for the detection of Cryptosporidium (CAS Registry number 137259-50-8) and in
       water by concentration, immunomagnetic separation (IMS), and immunofluorescence assay (FA)
       microscopy. Cryptosporidium may be verified using 4'.6-diamidino-2-phenylindole (DAPI)
       staining and differential interference contrast (DIG) microscopy. The method has been validated
       in surface water, but may be used in other waters, provided the laboratory demonstrates that the
       method's performance  acceptance criteria are met.
•j 2    This method is designed to meet the survey and monitoring requirements of the U.S.
       Environmental Protection Agency (EPA). It is based on laboratory testing of recommendations by
       a panel of experts convened by EPA. The panel was charged with recommending an improved
       protocol for recovery and detection of protozoa that could be tested and implemented with
       minimal additional research.
•j 3    This method identifies  the genera, Cryptosporidium, but not the species. The method cannot
       determine the host species of origin, nor can it determine the viability or infectivity of detected
       oocysts.
•j 4    This method is for use only by persons experienced in the determination of Cryptosporidium by
       filtration, IMS, and FA. Experienced persons are defined in Section 22.2 as analysts or principal
       analysts. Laboratories unfamiliar with analyses of environmental samples by the techniques in this
       method  should gain experience using water filtration techniques, IMS, fluorescent antibody
       staining with monoclonal antibodies, and microscopic examination of biological particulates using
       bright-field and DIG microscopy.
•j 5    Any modification of the method beyond those expressly permitted is subject to the application and
       approval of alternative  test procedures under 40  CFR Part 141.27.

2.0   Summary of Method
2i    A water sample is filtered and the oocysts and extraneous materials are retained on the filter.
       Although EPA has only validated the method using laboratory filtration of bulk water samples
       shipped from the field,  field-filtration also may be used.
2.2    Elution and separation
       2.2.1   Materials on the filter are eluted and the eluate is centrifuged to pellet the oocysts, and
               the supernatant fluid is aspirated.
       2.2.2   The oocysts are magnetized by attachment of magnetic beads conjugated to anti-
               Cryptosporidium antibodies. The magnetized oocysts are separated from the extraneous
               materials using a magnet, and the extraneous materials are discarded. The magnetic bead
               complex is then detached from the oocysts.
2.3    Enumeration
       2.3.1   The oocysts are stained on well slides with fluorescently labeled monoclonal antibodies
               and 4',6-diamidino-2-phenylindole (DAPI). The stained sample is examined using
               fluorescence and differential interference contrast (DIG) microscopy.
       2.3.2   Qualitative analysis is performed by scanning each slide well for objects that meet the
               size, shape, and fluorescence characteristics of Cryptosporidium oocysts.
                                                                               December 2005

-------
Method 1622 - Cryptosporidium
        2.3.3    Quantitative analysis is performed by counting the total number of objects on the slide
                confirmed as oocysts.
2 4     Quality is assured through reproducible calibration and testing of the filtration, immunomagnetic
        separation (IMS), staining, and microscopy systems. Detailed information on these tests is
        provided in  Section 9.0.

3.0    Definitions
3 -j     Cryptosporidium is a genus of protozoan parasites potentially found in water and other media.
        The recent taxonomy of the genus Cryptosporidium includes the following species and their
        potential hosts: C. hominis (humans; formerly C. parvum genotype I; Reference 20.1); C. parvum
        (bovine and other mammals including humans; formerly genotype II;); C. baileyi and C.
        meleagridis (birds); C.  muris (rodents); C. canis (dogs); C.felis (cats); C. serpentis (reptiles); and
        C. nasorum  (fish).  Cryptosporidium oocysts are defined in this method as objects exhibiting
        brilliant apple green fluorescence under UV light (FA-positive), typical size (4 to 6 urn) and shape
        (round to oval), and no atypical characteristics by FA, DAPI fluorescence, or DIG microscopy.
        Examination and characterization using fluorescence (FITC and DAPI stain) and DIG microscopy
        are required for exclusion of atypical organisms (e.g., those possessing spikes, stalks, appendages,
        pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc.).
3 2     Definitions for other terms used in this method are given in the glossary (Section 22.0).
4.0    Contamination, Interferences, and Organism Degradation
4 •]     Turbidity caused by inorganic and organic debris can interfere with the concentration, separation,
        and examination of the sample for Cryptosporidium oocysts. In addition to naturally-occurring
        debris, e.g. clays and algae, chemicals, e.g. iron, alum coagulants and polymers added to source
        waters during the treatment process may result in additional interference.
4 2     Organisms and debris that autofluoresce or demonstrate non-specific immunofluorescence, such
        as algal and yeast cells, when examined by epifluorescent microscopy, may interfere with the
        detection of oocysts and contribute to false positives by immunofluorescence assay (FA)
        (Reference 20.3).
4 3     Solvents, reagents, labware, and other sample-processing hardware may yield artifacts that may
        cause misinterpretation of microscopic examinations for oocysts. All materials used must be
        demonstrated to be free from interferences under the conditions of analysis by running a method
        blank (negative control sample) initially and a minimum of every week or after changes in source
        of reagent water. Specific selection of reagents and purification of solvents and other materials
        may be required.
4 4     Freezing samples, filters, eluates, concentrates, or slides may interfere with the detection and/or
        identification of oocysts.
45     All equipment should be cleaned according to manufacturers' instructions. Disposable supplies
        should be used wherever possible.
December 2005

-------
                                                                    Method 1622 - Cryptosporidium
5.0    Safety
5 -j     The biohazard associated with, and the risk of infection from, oocysts is high in this method
        because live organisms are handled. This method does not purport to address all of the safety
        problems associated with its use. It is the responsibility of the laboratory to establish appropriate
        safety and health practices prior to use of this method. In particular, laboratory staff must know
        and observe the safety procedures required in a microbiology laboratory that handles pathogenic
        organisms while preparing,  using, and disposing of sample concentrates, reagents and materials,
        and while operating sterilization equipment.
5 2     The toxicity or carcinogenicity of each compound or reagent used in this method has not been
        precisely determined; however, each chemical compound should be treated as a potential health
        hazard. Exposure to these compounds should be reduced to the lowest possible level. The
        laboratory is responsible for maintaining current knowledge of Occupational Safety and Health
        Administration regulations regarding the safe handling of the chemicals specified in this method.
        A reference file of material  safety data sheets should be made available to all personnel involved
        in these analyses. Additional information on laboratory safety can be found in References 20.4
        through 20.7.
5 3     Samples may contain high concentrations of biohazards and toxic compounds, and must be
        handled with gloves. Reference materials and standards containing oocysts must also be handled
        with gloves and laboratory staff must never place gloves in or near the face after exposure to
        solutions known or suspected to contain oocysts. Do not mouth-pipette.
5 4     Laboratory personnel must change gloves after handling filters and other contaminant-prone
        equipment and reagents. Gloves must be removed  or changed before touching any other laboratory
        surfaces or equipment.
5 5     Centers for Disease Control (CDC) regulations (42 CFR 72) prohibit interstate shipment of more
        than 4 L  of solution known to contain  infectious materials (see
        http://www.cdc.gov/od/ohs/biosftv/shipregs.htm for details). State regulations may contain similar
        regulations for intrastate commerce. Unless the sample is known or suspected to contain
        Cryptosporidium or other infectious agents (e.g., during an outbreak), samples should be shipped
        as noninfectious and should not be marked as infectious. If a sample is known or suspected to be
        infectious, and the sample must be shipped to a laboratory by a transportation means  affected by
        CDC  or state regulations, the sample should be shipped in accordance with these regulations.

6.0    Equipment and  Supplies	
        NOTE:  Brand names, suppliers, and part numbers are for illustrative purposes only. No
        endorsement is implied. Equivalent performance may be achieved using apparatus and
        materials other than those specified here, but demonstration of equivalent performance
        that meets the requirements of this method is the responsibility of the laboratory.
g <|     Sample collection equipment for shipment of bulk water samples for laboratory filtration.
        Collapsible LDPE cubitainer for collection of 10-L bulk sample(s)—Cole Farmer cat. no. U-
        06100-30 or equivalent. Fill completely to ensure collection of a full 10-L sample. Discard after
        one use.
g 2     Equipment for sample filtration. Four options have been demonstrated to be acceptable for use
        with Method 1622. Other options may be used if their acceptability is demonstrated according to
        the  procedures outlined in Section  9.1.2.


                                               3                                 December 2005

-------
 Method 1622 - Cryptosporidium
        6.2.1    Cubitainer spigot to facilitate laboratory filtration of sample (for use with any filtration
                option)—Cole Farmer cat. no. U-06061-01, or equivalent.
        6.2.2    Original Envirochek™ sampling capsule or Envirochek™ HV sampling capsule
                equipment requirements (for use with the procedure described in Section 12.2). The
                versions of the method using these filters were validated using 10-L and 50-L sample
                volumes, respectively. Alternate sample volumes may be used, provided the laboratory
                demonstrates acceptable performance on initial and ongoing spiked reagent water and
                source water samples (Section 9.1.2).
                6.2.2.1        Sampling capsule
                              6.2.2.1.1      Envirochek™, Pall Corporation, Ann Arbor, MI, part no.
                                            12110 (individual filter) and or part no. 12107 (box of 25
                                            filters) (www.pall.com or (800) 521-1520 ext. 2)
                              6.2.2.1.2      Envirochek™ HV, Pall Corporation, Ann Arbor, MI, part
                                            no. 12099 (individual filter) or part no. 12098 (box of 25
                                            filters) (www.pall.com or (800) 521-1520 ext. 2)
                6.2.2.2        Laboratory shaker with arms for agitation of sampling capsules
                              6.2.2.2.1      Laboratory shaker—Lab-Line model 3589 (available
                                            through VWR Scientific cat. no. 57039-055), Pall
                                            Corporation part no. 4821, Fisher cat. no. 14260-11, or
                                            equivalent
                              6.2.2.2.2      Side arms for laboratory shaker—Lab-Line Model 3587-
                                            4 (available through VWR Scientific cat. no. 57039-045),
                                            Fisher cat. no. 14260-13, or equivalent
        6.2.3    Filta-Max® foam filter equipment requirements  (for use with  the procedure described in
                Section 12.3). The version of the method using this filter was validated using 50-L
                sample volumes; alternate sample volumes may be used, provided the laboratory
                demonstrates acceptable performance on initial and ongoing spiked reagent water and
                matrix samples (Section 9.1.2).
                6.2.3.1        Foam filter—Filta-Max®, IDEXX, Westbrook, ME. Filter module cat.
                              no. FMC 10603
        NOTE:  Check at least one filter per batch to ensure that the filters have not been
        affected by improper storage or other factors that could result in brittleness or other
        problems. At a  minimum confirm that the test filter expands properly in water before
        using the batch or shipping filters to the field.

                6.2.3.2       Filter processing equipment—Filta-Max® starter kit, IDEXX,
                             Westbrook, ME, cat. no. FMC 11002. Starter kit  includes manual wash
                             station with clamp set (FMC 10101 or  10106) including plunger head
                             (FMC  12001), tubing set (FMC 10307), vacuum set (FMC 10401), MKII
                             filter housing with hose-tail fittings (FMC 10504) and green housing
                             tools (FMC 10506).  In addition, processing requires magnetic stirrer
                             (FMC  10901) and filter membranes, 100 pk, (FMC 10800).
        6.2.4    Portable Continuous-Flow Centrifuge (PCFC) requirements (for use with procedures
                described in Section 12.4). The version of the method using this technique was validated
                for Cryptosporidium in 50-L sample volumes; alternate sample volumes may be used,

December 2005                                 4

-------
                                                                   Method 1622 - Cryptosporidium
               provided the laboratory demonstrates acceptable performance on initial and ongoing
               spiked reagent water and matrix samples (Section 9.1.2). The technique is based on
               technology from Haemonetics Corporation, Braintree, MA.
g 3    Ancillary sampling equipment
       6.3.1   Tubing—Glass, polytetrafluoroethylene (PTFE), high-density polyethylene (HOPE), or
               other tubing to which oocysts will not easily adhere, Tygon formula R-3603, or
               equivalent. If rigid tubing (glass, PTFE, HDPE) is used and the sampling system uses a
               peristaltic pump, a minimum length of compressible tubing may be used in the pump.
               Before use, the tubing must be autoclaved, thoroughly rinsed with  detergent solution,
               followed by repeated rinsing with reagent water to minimize sample contamination.
               Alternately, decontaminate using hypochlorite solution, sodium thiosulfate, and multiple
               reagent water rinses. Dispose of tubing after one use whenever possible or when wear is
               evident.
       6.3.2   Flow control valve—0.5 gpm (0.03 L/s), Bertram Controls, Plast-O-Matic cat. no.
               FC050B1/2-PV, or equivalent; or 0.4- to 4-Lpm flow meter with valve, Alamo Water
               Treatment, San Antonio, TX, cat. no. R5310, or equivalent
       6.3.3   Pump— peristaltic, centrifugal, impeller, or diaphragm pump; MasterFlex I/P®
               EasyLoad® peristaltic pump (Cole-Parmer cat. No. EW-77963-10) with 77601-10
               pumphead, 77410-00 drive unit, and 06429-73 Tygon LFL tubing; Dayton, model
               number 3YU61 (available through Grainger), Jabsco Flexible Impeller Pump (Cole-
               Parmer cat. No. EW-75202-00); Simer, model number M40; or equivalent. It is
               recommended that the pump be placed on the effluent side of the filter, when possible, to
               reduce the risk of contamination and the amount of tubing replaced or cleaned.
       6.3.4   Flow meter—SaMeCo cold water totalizer, E. Clark and Associates, Northboro, MA,
               product no. WFU 10.110; Omega flow meter, Stamford, CT, model FTB4105; or
               equivalent. Alternatively,  use a graduated carboy(s) (See Section 6.18)
g 4    Equipment for spiking samples in the laboratory
       6.4.1   Collapsible 10-L LDPE cubitainer with cubitainer spigot—Cole Partner cat. no. U-
               06100-30 or equivalent and Cole Farmer cat. no. U-06061-01, or equivalent. Discard
               after one use to eliminate possible contamination. Alternatively, use clean, 10-L carboy
               with bottom delivery port ('/2"), Cole-Palmer cat. no. 06080-42, or  equivalent; calibrate
               to 10.0 L and mark level with waterproof marker
       6.4.2   Stir bar—Fisher cat. no. 14-513-66, or equivalent
       6.4.3   Stir plate—Fisher cat. no. 11-51049S,  S50461HP, or equivalent
       6.4.4   Hemacytometer—Neubauer type, Hausser Scientific, Horsham, PA, product no. 3200 or
               1475, or equivalent
       6.4.5   Hemacytometer coverslip—Hausser Scientific, product no. 5000 (for  hemacytometer cat.
               no. 3200) or 1461 (for hemacytometer cat. no 1475), or equivalent
       6.4.6   Lens paper without silicone—Fisher cat. no. 11 -995, or equivalent
       6.4.7   Polystyrene or polypropylene conical tubes with screw caps—15- and 50-mL
       6.4.8   Equipment required for enumeration of spiking suspensions using  membrane filters
               6.4.8.1       Glass microanalysis filter holder—25-mm-diameter, with  fritted glass
                             support, Fisher cat. no. 09-753E, or equivalent. Replace stopper with size
                             8, one-hole rubber stopper, Fisher Cat. No. 14-135M, or equivalent.
               6.4.8.2       Three-port vacuum filtration manifold and vacuum source—Fisher Cat.
                             No. 09-753-39A, or equivalent
                                                                                 December 2005

-------
Method 1622 - Cryptosporidium
                6.4.8.3       Cellulose acetate support membrane—1.2-um-pore-size, 25-mm-
                             diameter, Fisher cat. no. A12SP02500, or equivalent
                6.4.8.4       Polycarbonate track-etch hydrophilic membrane filter—1 -urn-pore-size,
                             25-mm-diametcr, Fisher cat. no. K10CP02500, or equivalent
                6.4.8.5       100 x 15 mm polystyrene petri dishes (bottoms only)
                6.4.8.6       60 x 15 mm polystyrene petri dishes
                6.4.8.7       Glass microscope slides—1 in. x 3 in or 2 in. x 3 in.
                6.4.8.8       Coverslips—25 mm2
g 5     Immunomagnetic separation (IMS) apparatus
        6.5.1    Sample mixer—Dynal Inc., Lake Success, NY, cat. no. 947.01, or equivalent
        6.5.2    Magnetic particle concentrator for 10-mL test tubes—Dynal MPC®-1 , cat. no. 120.01 or
                MPC®-6, cat. No 120.02, or equivalent
        6.5.3    Magnetic particle concentrator for microcentrifuge tubes—Dynal MPC®-M, cat. no.
                120.09 (no longer available); Dynal MPC®-S, cat. no. 120.20, or equivalent
        6.5.4    Flat-sided sample tubes—16 x 125 mm Leighton-type tubes with 60 x  10 mm flat-sided
                magnetic capture area, Dynal L10, cat. no. 740.03, or equivalent
g g     Powder-free latex gloves—Fisher cat no. 113945B, or equivalent
g 7     Graduated cylinders, autoclavable—10-, 100-, and 1000-mL
g g     Centrifuges
        6.8.1    Centrifuge capable of accepting 15- to 250-mL conical centrifuge tubes and achieving
                1500 x G—International Equipment Company, Needham Heights, MA, Centrifuge Size
                2, Model K with swinging bucket, or equivalent
        6.8.2    Centrifuge tubes—Conical, graduated, 1.5-, 50-, and 250-mL
g g     Microscope
        6.9.1    Epifluorescence/differential interference contrast (DIC) with stage and ocular
                micrometers and 20X (N.A.=0.4) to 100X (N.A.=1.3) objectives—Zeiss™ Axioskop,
                Olympus™ BH, or equivalent. Hoffman Modulation Contrast optics may be equivalent.
        6.9.2    Excitation/band-pass filters for immunofluorescence assay (FA)—Zeiss™ 487909 or
                equivalent, including, 450- to 490-nm exciter filter, 510-nm dicroic beam-splitting
                mirror, and 515- to 520-nm barrier or suppression filter
December 2005

-------
                                                                   Method 1622 - Cryptosporidium
       6.9.3
        Excitation/band-pass filters for DAPI-
        Brattleboro, VT), or equivalent
-Filters cited below (Chroma Technology,
6.10
6.11
6.12
6.13
6.14
6.15
6.16

6.17
Microscope
model
Zeiss™ -
Axioskop
Zeiss™ -IM35
Olympus™ BH
Olympus™ BX
Olympus™
IMT2
Fluoro-chrome
DAPI (UV)
DAPI (UV)
DAPI (UV)
Excitation
filter (nm)
340-380
340-380
340-380
Dichroic
beam- splitting
mirror (nm)
400
400
400
Barrier or
suppression
filter (nm)
420
420
420
Filter holder
DAPI (UV)
340-380
400
420
Filter holder
DAPI (UV)
340-380
400
420
Filter holder
Chroma
catalog
number
CZ902
CZ702
11000
91002
11000
91008
11000
91003
Ancillary equipment for microscopy
6.10.1  Well slides— Spot-On well slides, Dynal cat. no. 740.04; treated, 12-mm diameter well
        slides, Meridian Diagnostics Inc., Cincinnati, OH, cat. no. R2206; or equivalent
6.10.2  Glass coverslips—22 x 50 mm
6.10.3  Nonfluorescing immersion oil—Type FF, Cargille cat. no. 16212, or equivalent
6.10.4  Micropipette, adjustable:      0- to 10-uL with 0- to 10-uL tips
                                    10- to 100-uL, with 10- to 200-uL tips
                                    100- to 1000-uL with 100- to 1000-uL tips
6.10.5  Forceps—Splinter, fine tip
6.10.6  Forceps—Blunt-end
6.10.7  Desiccant—Drierite™ Absorbent, Fisher cat. no.  07-577-1 A, or equivalent
6.10.8  Humid chamber—A tightly sealed plastic container containing damp paper towels on top
        of which the slides are placed
Pipettes—Glass or plastic
6.11.1  5-, 10-, and 25-mL
6.11.2  Pasteur, disposable
Balances
6.12.1  Analytical—Capable of weighing 0.1 mg
6.12.2  Top loading—Capable of weighing 10 mg
pH meter
Incubator—Fisher Scientific Isotemp™, or equivalent
Vortex mixer—Fisons Whirlmixer, or equivalent
Vacuum source—Capable of maintaining 25 in. Hg, equipped with shutoff valve and vacuum
gauge
Miscellaneous labware and supplies
6.17.1  Test tubes and rack
6.17.2  Flasks—Suction, Erlenmeyer, and volumetric, various sizes
6.17.3  Beakers—Glass or plastic, 5-, 10-, 50-, 100-, 500-, 1000-, and 2000-mL
                                                                                 December 2005

-------
 Method 1622 - Cryptosporidium
        6.17.4  Lint-free tissues
6.18   10- to 15-L graduated container—Fisher cat. no. 02-961-50B, or equivalent; calibrate to 9.0, 9.5,
        10.0, 10.5, and 11.0 L and mark levels with waterproof marker
gig   Filters for filter-sterilizing reagents—Sterile Acrodisc, 0.45 (im, Pall Corporation, cat. no. 4184,
        or equivalent

7.0    Reagents and Standards
7 -j     Reagents for adjusting pH
        7.1.1    Sodium hydroxide (NaOH)—ACS reagent grade, 6.0 N and 1.0 N in reagent water
        7.1.2    Hydrochloric acid  (HC1)—ACS reagent grade, 6.0 N, 1.0 N, and 0.1 N in reagent water

        NOTE:  Due to the low volumes oj pH-adjusting reagents used in this method, and the
        impact that changes inpH have on the immunofluorescence assay, the laboratory must
       purchase standards at the required normality directly from a vendor. Normality must not
        be adjusted by the laboratory.

1 2     Solvents—Acetone, glycerol, ethanol, and methanol, ACS reagent grade
73     Reagent water—Water  in which oocysts and interfering materials and substances, including
        magnetic minerals, are not detected by this method. Sec Reference 20.8  (Section 9020) for reagent
        water requirements.
7 4     Reagents for eluting filters

        NOTE:  Laboratories should store prepared eluting solution far no more than 1 week or
        when noticeably turbid, whichever comes sooner.

        7.4.1    Reagents for eluting Envirochek™ and Envirochek™ HV sampling capsules (Section
                6.2.2)
                7.4.1.1       Laureth-12—PPG Industries, Gurnee, IL, cat. no. 06194, or equivalent.
                             Store Laureth-12 as a 10% solution in reagent water. Weigh 10 g of
                             Laureth-12 and dissolve using a microwave or hot plate in 90 mL of
                             reagent water. Dispense 10-mL aliquots into sterile vials and store at
                             room temperature for up to 2 months, or in the freezer for up to a year.
                7.4.1.2       1 M Tris, pH 7.4—Dissolve 121.1 g Tris (Fisher cat. no. BP152) in 700
                             mL of reagent water and adjust pH to 7.4 with 1 N HC1 or NaOH. Dilute
                             to a final  1000 mL with reagent water and adjust the final pH.  Filter-
                             sterilize through a 0.2-um membrane into a sterile plastic container and
                             store at room temperature. Alternatively, use prepared TRIS, Sigma
                             T6066  or equivalent.
                7.4.1.3       0.5  M EDTA, 2 Na, pH 8.0—Dissolve 186.1 g ethylenediamine
                             tetraacetic acid, disodium salt dihydrate (Fisher cat. no. S311) in 800 mL
                             of reagent water and adjust pH to 8.0 with 6.0 N HC1 or NaOH. Dilute to
                             a final volume of 1000 mL with reagent water and adjust to pH 8.0 with
                             1.0 N HC1 or NaOH. Alternatively, use prepared EDTA, Sigma E5134 or
                             equivalent.
                7.4.1.4       Antifoam A—Sigma Chemical Co. cat. no. A5758, or equivalent
December 2005

-------
                                                                   Method 1622 - Cryptosporidium
               7.4.1.5       Preparation of clution buffer solution—Add the contents of a pre-
                             prepared Laureth-12 vial (Section 7.4.1.1) to a 1000-mL graduated
                             cylinder. Rinse the vial several times to ensure the transfer of the
                             detergent to the cylinder. Add 10 mL of Tris solution (Section 7.4.1.2), 2
                             mL of EDTA solution (Section 7.4.1.3), and 150 uL Antifoam A (Section
                             7.4.1.4). Dilute to 1000 mL with reagent water.
       7.4.2   Reagents for eluting Filta-Max® foam filters (Section 6.2.3)
               7.4.2.1       Phosphate buffered saline (PBS), pH 7.4—Sigma Chemical Co.  cat. no.
                             P-3813, or equivalent. Alternately, prepare PBS by adding the following
                             to 1 L of reagent water: 8 g NaCl; 0.2 g KC1; 1.15 g Na,HPO4, anhydrous;
                             and 0.2 g KH2PO4.
               7.4.2.2       Tween® 20 —Sigma Chemical Co. cat. no. P-7949, or equivalent
               7.4.2.3       High-vacuum grease—BDH/Merck. cat. no. 636082B, or equivalent
               7.4.2.4       Preparation of PBST elution buffer. Add 100 uL of Tween® 20  to
                             prepared PBS (Section 7.4.2.1). Alternatively, add the contents of one
                             packet of PBS to  1.0 L of reagent water. Dissolve by stirring for 30
                             minutes. Add 100 uL of Tween® 20 . Mix by stirring  for 5 minutes.
       7.4.3   Reagents for Portable Continuous-Flow Centrifuge (Section 6.2.4)
               7.4.3.1       Sodium dodecyl sulfate—Sigma Chemical Co. cat. no. 71730 or
                             equivalent
               7.4.3.2       TWEEN 80— Sigma Chemical Co. cat. no. PI754 or equivalent
               7.4.3.3       Antifoam A—Sigma Chemical Co. cat. no. A5758, or equivalent
               7.4.3.4       Preparation of concentrated elution buffer. Add above reagents to obtain
                             a final concentration of 1% sodium dodecyl sulfate, 0.01% TWEEN 80,
                             and 0.001% Antifoam A in concentrated sample volume of ~250mL
7 5    Reagents for immunomagnetic separation (IMS)—Dynabeads® anti-Cryptosporidium beads,
       Dynal cat. nos. 730.01/730.11, or equivalent
7 g    Direct antibody labeling reagents for detection of oocysts. Store reagents between 1 °C and 10°C
       and return promptly to this temperature after each use. Do not allow any of the reagents to freeze.
       The reagents should be protected from exposure to light. Diluted, unused working reagents should
       be discarded after 48 hours. Discard reagents after the expiration date is reached. The labeling
       reagents in Sections 7.6.1-7.6.3 have been approved for use with this method.
       7.6.1   MeriFluor® Cryptosporidium/Giardia, Meridian Diagnostics cat. no. 250050,
               Cincinnati, OH, or equivalent
       7.6.2   Aqua-Glo™ G/C Direct FL, Waterborne cat. no. A100FLR, New Orleans, LA, or
               equivalent
       7.6.3   Crypt-a-Glo™, Waterborne cat. no. A400FLR, New Orleans, LA, or equivalent
       7.6.4   EasyStain™C&G, BTF Pty Limited, Sydney, Australia or equivalent

       NOTE: If a laboratory will use multiple  types of labeling reagents,  the laboratory must
       demonstrate acceptable performance through an initial precision and recovery test
       (Section 9.4) for each type, and must perform positive and negative staining controls for
       each batch of slides stained using each product. However, the laboratory is not required
       to analyze additional ongoing precision and recovery samples or method blank samples
       for each type.  The performance of each labeling reagent used also should be monitored in
       each source water type.

       7.6.5   Diluent for labeling reagents—Phosphate buffered saline (PBS) (Section 7.4.2).

                                               9                                 December 2005

-------
Method 1622 - Cryptosporidium
7 1    4',6-diamidino-2-phenylindole (DAPI) stain—Sigma Chemical Co. cat. no. D9542, or equivalent
       7.7.1    Stock solution—Dissolve 2 mg/mL DAPI in absolute methanol. Prepare volume
                consistent with minimum use. Store between 1°C and 10°C in the dark. Do not allow to
                freeze. Discard unused solution when positive staining control fails or after specified
                time determined by laboratory.
       7.7.2    Staining solution—Follow antibody kit manufacturer's instructions.  Add 10uLof2
                mg/mL DAPI stock solution to 50 mL of PBS for use with Aqua-Glo™ G/C Direct FL or
                MeriFluor® Cryptosporidium/Giardia.  Add 50 uL of 2 mg/mL DAPI stock solution to
                50 mL of PBS for use with EasyStain™. Prepare working solution daily and store
                between 1°C and 10°C (do not allow to freeze). DAPI is light sensitive; therefore, store
                in the dark except when staining. The DAPI concentration may be increased if
                fading/diffusion of DAPI staining is encountered, but the staining solution must be tested
                first on expendable environmental samples to confirm that staining intensity is
                appropriate.
7 g    Mounting medium
       7.8.1    DABCO/glycerol mounting medium (2%)—Dissolve 2 g of DABCO (Sigma Chemical
                Co. cat no. D-2522, or equivalent) in 95 mL of warm glycerol/PBS (60% glycerol, 40%
                PBS). After the DABCO has dissolved completely, adjust the solution volume to  100 mL
                by adding an appropriate volume of glycerol/PBS solution. Alternately, dissolve the
                DABCO in 40 mL of PBS, then add azide (1 mL of 100X,  or 10% solution), then 60 mL
                of glycerol.
       7.8.2    Mounting medium supplied with MeriFluor® Cryptosporidium/Giardia, Meridian
                Diagnostics cat. no. 250050, or equivalent (Section 7.6.1)
       7.8.3    Mounting medium supplied with Aqua-Glo™ G/C Direct FL kit, Waterborne cat. no.
                A100FLR, cat. no. Ml01, or equivalent (Section 7.6.2)
       7.8.4    Mounting medium supplied with EasyStain™C&G, BTF Pty Limited or equivalent
                (Section 7.6.4)
       7.8.5    Elvanol or equivalent permanent, non-fade archiving mounting medium

7 g    Clear fingernail polish or clear fixative, PGC Scientifics, Gaithersburg, MD, cat. no.  60-4890-00,
       or equivalent
710   Oocyst suspensions for  spiking
       7.10.1   Enumerated spiking suspensions prepared by flow cytometer—not formalin fixed.
                7.10.1.1      Live, flow cytometer-sorted oocysts —Wisconsin State Laboratory of
                             Hygiene Flow Cytometry Unit ([608]  224-6260), or equivalent
                7.10.1.2      Irradiated, flow cytometer-sorted oocysts —flow cytometer-sorted
                             oocysts —BTF EasySeed™ (contactfebtfbio.cotri). or equivalent
       7.10.2   Materials for manual  enumeration of spiking suspensions
                7.10.2.1      Purified Cryptosporidium oocyst stock suspension for manual
                             enumeration—not formalin-fixed:  Sterling Parasitology Laboratory,
                             University of Arizona, Tucson, or equivalent
                7.10.2.2      Tween® 20 , 0.01%—Dissolve 1.0 mL of a 10% solution ofTween® 20
                             in 1 L of reagent water
       7.10.3   Storage procedure—Store oocyst suspensions between 1°C and 10°C, until ready to use;
                do not allow to freeze
7 .j-j   Additional reagents for enumeration of spiking suspensions using membrane filtration (Section
       11.3.6)—Sigmacote® Sigma Company Product No. SL-2, or equivalent

December 2005                                10

-------
                                                                   Method 1622 - Cryptosporidium
8.0   Sample Collection and Storage
g -j    Sample collection, shipment, and receipt
       8.1.1   Sample collection. Samples are collected as bulk samples and shipped to the laboratory
               on ice for processing through the entire method, or are filtered in the field and shipped to
               the laboratory on ice for processing from elution (Section 12.2.6) onward.
       8.1.2   Sample shipment. Ambient water samples are dynamic environments and, depending on
               sample constituents and environmental conditions, Cryptosporidium oocysts present in a
               sample can degrade, potentially biasing analytical results. Samples should be chilled to
               reduce biological activity, and preserve the state of source water samples between
               collection and analysis. Samples analyzed by an off-site laboratory should be shipped on
               ice via overnight service on the day they are collected.
       NOTE: See transportation precautions in Section 5.5.

               8.1.2.1        If samples are collected early in the day,  chill samples by storing in a
                             refrigerator between 1°C and 10°C or pre-icing the sample in a cooler. If
                             the sample is pre-iced before shipping, replace with fresh ice immediately
                             before shipment.
               8.1.2.2       If samples are collected later in the day, these samples may be chilled
                             overnight in a refrigerator between 1°C and 10°C. This should be
                             considered for bulk water samples that will be shipped off-site, as this
                             minimizes the potential for water samples collected during the summer to
                             melt the ice in which they are packed and arrive at the laboratory at
                             >20°C.
               8.1.2.3       If samples are shipped  after collection at >20°C with no chilling, the
                             sample will not maintain the temperature during shipment at <20°C.
               8.1.2.4       Public water systems shipping samples to off-site laboratories for analysis
                             should include in the shipping  container a means for monitoring the
                             temperature of the sample during shipping to verify that the sample did
                             not freeze or exceed 20°C. Suggested approaches for monitoring sample
                             temperature during shipping are discussed in Section 8.1.4.
       8.1.3   Sample receipt. Upon receipt, the laboratory  must record the sample temperature.
               Samples that were not collected the same day  they were received, and that are received at
               >20°C or frozen, or samples that the laboratory has determined exceeded >20°C or froze
               during shipment, must be rejected. After receipt, samples must be stored at the laboratory
               between 1°C and 10°C, and not frozen, until processed.
       8.1.4   Suggestions on measuring sample temperature. Given the importance of maintaining
               sample temperatures for Cryptosporidium determination, laboratories performing
               analyses using this method must establish acceptance criteria for receipt of samples.
               transported to their laboratory. Several options are available to measure sample
               temperature upon receipt at the laboratory  and, in some cases, during shipment:
               8.1.4.1        Temperature sample. One option, for filtered samples only (not for 10-L
                             bulk samples), is for the sampler to fill a small, inexpensive sample bottle
                             with water and pack this "temperature sample" next to the filtered
                             sample. The temperature of this extra sample volume is measured upon
                             receipt to estimate the temperature of the filter. Temperature sample
                             bottles  are not appropriate for use with bulk samples because of the
                             potential effect that the difference in sample volume may have in
                             temperature equilibration in the sample cooler. Example product: Cole
                             Farmer cat. no. U-06252-20.


                                               11                                 December 2005

-------
 Method 1622 - Cryptosporidium
                8.1.4.2       Thermometer vial. A similar option is to use a thermometer that is
                              securely housed in a liquid-filled vial. Unlike temperature samples, the
                              laboratory does not need to perform an additional step to monitor the
                              temperature of the vial upon receipt, but instead just needs to read the
                              thermometer. The thermometer vial is appropriate for use with filtered
                              samples not bulk samples. Example product: Eagle-Picher  Sentry
                              Temperature Vial 3TR40CS-F or 3TR-40CS.
                8.1.4.3       iButton. Measures the sample temperature during shipment and upon
                              receipt.  An iButton is a small, waterproof device that contains a
                              computer chip that can be programmed to record temperature at different
                              time intervals.  The information is then downloaded from the iButton
                              onto a computer.  The iButton should be placed in a temperature sample,
                              rather than placed loose in the cooler, or attached to the sample container.
                              This option is appropriate for use with both filtered and bulk samples.
                              Information on Thermocron® iButtons is available from
                              http://www.ibutton.com/. Distributors include http://www.pointsix.com/.
                              http://www.rdsdistributing.com. and http://www.scigiene.com/.
                8.1.4.4       Stick-on temperature strips. Another option is for the laboratory to
                              apply a stick-on temperature strip to the outside of the sample container
                              upon receipt at the laboratory. This option does not measure temperature
                              as precisely as the other options, but provides an indication of sample
                              temperature to verify that the sample temperature is acceptable. This
                              option is appropriate for use with both filtered and bulk samples. Example
                             product: Cole Farmer cat. no. U-90316-00.
                8.1.4.5       Infrared thermometers. A final option is to measure the temperature of
                              the surface of the sample container or filter using an infrared
                              thermometer. The thermometer is pointed at the sample, and measures the
                              temperature without coming in contact with the sample volume. This
                              option is appropriate for use with both filtered and bulk samples. Example
                             product: Cole Farmer cat. no. EW-39641-00.
                As with other laboratory equipment, all temperature measurement devices must be
                calibrated routinely to ensure accurate measurements. See the EPA Manual for the
                Certification of Laboratories Analyzing Drinking Water (Reference 20.9) for more
                information.
22     Sample holding times. Samples must be processed or examined within each of the holding times
        specified in Sections 8.2.1 through 8.2.4. Sample processing should be completed as soon as
        possible by the laboratory. The laboratory should complete sample filtration, elution,
        concentration, purification, and staining the day the sample is received wherever possible.
        However, the laboratory is permitted to  split up the sample processing steps if processing a
        sample completely in one day is not possible. If this is necessary, sample processing can be halted
        after filtration, application of the purified sample onto the slide, or staining. Table 1, in Section
        21.0 provides abreakdown of the holding times for each set of steps. Sections 8.2.1 through 8.2.4
        provide descriptions  of these holding times.
        8.2.1    Sample collection and filtration. Sample  elution must be initiated within 96 hours of
                sample collection (if shipped to the laboratory as a bulk sample) or filtration (if filtered
                in the field).
        8.2.2    Sample elution, concentration,  and purification. The laboratory must complete
                elution, concentration, and purification  (Sections 12.2.6 through 13.3.3.11) in one work
                day. It is critical that these steps be completed in one work day to minimize the time that
                any target organisms present in the sample  sit in eluate or concentrated matrix. This
                process ends with the  application of the purified sample on the slide for drying.

December 2005                                 12

-------
                                                                    Method 1622 - Cryptosporidium
       8.2.3    Staining. The sample must be stained within 72 hours of application of the purified
                sample to the slide.
       8.2.4    Examination. Although immunofluorescence assay (FA) and 4',6-diamidino-2-
                phenylindole (DAPI) and differential interference contrast (DIG) microscopy
                examination and characterization should be performed immediately after staining is
                complete, laboratories have up to 168 hours (7 days) from the completion of sample
                staining to perform the examination and verification of samples. However, if
                fading/diffusion of FITC or DAPI staining is noticed, the laboratory must reduce this
                holding time. In addition the laboratory may adjust the concentration of the DAPI
                staining solution (Sections 7.7.2) so that fading/diffusion does not occur.
g 3    Spiking suspension enumeration holding times. Flow-cytometer-sorted spiking suspensions
       (Sections 7.10.1 and 11.2) used for spiked quality control (QC) samples (Section 9) must be used
       within the expiration date noted on the suspension. Manually enumerated spiking suspensions
       must be used within 24 hours of enumeration of the spiking suspension if the  hemacytometer
       chamber technique is used (Section 11.3.4); or within 24 hours of application of the spiking
       suspension to the slides if the well slide or membrane filter enumeration technique is used
       (Sections 11.3.5 and 11.3.6). Oocyst suspensions must be stored between 1°C and 10°C, until
       ready to use; do not allow to freeze.

9.0   Quality Control
g i    Each laboratory that uses this method is required to operate a formal quality assurance (QA)
       program that addresses and  documents data quality, instrument and equipment maintenance and
       performance, reagent quality and performance, analyst training and certification, and records
       storage and retrieval. General requirements and recommendations for QA and quality control
       (QC) procedures for microbiology laboratories are provided in References 20.8, 20.9, 20.10. The
       minimum analytical requirements of this program consist of an initial demonstration of laboratory
       capability (IDC) through performance of the initial precision and recovery (IPR) test  (Section
       9.4), and ongoing demonstration of laboratory capability and method performance through the
       matrix spike (MS) test (Section 9.5.1), the method blank test (Section 9.6), the ongoing precision
       and recovery (OPR) test (Section 9.7), staining controls (Section  14.1 and 15.2.1), and analyst
       verification tests (Section 10.6). Laboratory performance is compared to established performance
       criteria to determine if the results of analyses meet the performance characteristics of the method.
       9.1.1    A test of the microscope used for detection of oocysts is performed prior to examination
                of slides. This test is described in Section 10.0.
       9.1.2    In recognition of advances that are occurring in analytical technology, the laboratory is
                permitted to modify certain method procedures to improve recovery or lower the costs of
                measurements, provided that all required quality control (QC) tests are performed and all
                QC acceptance criteria are met. Method procedures that can be modified include front-
                end techniques, such as filtration or immunomagnetic separation (IMS). The laboratory
                is not permitted to use an alternate determinative technique to replace
                immunofluorescence assay  in this method (the use of different determinative techniques
                are considered to be different methods, rather than modified version of this method).
                However, the laboratory is permitted to modify the immunofluorescence assay
                procedure, provided that all required QC tests are performed (Section 9.1.2.1) and all QC
                acceptance criteria are met (see guidance on the use of multiple labeling reagents in
                Section 7.6).
                                               13                                 December 2005

-------
 Method 1622 - Cryptosporidium
        NOTE:  Method modifications should be considered only to improve method
        performance, reduce cost, or reduce sample processing time. Method modifications that
        reduce cost or sample processing time, but that result in poorer method performance
        should not be used.

                9.1.2.1      Method modification validation/equivalency demonstration requirements
                             9.1.2.1.1      Method modifications at a single laboratory. Each
                                            time a modification is made to this method for use in a
                                            single laboratory, the laboratory must, at a minimum,
                                            validate the modification according to Tier 1 of EPA's
                                            performance-based measurement system (PBMS) (Table
                                            2) to demonstrate that the modification produces results
                                            equivalent or superior to results produced by this method
                                            as written. Briefly, each time a modification is made to
                                            this method, the laboratory is required to demonstrate
                                            acceptable modified method performance through the
                                            IPR test (Section 9.4). IPR results must meet the QC
                                            acceptance criteria in Tables 3 and 4 in Section 21.0, and
                                            should be comparable to previous results using the
                                            unmodified procedure. Although not required, the
                                            laboratory also should perform a matrix spike/matrix
                                            spike duplicate (MS/MSD) test to demonstrate the
                                            performance of the modified method in at least one real-
                                            world matrix before analyzing field samples using the
                                            modified method. The laboratory is required to perform
                                            MS samples using the modified method at the frequency
                                            noted in Section 9.1.8. If the modified method involves
                                            changes that cannot be adequately evaluated through
                                            these tests, additional tests may be required to
                                            demonstrate acceptability.
                             9.1.2.1.2      Method modifications for nationwide approval. If the
                                            laboratory or a manufacturer seeks EPA approval of a
                                            method modification for nationwide use, the laboratory
                                            or manufacturer must, at a minimum, validate the
                                            modification according to Tier 2 of EPA's PBMS (Table
                                            2). Briefly, at least three laboratories must perform IPR
                                            tests (Section 9.4)  and MS/MSD (Section 9.5) tests using
                                            the modified method, and all tests must meet the QC
                                            acceptance criteria specified in Tables 3 and 4 in Section
                                            21.0. Upon nationwide approval, laboratories electing to
                                            use the modified method still must demonstrate
                                            acceptable performance in their own laboratory according
                                            to the requirements in Section 9.1.2.1.1. If the modified
                                            method involves changes that cannot be adequately
                                            evaluated through these tests, additional tests may be
                                            required to demonstrate acceptability.
                9.1.2.2      The laboratory is required to maintain records of modifications made to
                             this method. These records include the following, at a minimum:
                             9.1.2.2.1      The names, titles, addresses, and telephone numbers of
                                            the analyst(s) who performed the analyses and
December 2005                                 14

-------
                                                             Method 1622 - Cryptosporidium
                                     modification, and ofthe quality control officer who
                                     witnessed and will verify the analyses and modification.
                      9.1.2.2.2      A listing of the analyte(s) measured (Cryptosporidium).
                      9.1.2.2.3      A narrative stating reason(s) for the modification.
                      9.1.2.2.4      Results from all QC tests comparing the modified method
                                     to this method, including:
                                     (a)     IPR (Section 9.4)
                                     (b)     MS/MSD (Section 9.5)
                                     (c)     Analysis of method blanks (Section 9.6)
                      9.1.2.2.5      Data that will allow an independent reviewer to validate
                                     each determination by tracing the following processing
                                     and analysis steps leading to the final result:
                                     (a)     Sample numbers and other identifiers
                                     (b)     Source of spiking suspensions, as well as lot
                                            number and date received (Section 7.10)
                                     (c)     Spike enumeration date and time
                                     (d)     All spiking suspension enumeration counts and
                                            calculations (Section 11.0)
                                     (e)     Sample spiking dates and times
                                     (f)     Volume filtered (Section 12.2.5.2)
                                     (g)     Filtration and elution dates and times
                                     (h)     Pellet volume, resuspended concentrate volume,
                                            resuspended concentrate volume transferred to
                                            IMS, and all calculations required to verify the
                                            percent of concentrate examined (Section 13.2)
                                     (i)     Purification completion dates and times (Section
                                            13.3.3.11)
                                     (j)     Staining completion dates and times (Section
                                            14.10)
                                     (k)     Staining control results (Section 15.2.1)
                                     (1)     All required examination information (Section
                                            15.2.2)
                                     (m)    Examination completion dates and times  (Section
                                            15.2.4)
                                     (n)     Analysis sequence/run chronology
                                     (o)     Lot numbers of elution, IMS, and staining
                                            reagents
                                     (p)     Copies of bench sheets, logbooks, and other
                                            recordings of raw data
                                     (q)     Data system outputs, and other data to link the
                                            raw data to the results reported
9.1.3   The laboratory shall spike a separate sample aliquot from the same source to monitor
        method performance. The frequency of the MS test is described in Section 9.1.8 and the
        procedures are described in Section 9.5.1.
9.1.4   Analysis of method blanks is required to demonstrate freedom from contamination. The
        frequency of the analysis of method blanks is described in Section 9.1.7 and the
        procedures and criteria for analysis of a method blank are described in Section 9.6.
9.1.5   The laboratory shall, on an ongoing basis, demonstrate through analysis of the ongoing
        precision and recovery (OPR) sample that the analysis system is in control. Frequency of
        OPR samples is described in Section 9.1.7 and the procedures are described in Section
        9.7.

                                        15                                 December 2005

-------
Method 1622 - Cryptosporidium
        9.1.6   The laboratory shall maintain records to define the quality of data that are generated.
                Development of accuracy statements is described in Sections 9.5.1.4 and 9.7.6.
        9.1.7   The laboratory shall analyze one method blank (Section 9.6) and one OPR sample
                (Section 9.7) each week (7 day or 168 hours time period which begins with processing
                the OPR) in which samples arc analyzed if 20 or fewer field samples are analyzed during
                this period. The laboratory shall analyze one laboratory blank and one OPR sample for
                every 20 samples if more than 20 samples  are analyzed in a one week (7 day or 168
                hours) period.
        9.1.8   The laboratory shall analyze MS samples (Section 9.5.1) at a minimum frequency of 1
                MS sample per 20 field samples from each source analyzed. The laboratory should
                analyze an MS sample when samples are first received from a PWS for which the
                laboratory has never before analyzed samples to identify potential method performance
                issues with the matrix (Section 9.5.1; Tables 3 and 4). If an MS sample cannot be
                analyzed on the first sampling event, the first MS sample should be analyzed as soon as
                possible to identify potential method performance issues with the matrix.
g 2     Micropipette calibration
        9.2.1    Micropipettes must be sent to the manufacturer for calibration annually. Alternately, a
                qualified independent technician specializing in micropipette calibration can be used, or
                the calibration can be performed by the laboratory, provided the laboratory maintains a
                detailed procedure that can be evaluated by an independent auditor. Documentation on
                the precision of the recalibrated micropipette must be obtained  from the manufacturer or
                technician.
        9.2.2   Internal and external  calibration records must be kept on file in the laboratory's QA
                logbook.
        9.2.3   If a micropipette calibration problem is suspected, the laboratory shall tare an empty
                weighing boat on the  analytical balance and pipette the following volumes of reagent
                water into the weigh boat using the pipette in question:  100% of the maximum
                dispensing capacity of the micropipette,  50% of the capacity, and 10% of the capacity.
                Ten replicates should be performed at each weight. Record the  weight of the water
                (assume that 1.00 mL of reagent water weighs  1.00 g) and calculate the relative standard
                deviation (RSD) for each. If the weight of the reagent water is within  1% of the desired
                weight (mL) and the RSD of the replicates at each weight  is within 1%, then the pipette
                remains acceptable for use.
        9.2.4   If the weight of the reagent water is outside the acceptable limits, consult the
                manufacturer's instruction manual troubleshooting section and  repeat steps described in
                Section 9.2.3. If problems with the pipette  persist, the laboratory must send the pipette to
                the manufacturer for recalibration.
g 3     Microscope adjustment and calibration—Adjust the microscope as specified in Section 10.0.  All
        of the requirements in Section  10.0 must be met prior to analysis of IPRs, method blanks, OPRs,
        field samples, and MS/MSDs.
g 4     Initial precision and recovery (IPR)—To establish the ability to demonstrate control over the
        analytical system and to generate acceptable precision and recovery, the  laboratory shall perform
        the following operations:
        9.4.1    Using the spiking procedure in Section 11.4 and enumerated spiking suspensions
                (Section 7.10.1 or Section 11.3), spike, filter, elute, concentrate, separate (purify), stain,
                and examine the four reagent water samples spiked with -100-500 oocysts.
                9.4.1.1       The laboratory is permitted to analyze the four spiked reagent samples on
                             the same day or on as many as four different days (provided that the
                             spiked reagent samples are  analyzed consecutively), and also may use


December 2005                                  16

-------
                                                                   Method 1622 - Cryptosporidium
                             different analysts and/or reagent lots for each sample (however, the
                             procedures used for all analyses must be identical). Laboratories should
                             note that the variability of four measurements performed on multiple days
                             or using multiple analysts or reagent lots may be greater than the
                             variability of measurements performed on the same day with the same
                             analysts and reagent lots. As a result, the laboratory is at a greater risk of
                             generating unacceptable IPR results if the test is performed across
                             multiple days, analysts, and /or reagent lots.
               9.4.1.2       If more than one modification will be used for filtration and/or separation
                             of samples, a separate set of IPR samples must be prepared for each
                             modification.
               9.4.1.3       The set of four IPR samples must be accompanied by analysis of an
                             acceptable method blank (Section 9.6).
       9.4.2   Calculate the percent recovery (R) using the following equation:
                                N
                  R=   100 x  	
               where:
                      R = the percent recovery
                      N = the number of oocysts counted
                      T = the number of oocysts spiked

               This calculation assumes that the total volume spiked was processed and examined.
       9.4.3   Using percent recovery (R) of the four analyses, calculate the mean percent recovery and
               the relative standard deviation (RSD) of the recoveries for Cryptosporidium. The RSD is
               the standard deviation divided by the mean, times 100.
       9.4.4   Compare the mean and RSD to the corresponding method performance acceptance
               criteria for initial precision and recovery in Table 3 in Section 21.0. If the mean and RSD
               for recovery meet the acceptance criteria, system performance is acceptable and analysis
               of blanks and samples may begin. If the mean or RSD falls outside the range for
               recovery, system performance is unacceptable. In this event, trouble-shoot the problem
               by starting at the end of the method (see guidance in Section 9.7.5), correct the problem
               and repeat  the IPR test (Section 9.4.1).
       9.4.5   Examine and document the IPR slides following the procedure in Section 15.0. The first
               three Cryptosporidium oocysts identified in each IPR sample must be characterized (size,
               shape, DAPI category, and DIG category) and documented on the examination form, as
               well as any additional comments on organisms appearance, if notable.
       9.4.6   Using 200X to 400X magnification, more than 50% of the oocysts must appear
               undamaged and morphologically intact; otherwise, the organisms in the spiking
               suspension may be of unacceptable quality or the analytical process may be damaging
               the organisms. If the quality of the organisms on the IPR test  slides is unacceptable,
               examine the spiking suspension organisms directly (by centrifuging, if possible, to
               concentrate the organisms  in a volume that can be applied directly to a slide). If the
               unprocessed organisms appear undamaged and morphologically intact under DIC,
               determine the step or reagent that is causing damage to the organisms. Correct the
               problem (see Section 9.7.5) and repeat the IPR test.
g 5    Matrix spike (MS) and matrix spike duplicate (MSD)
                                               17                                December 2005

-------
Method 1622 - Cryptosporidium
        9.5.1    Matrix spike— The laboratory shall spike and analyze a separate field sample aliquot to
                determine the effect of the matrix on the method's oocyst  recovery. The MS and field
                sample must be that was collected from the same sampling location as split samples or as
                samples sequentially collected immediately after one another. The MS sample volume
                analyzed must be within 10% of the field sample volume. The MS shall be analyzed
                according to the frequency in Section 9.1.8.
                9.5.1.1       Analyze an unspiked field sample according to the procedures in Sections
                             12.0 to 15.0. Using the spiking procedure in Section 11.4 and enumerated
                             spiking suspensions (Section 7.10.1 or Section 11.3), spike, filter, elute,
                             concentrate, separate (purify), stain, and examine a second field sample
                             aliquot with a similar number of organisms as that used in the IPR or
                             OPR tests (Sections 9.4 and 9.7).
                9.5.1.2      Calculate the percent recovery (R) using the following equation.
                                R=    100 x
                             where
                                     R is the percent recovery
                                     Nsp is the number of oocysts counted in the spiked sample
                                     Ns is the number of oocysts counted in the unspiked sample
                                     T is the true value of the oocysts spiked

                9.5.1.3      Compare the recovery with the acceptance criteria in Table 3 in Section
                             21.0.
        NOTE:  Some sample matrices may prevent the acceptance criteria  in Tables 3 from
        being met. An assessment of the distribution of MS recoveries across 430 MS samples
       from 87 sites during the ICR Supplemental Surveys is provided in Table 5.

                9.5.1.4      As part of the QA program for the laboratory, method precision for
                             samples should be assessed and records maintained. After the analysis of
                             five samples, the  laboratory should calculate the mean percent recovery
                             (P) and the standard deviation of the percent recovery (sr). Express the
                             precision assessment as a percent recovery interval from P - 2 sr to P + 2
                             sr for each matrix. For example, if P = 80% and sr = 30%, the accuracy
                             interval is expressed as 20% to 140%. The precision assessment should
                             be updated regularly across all MS samples and stratified by MS samples
                             for each source.
        9.5.2    Matrix spike duplicate—MSD analysis is required as part of Tier 2 or nationwide
                approval of a modified version of this method to demonstrate that the modified version
                of this method produces results equal or superior to  results produced by the method as
                written (Section 9.1.2.1.2). At the same time the laboratory spikes and analyzes the
                second field sample aliquot in  Section 9.5.1.1, the laboratory shall spike and analyze a
                third, identical field sample aliquot.
        NOTE:  Matrix spike duplicate samples are only required for Tier 2 validation studies.
        They are recommended for Tier I validation, but not required.

                9.5.2.1       Calculate the percent recovery (R) using the equation in  Section 9.5.1.2.
December 2005                                 18

-------
                                                                  Method 1622 - Cryptosporidium
              9.5.2.2       Calculate the mean of the number of oocysts in the MS and MSD
                           (= [MS+MSDJ/2).
              9.5.2.3       Calculate the relative percent difference (RPD) of the recoveries using the
                           following equation:
                                                 N   - N
                                                 INMS  '"MSD
                              RPD =    100 x
                           where
                                  RPD is the relative percent difference
                                  NMS is the number of oocysts counted in the MS
                                  NMSD is the number of oocysts counted in the MSD
                                  Xmean is the mean number of oocysts counted in the MS and MSD
              9.5.2.4       Compare the mean MS/MSD recovery and RPD with the acceptance
                           criteria in Table 3 in Section 21.0.
g    Method blank (negative control sample, laboratory blank)—Reagent water blanks are routinely
     analyzed to demonstrate freedom from contamination. Analyze the blank immediately after
     analysis of the IPR test (Section 9.4) and OPR test (Section 9.7) and prior to analysis of samples
     for the week to demonstrate freedom from contamination.
     9.6.1    Filter, elute, concentrate, separate (purify), stain, and examine at least one reagent water
              method blank per week (Section 9.1.7) according to the procedures in Sections 12.0 to
              15.0. A method blank must be analyzed each week (7 day or 168 hours time period that
              begins with processing the OPR) in which samples are analyzed if 20 or fewer field
              samples are analyzed during this period. If more than 20 samples are analyzed in a week
              (7 days or 168 hours), process and analyze one reagent water method blank for every 20
              samples.
     9.6.2    Actions
              9.6.2.1       If Cryptosporidium oocysts or potentially interfering organisms or
                           materials that may be misidentified as oocysts are not found in the
                           method blank, the method blank test is acceptable and analysis of samples
                           may proceed.
              9.6.2.2       ^Cryptosporidium oocysts (as defined in Section 3) or any potentially
                           interfering organism or materials that may be misidentified as oocysts are
                           found in the method blank, the method blank test is unacceptable. Any
                           field sample in a batch associated with an unacceptable method blank is
                           assumed to be contaminated and should be recollected. Analysis of
                           additional samples is halted until the source of contamination is
                           eliminated, the method blank test is performed again, and no evidence of
                           contamination is detected.
7    Ongoing precision and recovery (OPR; positive control sample; laboratory control
     sample)—Using the spiking procedure in Section 11.4 and enumerated spiking suspensions
     (Section  7.10.1 or Section 11.3), filter, elute, concentrate, separate (purify), stain, and examine at
     least one reagent water sample spiked with -100  to 500 oocysts each week to verify all
     performance criteria. The laboratory must analyze one OPR sample for every 20 samples if more
     than 20 samples are analyzed in a week. If multiple method variations are used, separate OPR
     samples must be prepared for each method variation. Adjustment and/or recalibration of the
     analytical system shall be performed until all performance criteria are met. Only after all
     performance criteria are met should samples be analyzed.
     9.7.1    Examine the  slide from the OPR prior to analysis of samples from the same batch.


                                             19                                December 2005

-------
Method 1622 - Cryptosporidium
                9.7.1.1       Using 200X to 400X magnification, more than 50% of the oocysts must
                             appear undamaged and morphologically intact; otherwise, the organisms
                             in the spiking suspension may be of unacceptable quality or the analytical
                             process may be damaging the organisms. Examine the spiking suspension
                             organisms directly (by centrifuging, if possible, to concentrate the
                             organisms in a volume that can be applied directly to a slide). If the
                             organisms appear undamaged and morphologically intact under DIG,
                             determine the step or reagent that is causing damage to the organisms.
                             Correct the problem and repeat the OPR test.
                9.7.1.2       Identify and enumerate each organism using epifluorescence microscopy.
                             The first three Cryptosporidium oocysts identified in the OPR sample
                             must be examined using FITC, DAPI, and DIC, as per Section 15.2, and
                             the detailed characteristics (size, shape, DAPI category, and DIC
                             category) reported on the Cryptosporidium report form, as well as any
                             additional comments on organism appearance, if notable.
        9.7.2    Calculate the percent recovery (R)  using the following equation:


                                N
                  R=   100 x 	
                                T

                where:
                      R = the percent recovery
                      N = the number of oocysts detected
                      T = the number of oocysts spiked
        9.7.3    Compare the recovery with the acceptance criteria for ongoing precision and recovery in
                Table  3 in Section 21.0.
        9.7.4    Actions
                9.7.4.1       If the recoveries for Cryptosporidium meet the acceptance criteria, system
                             performance  is acceptable and analysis of samples may proceed.
                9.7.4.2       If the recovery for Cryptosporidium falls outside of the criteria, system
                             performance  is unacceptable. Any sample in a batch associated with an
                             unacceptable OPR sample is unacceptable. Analysis of additional samples
                             is halted until the analytical system is brought under control.
                             Troubleshoot the problem using the procedures at Section 9.7.5 as a
                             guide. After assessing the issue, perform another OPR test and verify that
                             Cryptosporidium recoveries meet the  acceptance criteria.
        9.7.5    Troubleshooting. If an OPR sample has failed, and the cause of the failure is not known,
                the laboratory generally should identify the problem working backward  in the analytical
                process from the microscopic examination to filtration.
                9.7.5.1       Quality of spiked organisms. Examine the spiking suspension organisms
                             directly (by centrifuging, if possible, to concentrate the organisms in a
                             volume that can be applied directly to a slide). If the organisms appear
                             damaged under DIC, obtain fresh spiking materials. If the organisms
                             appear undamaged and morphologically intact, determined whether the
                             problem is associated with the microscope system or antibody stain
                             (Section  9.7.5.2).
                9.7.5.2       Microscope system and antibody  stain: To determine if the failure of
                             the OPR test  is due to changes in the microscope or problems with the
                             antibody stain, re-examine the positive staining control (Section 15.2.1),

December 2005                                 20

-------
                                                                    Method 1622 - Cryptosporidium
                             check Kohler illumination, and check the fluorescence of the fluorescein-
                             labelcd monoclonal antibodies (Mabs) and 4',6-diamidino-2-phenylindole
                             (DAPI). If results are unacceptable, re-examine a previously-prepared
                             positive staining control to determine whether the problem is associated
                             with the microscope or the antibody stain.
                9.7.5.3       Separation (purification) system: To determine if the failure of the
                             OPR test is attributable to the separation system, check system
                             performance by spiking a 10-mL volume of reagent water with -100 - 500
                             oocysts and processing the sample through the MS, staining, and
                             examination procedures in Sections 13.3 through 15.0. Recoveries should
                             be greater than 70%.
                9.7.5.4       Filtration/elution/concentration system: If the failure of the OPR test is
                             attributable to the filtration/elution/concentration system, check system
                             performance by processing spiked reagent water according to the
                             procedures in Section 12.2 through 13.2.2, and filter, stain, and examine
                             the sample concentrate according to Section 11.3.6.
       9.7.6    The laboratory should add results that pass the specifications in Section 9.7.3 to initial
                and previous ongoing data and update the QC chart to form a graphic representation of
                continued laboratory performance.  The laboratory should develop a statement of
                laboratory accuracy (reagent water, raw surface water) by calculating the mean percent
                recovery (R) and the standard deviation of percent recovery (sr). Express the accuracy as
                a recovery interval from R - 2 sr to R + 2 sr. For example, if R = 95%  and sr = 25%, the
                accuracy is 45% to 145%.
9 g    The laboratory should periodically analyze an external QC sample, such as a performance
       evaluation or standard reference material, when available. The laboratory also should periodically
       participate in interlaboratory comparison studies using the method.
g g    The specifications contained in this method  can be met if the analytical system is under control.
       The standards used for initial (Section 9.4) and ongoing (Section 9.7) precision and recovery
       should be identical, so that the most precise results will be obtained. The microscope in particular
       will provide the most reproducible results if dedicated to the settings and conditions required for
       the determination of Cryptosporidium by this method.
g -|Q   Depending on specific program requirements, field replicates may be collected to determine the
       precision of the sampling technique, and duplicate spiked samples may be required to determine
       the precision of the analysis.

10.0 Microscope Calibration and Analyst Verification
101   In a room capable of being darkened to near-complete darkness,  assemble the microscope, all
       filters, and attachments. The microscope should be placed on a solid surface free from vibration.
       Adequate workspace should be provided on  either side of the microscope for taking notes and
       placement of slides and ancillary materials.
102   Using the manuals provided with the microscope, all analysts must familiarize themselves with
       operation of the microscope.
•J 0 3   Microscope adjustment and calibration (adapted from Reference 20.10)
       10.3.1   Preparations for adjustment
                10.3.1.1      The microscopy portion of this procedure depends upon proper alignment
                             and adjustment of very sophisticated optics. Without proper alignment
                             and adjustment, the  microscope will not function at maximal efficiency,
                             and reliable identification and enumeration of oocysts will not be
                                               21                                 December 2005

-------
Method 1622 - Cryptosporidium
                              possible. Consequently, it is imperative that all portions of the
                              microscope from the light sources to the oculars are properly adjusted.
                10.3.1.2      While microscopes from various vendors are configured somewhat
                              differently, they all operate on the same general physical principles.
                              Therefore, slight deviations or adjustments may be required to make the
                              procedures below work for a particular instrument.
                10.3.1.3      The sections below assume that the mercury bulb has not exceeded time
                              limits of operation, that the lamp socket is connected to the lamp house,
                              and that the condenser is adjusted to produce Kohler illumination.
                10.3.1.4      Persons with astigmatism should always wear  contact lenses or glasses
                              when using the microscope.

        CAUTION:    In the procedures below, do not touch the quartz portion of the mercury

        bulb with your bare fingers. Finger oils can cause rapid degradation of the quartz and
        premature failure of the bulb.

        WARNING:   Never look at the ultraviolet (UV) light from the mercury lamp, lamp

        house, or the UV image without a barrier filter in place. UV radiation  can cause serious
        eye damage.

        10.3.2  Epifluorescent mercury bulb adjustment: The purpose of this procedure is to ensure even
                field illumination. This procedure must be followed when the microscope is first used,
                when replacing bulbs, and if problems such as diminished fluorescence or uneven field
                illumination are experienced.
                10.3.2.1      Remove the diffuser lens between the lamp and microscope or swing it
                              out of the transmitted light path.
                10.3.2.2      Using a prepared microscope slide, adjust the focus so the image in the
                              oculars is sharply defined.
                10.3.2.3      Replace the slide with a business card or a piece of lens paper.
                10.3.2.4      Close the field diaphragm (iris diaphragm in the microscope base) so only
                              a small point of light is visible on the card. This dot of light indicates the
                              location of the center of the field of view.
                10.3.2.5      Mount the  mercury lamp house on the microscope without the UV
                              diffuser lens in place and turn on the mercury bulb.
                10.3.2.6      Remove the objective in the light path from the nosepiece. A primary
                              (brighter) and secondary image (dimmer) of the mercury bulb arc should
                              appear on the card after focusing the image with the appropriate
                              adjustment.
                10.3.2.7      Using the lamp house adjustments, adjust the primary and secondary
                             mercury bulb images so they are side by side (parallel to each other)  with
                             the transmitted light dot in between them.
                10.3.2.8     Reattach the objective to the nosepiece.
                10.3.2.9     Insert the diffuser lens into the light path between the mercury lamp
                             house and the microscope.
                10.3.2.10    Turn off the transmitted light and replace the card with a slide of
                             fluorescent material. Check the field for even fluorescent illumination.
                             Adjustment of the diffuser lens probably will be required. Additional
                             slight adjustments as in Section  10.3.2.7 above may be required.


December 2005                                 22

-------
                                                            Method 1622 - Cryptosporidium
        10.3.2.11     Maintain a log of the number of hours the UV bulb has been used. Never
                      use the bulb for longer than it has been rated. Fifty-watt bulbs should not
                      be used longer than 100 hours; 100-watt bulbs should not be used longer
                      than 200 hours.
10.3.3  Transmitted bulb adjustment: The purpose of this procedure is to center the filament and
        ensure e'ven field illumination. This procedure must be followed when the bulb is
        changed.
        10.3.3.1      Remove the diffuser lens between the lamp and microscope or swing it
                      out of the transmitted light path.
        10.3.3.2      Using a prepared microscope slide and a 40X (or similar) objective,
                      adjust the focus so the image in the oculars is sharply defined.
        10.3.3.3      Without the ocular or Bertrand optics in place, view the pupil and
                      filament image at the bottom of the tube.
        10.3.3.4      Focus the lamp filament image with the appropriate adjustment on the
                      lamp house.
        10.3.3.5      Similarly, center the lamp filament image within the pupil with the
                      appropriate adjustment(s) on the lamp house.
        10.3.3.6      Insert the diffuser lens into the light path between the transmitted lamp
                      house and the microscope.
10.3.4  Adjustment of the interpupillary distance and oculars for each eye: These adjustments
        are necessary so that eye strain is reduced to a minimum, and must be made for each
        individual using the microscope. Section 10.3.4.2 assumes use of a microscope with both
        oculars adjustable; Section 10.3.4.3 assumes use of a microscope with a single adjustable
        ocular. The procedure must be followed each time an analyst uses the microscope.
        10.3.4.1      Interpupillary distance
                      10.3.4.1.1      Place a prepared slide on the microscope stage, turn on
                                     the transmitted light, and focus the specimen image using
                                     the coarse and fine adjustment knobs.
                      10.3.4.1.2     Using both hands, move the oculars closer together or
                                     farther apart until a single  circle of light is observed
                                     while looking through the  oculars with both eyes. Note
                                     interpupillary distance.
        10.3.4.2      Ocular adjustment for microscopes capable of viewing a photographic
                      frame through the viewing binoculars: This procedure assumes both
                      oculars are adjustable.
                      10.3.4.2.1      Place a card between the right ocular and eye keeping
                                     both eyes open. Adjust the correction (focusing) collar on
                                     the left ocular by focusing the left ocular until it  reads the
                                     same as the interpupillary distance. Bring an image
                                     located in the center of the field of view into as sharp a
                                     focus as possible.
                      10.3.4.2.2     Transfer the card to between the left eye and ocular.
                                     Again keeping both eyes open, bring the same image into
                                     as sharp a focus for the right eye as possible by adjusting
                                     the ocular correction (focusing) collar at the top  of the
                                     right ocular.
                                       23                                 December 2005

-------
 Method 1622 - Cryptosporidium
                10.3.4.3      Ocular adjustment for microscopes without binocular capability: This
                              procedure assumes a single focusing ocular. The following procedure
                              assumes that only the right ocular is capable of adjustment.
                              10.3.4.3.1     Place a card between the right ocular and eye keeping
                                            both eyes open. Using the fine adjustment, focus the
                                            image for the left eye to its sharpest point.
                              10.3.4.3.2     Transfer the card to between the left eye and ocular.
                                            Keeping both eyes open, bring the image for the right eye
                                            into sharp focus by adjusting the ocular collar at the top
                                            of the ocular without touching the coarse or fine
                                            adjustment.
        10.3.5  Calibration of an ocular micrometer: This section assumes that a reticle has been
                installed in one of the oculars by a microscopy specialist and that a stage micrometer is
                available for calibrating the ocular micrometer (reticle). Once installed, the ocular reticle
                should be left in place. The more an ocular is manipulated the greater the probability is
                for it to become contaminated with dust particles. This calibration should be done for
                each objective in use on the microscope. If there is a top lens on the microscope, the
                calibration procedure must be done for the respective objective at each top lens setting.
                The procedure must be followed when the microscope is first used and each time the
                objective is changed.
                10.3.5.1       Place the stage micrometer on the microscope stage, turn on the
                              transmitted  light, and focus the micrometer image using the coarse and
                              fine adjustment knobs for the objective to be calibrated. Continue
                              adjusting the focus on the stage micrometer so you can distinguish
                              between the large (0.1 mm) and the small (0.01 mm) divisions.
                10.3.5.2      Adjust the stage and ocular with the micrometer so the "0" line on the
                              ocular micrometer is  exactly superimposed on the "0" line on the stage
                              micrometer.
                10.3.5.3      Without changing the stage adjustment, find a point as distant as possible
                              from the two 0 lines where two other lines are exactly superimposed.
                10.3.5.4      Determine the number of ocular micrometer spaces as well as the number
                              of millimeters on the stage micrometer between the two points of
                              superimposition. For example: Suppose 48  ocular micrometer spaces
                              equal 0.6 mm.
                10.3.5.5      Calculate the number of mm/ocular micrometer space. For example:


                                            0.6mm                            0.0125mm
                                   48 ocular micrometer spaces             ocular micrometer space

                10.3.5.6     Because most measurements of microorganisms are given in um rather
                             than mm, the value calculated above must be converted to um by
                             multiplying it by 1000 urn/mm. For example:

                                      0.0125mm              1,000 urn             12.5 urn
                                                         x             =
                                 ocular micrometer space          mm         ocular micrometer space
December 2005                                 24

-------
                                                            Method 1622 - Cryptosporidium
        10.3.5.7
Follow the procedure below for each objective. Record the information as
shown in the example below and keep the information available at the
microscope.
Item
no.
1
2
3
4
Objective
power
10X
20X
40X
100X
Description
N.A.3=
N.A.=
N.A.=
N.A.=
No. of ocular
micrometer
spaces




No. of stage
micrometer
mm'




pm/ocular
micrometer
space2




                      11000 (jm/mm
                      2(Stage micrometer length in mm * (1000 urn/mm)) + no. ocular micrometer
                      spaces
                      3N.A. refers to numerical aperature. The numerical aperature value is engraved
                      on the barrel of the objective.
10.3.6  Kohler illumination: This section assumes that Kohler illumination will be established
        for only the 100X oil DIC objective that will be used to identify internal morphological
        characteristics in Cryptosporidium oocysts. If more than one objective is to be used for
        DIC, then each time the objective is changed, Kohler illumination must be reestablished
        for the new objective lens. Previous sections have adjusted oculars and light sources.
        This section aligns and focuses the light going through the condenser underneath the
        stage at the specimen to be observed. If Kohler illumination is not properly established,
        then DIC will not work to its maximal potential. These steps need to become second
        nature and must be practiced regularly until they are a matter of reflex rather than a
        chore. The procedure must be followed each time  an analyst uses the microscope and
        each time the objective is changed.
        10.3.6.1      Place a prepared slide on the microscope stage, place oil on the slide,
                      move the 100X oil objective  into place, turn  on the transmitted light, and
                      focus the specimen image using the coarse and fine adjustment knobs.
        10.3.6.2      At this point both the radiant field  diaphragm in the microscope base and
                      the aperture diaphragm in the condenser should be wide open. Now close
                      down the radiant field diaphragm in the microscope base until the lighted
                      field is reduced to a small opening.
        10.3.6.3      Using the condenser centering screws on the front right and left of the
                      condenser, move the small lighted  portion of the field to the center of the
                      visual field.
        10.3.6.4      Now look to see  whether the leaves of the iris field diaphragm are sharply
                      defined (focused) or not. If they are not sharply defined,  then they can be
                      focused distinctly by changing the  height of the condenser up and down
                      with the condenser focusing knob while you are looking through the
                      binoculars. Once you have accomplished the precise focusing of the
                      radiant field diaphragm leaves, open the radiant field diaphragm until the
                      leaves just disappear from view.
        10.3.6.5      The aperture diaphragm of the condenser should now be adjusted to make
                      it compatible with the total numerical aperture of the optical system. This
                      is done by removing an ocular, looking into the tube at the rear focal
                      plane of the objective, and stopping down the aperture diaphragm iris
                      leaves until they are visible just inside the rear plane of the objective.
                                        25
                                                     December 2005

-------
Method 1622 - Cryptosporidium
                10.3.6.6     After completing the adjustment of the aperture diaphragm in the
                             condenser, return the ocular to its tube and proceed with the adjustments
                             required to establish DIG.
104   Microscope cleaning procedure
        10.4.1   Use canned air to remove dust from the lenses, filters, and microscope body.
        10.4.2   Use a Kimwipe-dampened with a microscope cleaning solution (MCS) (consisting of 2
                parts 90% isoproponal and 1 part acetone) to wipe down all surfaces of the microscope
                body. Dry off with a clean, dry Kimwipe.
        10.4.3   Protocol for cleaning oculars and condenser
                10.4.3.1      Use a new, clean Q-tip dampened with MCS to clean each lense. Start at
                             the center of the lens and spiral the Q-tip outward using little to no
                             pressure. Rotate the Q-tip head while spiraling to ensure a clean surface is
                             always contacting the lens.
                10.4.3.2     Repeat the procedure using a new, dry Q-tip.
                10.4.3.3     Repeat Sections 10.4.3.1  and  10.4.3.2.
                10.4.3.4     Remove the ocular and repeat the cleaning procedure on the bottom lens
                             of the ocular.
        10.4.4   Protocol for cleaning objective lenses
                10.4.4.1      Wipe 100X oil  objective  with lens paper to remove the bulk of the oil
                             from the objective.
                10.4.4.2     Hold a new Q-tip dampened with MCS at a 45° angle on the objective
                             and twirl.
                10.4.4.3     Repeat Sections 10.4.4.2  with a new, dry Q-tip.
                10.4.4.4     Repeat Sections 10.4.4.2  and 10.4.4.3.
                10.4.4.5     Clean all objectives whether they are used or not.
        10.4.5   Protocol for cleaning light source lens and filters
                10.4.5.1      Using a Kimwipe dampened with microscope cleaning solution, wipe off
                             the surface of each lens and filter.
                10.4.5.2     Repeat the procedure using a dry Kimwipe.
                10.4.5.3     Repeat Sections 10.4.5.1  and 10.4.5.2.
        10.4.6   Protocol for cleaning microscope stage
                10.4.6.1      Using a Kimwipe dampened with microscope cleaning solution, wipe off
                             the stage and stage clip. Be sure to clean off any residual immersion oil or
                             fingernail polish. Remove the stage clip if necessary to ensure that it is
                             thoroughly cleaned.
        10.4.7   Use 409 and a paper towel to clean the bench top surrounding the microscope.
        10.4.8   Frequency
                10.4.8.1      Perform Sections 10.4.2,  10.4.3, 10.4.4, 10.4.5 and 10.4.7 after each
                             microscope session.
                10.4.8.2      Perform complete cleaning each week.
•jQ_5   Protozoa libraries: Each laboratory is encouraged  to develop libraries of photographs and
        drawings for identification of protozoa.
        10.5.1  Take color photographs of Cryptosporidium oocysts by FA, 4',6-diamidino-2-
               phenylindole (DAPI), and DIG that the analysts (Section 22.2) determine are accurate
                (Section 15.2).

December 2005                                 26

-------
                                                                    Method 1622 - Cryptosporidium
       10.5.2  Similarly, take color photographs of interfering organisms and materials by FA, DAPI,
                and DIG that the analysts believe are not Cryptosporidium oocysts. Quantify the size,
                shape, microscope settings, and other characteristics that can be used to differentiate
                oocysts from interfering debris and that will result in accurate identification of positive
                or negative organisms.
106   Verification of analyst performance: Until standard reference materials, such as National Institute
       of Standards and Technology standard reference materials, are available that contain a reliable
       number of DAPI positive or negative oocysts,  this method shall rely upon the ability of the analyst
       for identification and enumeration of oocysts. The goal of analyst verification is to encourage
       comparison and discussion among analysts to continually refine the consistency of
       characterizations between analysts.
       10.6.1   At least monthly when microscopic examinations are being performed, the laboratory
                shall prepare a slide containing 40 to 200 oocysts. More than 50% of the oocysts must be
                DAPI positive and undamaged under DIC.
       10.6.2  Each analyst shall determine the total number of oocysts detected by FITC on the entire
                slide meeting the criteria in 10.6.1. For the same 10 oocysts, each analyst shall  determine
                the DAPI category (DAPI negative, DAPI positive internal intense blue and DAPI
                positive number of nuclei) and the DIC category (empty, containing amorphous
                structures,  or containing identifiable internal structures) of each. The DAPI/DIC
                comparisons maybe performed on the slide prepared in 10.6.1, OPR slide, MS slide, or a
                positive staining control slide.
       10.6.3  Requirements for laboratories with multiple analysts
                10.6.3.1       The total number of oocysts determined by each analyst (Section 10.6.2.)
                              must be within ±10%  of each other. If the number is not within this range,
                              the analysts must identify the source of any variability between  analysts'
                              examination criteria, prepare a new slide, and repeat the performance
                              verification (Sections 10.6.1 to 10.6.2). It is recommended that the DAPI
                              and DIC categorization of the same 10 oocysts occur with all analysts at
                              the same time, i.e. each analyst determines the categorizations
                              independently, then the differences in the DAPI and DIC  categorizations
                              among analysts are discussed and resolved, and these resolutions
                              documented. Alternatively, organism coordinates may be recorded for
                              each analyst to locate  and categorize the organisms at different times.
                              Differences among analysts must be discussed and resolved.
                10.6.3.2       Document the date, name(s) of analyst(s), number of total oocysts, and
                              DAPI and DIC categories determined by the analyst(s), whether the test
                              was passed/failed and the results of attempts  before the test was passed.
                10.6.3.3       Only after an analyst has passed the criteria in Section 10.6.3, may
                              oocysts in QC samples and field samples be identified and enumerated.
       10.6.4  Laboratories with only one analyst should maintain a protozoa library (Section 10.5) and
                compare the results of the examinations performed in Sections 10.6.1 and 10.6.2 to
                photographs of oocysts and interfering organisms to verify that examination results are
                consistent with these references. These laboratories also should perform  repetitive counts
                of a single  verification slide for FITC. These laboratories should also coordinate with
                other laboratories to share slides and  compare counts.
                                               27                                 December 2005

-------
Method 1622 - Cryptosporidium
11.0  Oocyst Suspension Enumeration and Sample Spiking
111    This method requires routine analysis of spiked QC samples to demonstrate acceptable initial and
        ongoing laboratory and method performance (initial precision and recovery samples [Section 9.4],
        matrix spike and matrix spike duplicate samples [Section 9.5], and ongoing precision and
        recovery samples [Section 9.7]). The organisms used for these samples must be enumerated to
        calculate recoveries (and precision) and monitor method performance. EPA recommends that flow
        cytometry be used for this enumeration, rather than manual techniques. Flow cytometer-sorted
        spikes generally are characterized by a relative standard deviation of <2.5%, versus greater
        variability for manual enumeration techniques (Reference 20.11). Guidance on preparing spiking
        suspensions using a flow cytometer is provided in Section 11.2. Manual enumeration procedures
        are provided in Section 11.3. The procedure for spiking bulk samples in the laboratory is provided
        in Section 11.4.
11 2    Flow cytometry enumeration guidelines. Although it is unlikely that many laboratories
        performing Method 1622  will have direct access to a flow cytometer for preparing spiking
        suspensions, flow-sorted suspensions are available from commercial vendors and other sources
        (Section 7.10.1). The information provided in Sections 11.2.1 through 11.2.4 is simply meant as a
        guideline for preparing spiking suspensions using a flow cytometer. Laboratories performing flow
        cytometry must develop and implement detailed standardized protocols for calibration and
        operation of the flow cytometer.
        11.2.1  Spiking suspensions should be prepared using unstained organisms that have not been
               formalin-fixed.
        11.2.2  Spiking suspensions should be prepared using Cryptosporidium parvum oocysts <3
               months old.
        11.2.3  Initial calibration. Immediately before sorting spiking suspensions, an initial calibration
               of the flow cytometer should be performed by conducting 10 sequential  sorts directly
               onto membranes or well slides. The oocyst levels used for the initial calibration should
               be the same as the levels used for the spiking suspensions. Each initial calibration sample
               should be stained and manually counted microscopically and the manual counts used to
               verify the accuracy of the system. The relative standard deviation (RSD) of the 10 counts
               should be < 2.5%. If the RSD is > 2.5%, the laboratory should perform the initial
               calibration again, until the RSD of the 10 counts is < 2.5%. In addition to counting the
               organisms, the laboratory also should evaluate the quality of the organisms using DAPI
               fluorescence and DIG to confirm that the organisms are in good condition.
        11.2.4  Ongoing calibration. When sorting the spiking suspensions for use in QC samples, the
               laboratory should perform ongoing calibration samples at a 10% frequency, at a
               minimum. The laboratory should sort the first run and every eleventh sample directly
               onto a membrane or well slide. Each ongoing calibration sample should be stained and
               manually counted microscopically and the manual counts used to verify  the accuracy of
               the system. The mean of the ongoing calibration counts also should be used as the
               estimated spike dose, if the relative standard deviation (RSD) of the ongoing calibration
               counts is < 2.5%. If the RSD is > 2.5%, the laboratory should discard the batch.
        11.2.5  Method blanks. Depending on the operation of the flow cytometer, method blanks
               should be prepared and examined at the same frequency as the ongoing calibration
               samples (Section 11.2.4).
        11.2.6  Holding time criteria. Flow-cytometer-sorted spiking suspensions (Sections 7.10.1 and
               11.2)  used for spiked quality control (QC) samples (Section 9) must be used within the
               expiration date noted on the suspension. The holding time specified by the flow
               cytometry laboratory should be determined based on a holding time study.
113   Manual enumeration procedures. Two sets of manual enumerations are required per organism
       before purified Cryptosporidium oocyst (Section 7.10.2) received from suppliers can be used to

December 2005                                28

-------
                                                            Method 1622 - Cryptosporidium
spike samples in the laboratory. First, the stock suspension must be diluted and enumerated
(Section 11.3.3) to yield a suspension at the appropriate oocyst concentration for spiking (spiking
suspension). Then, 10 aliquots of spiking suspension must be enumerated to calculate a mean
spike dose. Spiking suspensions can be enumerated using hemacytometer chamber counting
(Section 11.3.4), well slide counting (Section 11.3.5), or membrane filter counting (Section
11.3.6).
11.3.1  Precision criteria. The relative standard deviation (RSD) of the calculated mean spike
        dose for manually enumerated spiking suspensions must be < 16% for Cryptosporidium
        before proceeding (these criteria are based on the pooled RSDs of 105 manual
        Cryptosporidium enumerations enumerations submitted by 20 different laboratories
        under the EPA Protozoa Performance Evaluation Program).
11.3.2  Holding  time criteria. Manually enumerated spiking suspensions must be used within
        24 hours  of enumeration of the spiking suspension if the hemacytometer chamber
        technique is used (Section 11.3.4); or within 24 hours of application of the spiking
        suspension or membrane filter to the slides if the well slide or membrane filter
        enumeration technique is used (Sections 11.3.5 and 11.3.6).
11.3.3  Enumerating and diluting stock suspensions
        11.3.3.1       Purified, concentrated stock suspensions (Sections 7.10.2.1 and 7.10.2.2)
                      must be diluted and enumerated before the diluted suspensions are used
                      to spike samples in the laboratory. Stock suspensions should be diluted
                      with reagent water/Tween® 20 , 0.01% (Section 7.10.2.3), to a
                      concentration of 20 to 50 organisms per large hemacytometer square
                      before proceeding to Section  11.3.3.2.
        11.3.3.2      Apply a clean hemacytometer coverslip (Section 6.4.5) to the
                      hemacytometer and load the hemacytometer chamber with 10 uL of
                      vortexed suspension per chamber. If this operation has been properly
                      executed, the liquid should amply fill the entire chamber without bubbles
                      or overflowing into the surrounding moats. Repeat this step with a clean,
                      dry hemacytometer and coverslip if loading has been incorrectly
                      performed. See Section 11.3.3.13, below,  for the hemacytometer cleaning
                      procedure.
        11.3.3.3      Place the hemacytometer on the microscope stage and allow the oocysts
                      to settle for 2 minutes. Do not attempt to adjust the coverslip, apply clips,
                      or in any way disturb the chamber after it has been filled.
        11.3.3.4      Use 200X  magnification.
        11.3.3.5      Move the chamber so the ruled area is centered underneath the objective.
        11.3.3.6      Move the objective close to the coverslip while watching it from the side
                      of the microscope, rather than through the microscope.
        11.3.3.7      Focus up from the coverslip until the hemacytometer ruling appears.
        11.3.3.8      At each of the four corners of the chamber is a 1 -square-mm area divided
                      into 16 squares in which organisms are to be counted (Figure 1).
                      Beginning with the top row of four squares, count with a hand-tally
                      counter in  the directions indicated in Figure 2. Avoid counting organisms
                      twice by counting only those  touching the top and left boundary lines.
                      Count each square millimeter in this fashion.
                                       29                                 December 2005

-------
 Method 1622 - Cryptosporidium
                11.3.3.9      Use the following formula to determine the number of organisms per uL
                              of suspension:

                              number of            -„         dilution         .    3         number of
                          organisms counted                   factor          mm         organisms
                11.3.3.10     Record the result on a hemacytometer data sheet.
                11.3.3.11     A total of six different hemacytometer chambers must be loaded, counted,
                              and averaged for each suspension to achieve optimal counting accuracy.
                11.3.3.12     Based on the hemacytometer counts, the stock suspension should be
                              diluted to a final concentration of between 8 to 12 organisms per u.L;
	however, ranges as great as 5 to 15 organisms per uL can be used.	
        NOTE:  If the diluted stock suspensions (the spiking suspensions) will be enumerated
        using hemacytometer chamber counts (Section 11.3.4) or membrane filter counts (Section
        11.3.6), then the stock suspensions should be diluted with 0.01% Tween® 20 . If the
        spiking suspensions will be enumerated using well slide counts (Section 11.3.5), then the
        stock suspensions should be diluted in reagent water.

                              To calculate the volume (in uL) of stock suspension required per uL of
                              reagent water (or reagent water/Tween® 20 , 0.01%), use the following
                              formula:

                                                                required number of organisms
                   volume of stock suspension (uL) required =
                                                          number of organisms/ uL of stock suspension

                              If the volume is less than 10 uL, an additional dilution of the stock
                              suspension is recommended before proceeding.
                              To calculate the dilution factor needed to achieve the required number of
                              organisms per 10 u.L, use the following formula:

                                                          number of organisms required x 10uL
                              total volume (uL) =  	
                                                   predicted number of organisms per 10uL (8 to 12)

                              To calculate the volume of reagent water (or reagent water/Tween® 20 ,
                              0.01%) needed, use the following formula:

                    reagent water volume (ul_) =   total volume (uL) -   stock suspension volume required (uL)


                11.3.3.13     After each use, the hemacytometer and coverslip must be cleaned
                              immediately to prevent the organisms and debris from drying on it.  Since
                              this apparatus is precisely machined, abrasives cannot  be used to clean it,
                              as they will disturb the flooding and volume relationships.
                              11.3.3.13.1    Rinse the hemacytometer and cover glass first with tap
                                            water, then 70% ethanol, and finally with acetone.


December 2005                                 30

-------
                                                            Method 1622 - Cryptosporidium
                      11.3.3.13.2   Dry and polish the hemacytometer chamber and cover
                                    glass with lens paper. Store it in a secure place.
        11.3.3.14     Several factors are known to introduce errors into hemacytometer counts,
                      including:
                      •      Inadequate mixing of suspension before flooding the chamber
                      •      Irregular filling of the  chamber, trapped air bubbles, dust, or oil
                             on the chamber or coverslip
                      •      Total number of organisms counted is too low to provide
                             statistical confidence in the result
                      •      Error in recording tally
                      •      Calculation error; failure to consider dilution factor, or area
                             counted
                      •      Inadequate cleaning and removal of organisms from the previous
                             count
                      •      Allowing filled chamber to sit too long, so that the chamber
                             suspension dries and concentrates.
11.3.4  Enumerating spiking suspensions using a hemacytometer chamber

NOTE: Spiking suspensions enumerated using a hemacytometer chamber must be used
within 24 hours of enumeration.

        11.3.4.1      Vortex the tube containing the spiking suspension (diluted stock
                      suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert
                      the tube three times.
        11.3.4.2      To an appropriate-size beaker containing a stir bar, add enough spiking
                      suspension to perform all spike testing and the enumeration as described.
                      The liquid volume and beaker relationship should be such that a spinning
                      stir bar does not splash the sides of the beaker, the stir bar has unimpeded
                      rotation, and there is enough room to draw sample from the beaker with a
                      10-uL micropipette without touching the stir bar. Cover the beaker with a
                      watch glass or petri dish to prevent evaporation between sample
                      withdrawals.
        11.3.4.3      Allow the beaker contents to stir for a minimum of 30 minutes before
                      beginning enumeration.
        11.3.4.4      While the stir bar is still spinning, remove a 10-uL aliquot and carefully
                      load one side of the hemacytometer. Count all organisms on the platform,
                      at 200X magnification using phase-contrast or darkfield microscopy. The
                      count must include the entire area under the hemacytometer, not just the
                      four outer 1-mnr squares. Repeat this procedure nine times. This step
                      allows confirmation of the number of organisms per 10 uL (Section
                      11.3.3.12). Based on the 10 counts, calculate the mean, standard
                      deviation, and RSD of the counts.  Record the counts and the calculations
                      on a spiking suspension enumeration form. The relative  standard
                      deviation (RSD) of the calculated mean spike dose must be < 16% for
                      Cryptosporidium before proceeding. If the RSD is unacceptable, or the
                      mean number is outside the expected range, add additional oocysts from
                      stock suspension or dilute the contents of the beaker appropriately with
                      reagent water. Repeat the process to confirm counts. Refer to Section
                      11.3.3.14 for factors that may introduce errors.

                                       31                                 December 2005

-------
 Method 1622 - Cryptosporidium
        11.3.5  Enumerating spiking suspensions using well slides
        NOTE: Spiking suspensions enumerated using well slides must be used within 24 hours
        of application of the spiking suspension to the slides.

                11.3.5.1      Prepare well slides for sample screening and label the slides.
                11.3.5.2      Vortex the tube containing the spiking suspension (diluted stock
                              suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert
                              the tube three times.
                11.3.5.3      Remove a 10-uL aliquot from the spiking suspension and apply it to the
                              center of a well.
                11.3.5.4      Before removing subsequent aliquots, cap the tube and gently invert it
                              three times to ensure that the oocysts are in suspension.
                11.3.5.5      Ten wells must be prepared and counted, and the counts averaged, to
                              sufficiently enumerate the spike dose. Air-dry the well slides. Because
                              temperature and humidity varies from laboratory to laboratory, no
                              minimum time is specified. However, the laboratory must take care to
                              ensure that the sample has dried completely before staining to prevent
                              losses during the rinse steps. A slide warmer set at 35°C to 42°C also can
                              be used.
                11.3.5.6      Positive and negative controls must be prepared.
                              11.3.5.6.1     For the positive control, pipette 10 uL of positive antigen
                                             or 200 to 400 intact oocysts to the center of a well and
                                             distribute evenly over the well area.
                              11.3.5.6.2     For the negative control, pipette 50 uL of PBS onto the
                                             center of a well and spread it over the well area with a
                                             pipette tip.
                              11.3.5.6.3     Air-dry the control slides.
                11.3.5.7      Follow the manufacturer's instructions (Section 7.6) in applying the stain
                              to the slide.
                11.3.5.8      Place the slides in a humid chamber in the dark and incubate according to
                              manufacturer's directions. The humid chamber consists of a tightly sealed
                              plastic container containing damp paper towels on top of which the slides
                              are placed.
                11.3.5.9      Apply one drop of wash buffer (prepared according to the manufacturer's
                              instructions [Section  7.6]) to each well. Tilt each slide on a clean paper
                              towel, long edge down. Gently aspirate the excess detection reagent from
                              below the well using a clean Pasteur pipette or absorb with a paper towel
       	or other absorbent material. Avoid disturbing the sample.	
        NOTE: If using the MeriFluor® Cryptosporidium/Giardia stain (Section 7.6.1), do not
        allow slides to dry completely.


                11.3.5.10     Add mounting medium (Section 7.8) to each well.
                11.3.5.11     Apply a cover slip.  Use a tissue to remove excess mounting fluid from the
                              edges of the coverslip. Seal the edges of the coverslip onto the slide using
                              clear nail polish.
December 2005                                 32

-------
                                                             Method 1622 - Cryptosporidium
        11.3.5.12    Record the date and time that staining was completed. If slides will not be
                      read immediately, store in a humid chamber in the dark between 1°C and
                      10°C until ready for examination.
        11.3.5.13    After examination of the 10 wells, calculate the mean, standard deviation,
                      and RSD of the 10 replicates. Record the counts and the calculations on a
                      spiking suspension enumeration form. The relative standard deviation
                      (RSD) of the calculated mean spike dose must be < 16% for
                      Cryptosporidium before proceeding. If the RSD is unacceptable, or the
                      mean number is outside the expected range, add additional oocysts from
                      stock suspension or dilute the contents of the beaker appropriately with
                      reagent water. Repeat the process to confirm counts.
11.3.6  Enumeration of spiking suspensions using membrane filters

NOTE: Spiking suspensions enumerated using membrane filters must be used within 24
hours of application of the filters  to the slides.

        11.3.6.1      Precoat the glass funnels with Sigmacote®  by placing the funnel in a
                      large petri dish and applying 5-mL of Sigmacoat® to the funnel opening
                      using a pipette and allowing it to run down  the inside of the funnel.
                      Repeat for all funnels to be used. The pooled Sigmacoat® may be
                      returned to the bottle for re-use. Place the funnels at 35°C or 41°C for
                      approximately 5  minutes to dry.
        11.3.6.2      Place foil around the bottoms of the  100 x 15 mm petri  dishes.
        11.3.6.3      Filter-sterilize (Section 6.19) approximately 10 mL of PBS (Section
                      7.4.2.1). Dilute detection reagent (Section 7.6) as per manufacturer's
                      instructions using sterile PBS. Multiply the anticipated  number of filters
                      to be stained by 100 mL to calculate total volume of stain required.
                      Divide the total volume required by 5 to obtain the microliters of
                      antibody necessary. Subtract the volume of antibody from the  total stain
                      volume to obtain the required microliters of sterile PBS to add to the
                      antibody.
        11.3.6.4      Label the tops of foil-covered, 60 x 15 mm petri dishes for 10 spiking
                      suspensions plus positive and negative staining controls and multiple
                      filter blanks controls (one negative control, plus a blank after every five  •
                      sample filters to  control for carry-over). Create a humid chamber by
                      laying damp paper towels on the bottom of a stain tray (the inverted foil-
                      lined petri dishes will protect filters from light and prevent evaporation
                      during incubation).
        11.3.6.5      Place a decontaminated and cleaned filter holder base (Section 6.4.8.1)
                      into each of the three ports of the vacuum manifold (Section 6.4.8.2).
        11.3.6.6      Pour approximately 10 mL of 0.01% Tween® 20  into a 60 x  15 mm petri
                      dish.
        11.3.6.7      Using  forceps, moisten a 1.2-um cellulose-acetate support membrane
                      (Section 6.4.8.3) in the 0.01% Tween® 20  and place it on the fritted
                      glass support of one of the filter bases. Moisten a polycarbonate filter
                      (Section 6.4.8.4) the same way  and position it on top of the cellulose-
                      acetate support membrane. Carefully clamp the glass funnel to the loaded
                      filter support. Repeat for the other two filters.
        11.3.6.8      Add 5  mL of 0.01 % Tween® 20 to each of the three filtration units and
                      allow to stand.


                                        33                                 December 2005

-------
Method 1622 - Cryptosporidium
                11.3.6.9      Vortex the tube containing the spiking suspension (diluted stock
                              suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert
                              the tube three times.
                11.3.6.10     Using a micropipettor, sequentially remove two, 10-uL aliquots from the
                              spiking suspension and pipet into the 5 mL of 0.01% Tween® 20
                              standing in the unit. Rinse the pipet tip twice after each addition. Apply
                              10 uL of 0.01% Tween® 20  to the third unit to serve as the negative
                              control. Apply vacuum at 2" Hg and allow liquid to drain to miniscus,
                              then close off vacuum. Pipet  10 mL of reagent water into each funnel and
                              drain to miniscus, closing off the vacuum. Repeat the rinse and drain all
                              fluid, close off the vacuum.
                11.3.6.11     Pipet 100 mL of diluted antibody to the center of the bottom of a 60 x 15
                              mm petri dish for each sample.
                11.3.6.12     Unclamp the top funnel and transfer each cellulose acetate support
                              membrane/ polycarbonate filter combination onto the drop of stain using
                              forceps (apply each membrane/filter combination to a different petri dish
                              containing stain). Roll the filter into the drop to exclude air. Place the
                              small petri dish containing the filter onto the damp towel and cover with
                              the corresponding labeled foil-covered top. Incubate for approximately 45
                              minutes at room temperature.
                11.3.6.13     Reclamp the top funnels, apply vacuum and rinse each three times, each
                              time with 20 mL of reagent water.
                11.3.6.14     Repeat Sections 11.3.6.4 through 11.3.6.10 for the next three samples (if
                              that the diluted spiking suspension has sat less than 15 minutes, reduce
                              the suspension vortex time to 60 seconds). Ten, 10-uL spiking suspension
                              aliquots must be prepared and counted, and the counts averaged, to
                              sufficiently enumerate the spike dose. Include a filter blank sample at a
                              frequency of every five samples; rotate the position of filter blank to
                              eventually include all three filter placements.
                11.3.6.15     Repeat Sections 11.3.6.4 through 11.3.6.10 until the 10-uL spiking
                              suspensions have been filtered.  The last batch should include a 10-uL
                              0.01 Tween® 20 blank control and 20 uL of positive control antigen as a
                              positive staining control.
                11.3.6.16     Label slides. After incubation is complete, for each sample, transfer the
                              cellulose acetate filter support and polycarbonate filter from drop of stain
                              and place on fritted glass support. Cycle vacuum on and off briefly to
                              remove excess fluid. Peel the top polycarbonate filter off the supporting
                              filter and place on labeled slide. Discard cellulose acetate filter support.
                              Mount  and apply coverslips to the filters immediately to avoid drying.
                11.3.6.17     To each slide, add 20 uL of mounting medium (Section 7.8).
                11.3.6.18     Apply a coverslip. Seal the edges of the coverslip onto the slide using
                              clear nail polish. (Sealing may be delayed until cover slips  are applied to
                              all slides.)
                11.3.6.19     Record the date and time that staining was completed. If slides will not be
                              read immediately,  store sealed slides in a closed container in the dark
                              between 1°C and 10°C until ready for examination.
                11.3.6.20     After examination of the 10 slides, calculate the mean, standard
                              deviation, and RSD of the 10 replicates. Record the counts and the
                              calculations on a spiking suspension enumeration form. The relative


December 2005                                  34

-------
                                                                   Method 1622 - Cryptosporidium
                             standard deviation (RSD) of the calculated mean spike dose must be
                             < 16% for Cryptosporidium before proceeding. If the RSD is
                             unacceptable, or the mean number is outside the expected range, add
                             additional oocysts from stock suspension or dilute the contents of the
                             beaker appropriately with reagent water. Repeat the process to confirm
                             counts.
                11.3.6.21     If oocysts are detected on the filter blanks, modify the rinse procedure to
                             ensure that no carryover occurs and  repeat enumeration.
-j 1 4   Procedure for spiking samples in the laboratory with enumerated spiking suspensions.
       11.4.1   Arrange a disposable cubitainer or bottom-dispensing container to feed the filter or insert
                the influent end of the tube connected to the filter through the top of a carboy to allow
                siphoning of the sample.
       11.4.2   For initial precision and recovery (Section 9.4) and ongoing precision and recovery
                (Section 9.7) samples, fill the container with 10 L of reagent water or a volume of
                reagent water equal to the volume of the field samples analyzed in the analytical batch.
                For matrix spike samples (Section 9.5), fill the container with the field sample to be
                spiked. Continuously mix the sample (using a stir bar and stir plate  for smaller-volume
                samples and alternate means  for larger-volume samples).
       11.4.3   Follow the procedures in Section  11.4.3.1 or manufacturer's instructions for flow
                cytometer-enumerated suspensions and the procedures in Section 11.4.3.2 for manually
                enumerated suspensions.
                11.4.3.1      For flow cytometer-enumerated suspensions (where the entire volume of
                             a spiking suspension tube will be used):
                             11.4.3.1.1      Add 400 uL of Antifoam A to 100 mL of reagent water,
                                            and mix well to emulsify.
                             11.4.3.1.2      Add 500 uL of the diluted antifoam to the tube
                                            containing the spiking suspension and vortex for 30
                                            seconds.
                             11.4.3.1.3      Pour the suspension into the sample container.
                             11.4.3.1.4      Add 20 mL of reagent water to the empty tube, cap,
                                            vortex 10 seconds to rinse, and add the rinsate to the
                                            carboy.
                             11.4.3.1.5      Repeat this rinse using another 20 mL of reagent water.
                             11.4.3.1.6      Record the estimated number of organisms spiked, the
                                            date and time the sample was spiked, and the sample
                                            volume spiked on a  bench sheet. Proceed to Section
                                            n.4.4.
                11.4.3.2      For manually enumerated spiking suspensions:
                             11.4.3.2.1      Vortex the spiking suspension(s) (Section 11.2 or Section
                                            11.3) for a minimum of 30 seconds.
                             11.4.3.2.2      Rinse a pipette tip with 0.0 l%Tween® 20  once, then
                                            repeatedly pipette the well-mixed spiking suspension a
                                            minimum of five times before withdrawing an aliquot to
                                            spike the sample.
                             11.4.3.2.3      Add the spiking suspension(s) to the carboy, delivering
                                            the aliquot below the surface of the sample.
                             11.4.3.2.4      Record the estimated number of organisms spiked, the
                                            date and time the sample was spiked, and the sample

                                              35                                 December 2005

-------
 Method 1622 - Cryptosporidium
                                            volume spiked on a bench sheet. Proceed to Section
                                            11.4.4
        11.4.4  Allow the spiked sample to mix for approximately  1 minute in the container.
        11.4.5  Turn on the pump and allow the flow rate to stabilize. Set flow at the rate designated for
                the filter being used. As the carboy is depleted, check the flow rate and adjust if
                necessary.
        11.4.6  When the water level approaches the discharge port of the carboy, tilt the container so
                that it is completely emptied. At that time, turn off the pump and add 1-L PBST or
                reagent water to the 10-L carboy to rinse (5 L PBST or reagent water rinse to 50-L
                carboy). Swirl the contents to rinse down the sides. Additional rinses may be performed.
        11.4.7  Turn on the pump. Allow all of the water to flow through the filter and turn off the
                pump.
        11.4.8  Proceed to filter disassembly.


12.0  Sample Filtration and Elution
121    A water sample is filtered according to the procedures in Section 12.2, 12.3, or 12.4. Alternate
        procedures may be used if the laboratory first demonstrates that the alternate procedure provides
        equivalent or superior performance per Section 9.1.2.
        NOTE:  Sample elation must be initiated within 96 hours of sample collection (if shipped
        to the laboratory as a bulk sample)  or filtration (iffiltered in the field).

122    Capsule filtration (adapted from Reference 20.12). This procedure was validated using 10-L
        sample volumes  (for the original Envirochek ™ filter) and 50-L sample volumes (for the
        Envirochek™ HV filter).  Alternate sample volumes may be used, provided the laboratory
        demonstrates acceptable performance on initial and ongoing spiked reagent water and source
        water samples (Section 9.1.2).
        12.2.1   Flow rate adjustment
                12.2.1.1      Connect the sampling system, minus the capsule, to a carboy filled with
                             reagent water (Figure 3).
                12.2.1.2      Turn on the pump and adjust the flow rate to 2.0 L/min.
                12.2.1.3      Allow 2 to  10 L of reagent water to flush the system. Adjust the pump
                             speed as required during this period. Turn off the pump when the flow
                             rate has been adjusted.
        12.2.2  Install the capsule filter in the line, securing the inlet and outlet ends with the appropriate
                clamps/fittings.
        12.2.3  Record the sample number, sample turbidity (if not provided with the field sample),
                sample type, and sample filtration start date and time on a bench sheet.
        12.2.4  Filtration
                12.2.4.1      Mix the sample well by shaking, add stir bar and place on stir plate. Turn
                             on stir plate to lowest setting needed to keep sample thoroughly mixed.
                             Connect the sampling system to the  field carboy of sample water, or
                             transfer the sample water to the laboratory carboy used in Section
                             12.2.1.1. If the sample will be filtered from a field carboy, a spigot
                             (Section 6.2.1) can be used with the carboy to facilitate sample filtration.
        NOTE: If the bulk field sample is transferred to a laboratory carboy, the laboratory
        carboy must be cleaned and disinfected before it is used with another field sample.
December 2005                                36

-------
                                                             Method 1622 - Cryptosporidium
         12.2.4.2
         12.2.4.3
         12.2.4.4
12.2.5
12.2.6
12.2.4.5


12.2.4.6

Disassembly
12.2.5.1



12.2.5.2


12.2.5.3
Elution
Place the drain end of the sampling system tubing into an empty
graduated container with a capacity of 10 to 15 L, calibrated at 9.0, 9.5,
10.0,  10.5, and 11.0 L (Section 6.18). This container will be used to
determine the sample volume filtered. Alternately, connect a flow meter
(Section 6.3.4) downstream of the filter, and record the initial meter
reading.
Allow the carboy discharge tube and capsule to fill with  sample water by
gravity. Vent residual air using the bleed valve/vent port, gently shaking
or tapping the capsule, if necessary. Turn on the pump to start water
flowing through the filter. Verify that the flow rate is 2 L/min.
After all of the sample has passed through the filter, turn off the pump.
Allow the pressure to decrease until flow stops. (If the sample was
filtered in the field, and excess sample remains in the filter capsule upon
receipt in the laboratory, pull the remaining sample volume through the
filter before eluting the filter [Section 12.2.6].)
Turn off stir plate; add 1 L PBST or reagent water rinse (to 10-L carboy)
or 5 L PBST or reagent water rinse (to 50-L carboy).  Swirl or shake  the
carboy to rinse down the side walls.
Reconnect to pump, turn on pump and allow pump to pull all water
through filter; turn off pump.

Disconnect the inlet end of the capsule filter assembly while maintaining
the level of the inlet fitting above the level of the outlet fitting to prevent
backwashing and the loss of oocysts  from the filter. Restart the pump and
allow as much water to drain as possible. Turn off the pump.
Based on the water level in the graduated container and 1/2-L hash marks
or meter reading, record the volume filtered on the bench sheet to the
nearest quarter liter. Discard the contents of the graduated container.
Loosen the outlet fitting, then  cap the inlet and outlet fittings.
NOTE:  The laboratory must complete the elution, concentration, and purification
(Sections 12.2.6 through 13.3.3.11) in one work day. It is critical that these steps be
completed in one work day to minimize the time that any target organisms present in the
sample sit in eluate or concentrated matrix. This process ends with the application of the
purified sample on the slide for drying.
         12.2.6.1
              Setup
              12.2.6.1.1
                      12.2.6.1.2
                      12.2.6.1.3
               Assemble the laboratory shaker with the clamps aligned
               vertically so that the filters will be aligned horizontally.
               Extend the clamp arms to their maximum distance from
               the horizontal shaker rods to maximize the shaking
               action.
               Prepare sufficient quantity of elution buffer to elute all
               samples that are associated with the OPR/MB which used
               that batch of elution buffer. Elution may require up to
               275 mL of buffer per sample.
               Designate at least one 250-mL conical centrifuge tube for
               each sample and label with the sample number.
                                        37
                                                                  December 2005

-------
Method 1622 - Cryptosporidium
                12.2.6.2      Elution
                              12.2.6.2.1     Record the elution date and time on the bench sheet.
                                             Using a ring stand or other means, clamp each capsule in
                                             a vertical position with the inlet end up.
                              12.2.6.2.2     Remove the inlet cap, pour elution buffer through the
                                             inlet fitting, and allow the liquid level to stabilize.
                                             Sufficient elution buffer must be added to cover the
                                             pleated white membrane with buffer solution or elution
                                             buffer may be measured to ensure the use of one 250-mL
                                             centrifuge tube. Replace the inlet cap.
                              12.2.6.2.3     Securely clamp the capsule in one of the clamps on the
                                             laboratory shaker with the bleed valve positioned at the
                                             top on a vertical axis (in the 12 o'clock position). Turn on
                                             the shaker and set the speed to maximum (approximately
                                             900 rpm or per manufacturer's instructions). Agitate the
                                             capsule for approximately 5 minutes. Time the agitation
                                             using a lab timer, rather than the timer on the shaker to
                                             ensure accurate time measurement.
                              12.2.6.2.4     Remove the filter from the shaker, remove the inlet cap,
                                             and pour the contents  of the  capsule into the 250-mL
                                             conical centrifuge tube.
                              12.2.6.2.5     Clamp the capsule vertically with the inlet end up and
                                             add sufficient volume of elution buffer through the inlet
                                             fitting to cover the pleated membrane. Replace the inlet
                                             cap.
                              12.2.6.2.6     Return the capsule to the shaker with the bleed valve
                                             positioned at the 4 o'clock position. Turn on the shaker
                                             and agitate the capsule for approximately 5 minutes.
                              12.2.6.2.7     Remove the filter from the shaker, but leave the elution
                                             buffer in the capsule. Re-clamp the capsule to the shaker
                                             at the 8 o'clock position. Turn on the shaker and agitate
                                             the capsule for a final 5 minutes.
                              12.2.6.2.8     Remove the filter from the shaker and pour the contents
                                             into the 250-mL centrifuge tube. Rinse down the inside
                                             of the capsule filter walls with reagent water or elution
                                             buffer using a squirt bottle inserted in the inlet end of the
                                             capsule. Invert the capsule filter over the centrifuge tube
                                             and ensure that as much of the eluate as possible has been
                                             transferred.
        12.2.7  Proceed to Section 13.0 for concentration and separation (purification).
12.3    Sample filtration using the Filta-Max® foam filter. This procedure was validated using 50-L
        sample volumes. Alternate sample volumes may be used, provided the laboratory demonstrates
        acceptable performance on initial and ongoing spiked reagent water and source water samples
        (Section 9.1.2).
        NOTE:  The filtration procedures specified in Sections 12.3.1.2 - 12.3.1.6.3 are specific
        to laboratory filtration of a bulk sample. These procedures may require modification if
        samples will be filtered in the field.
December 2005                                 38

-------
                                                                    Method 1622 - Cryptosporidium
12.3.1 Filtration
               12.3.1.1      Flow rate adjustment
                             12.3.1.1.1     Connect the sampling system, minus the filter housing, to
                                            a carboy filled with reagent water.
                             12.3.1.1.2     Place the peristaltic pump upstream of the filter housing.
                             12.3.1.1.3     Turn on the pump and adjust the flow rate to 1 to 4 L per
                                            minute.
       NOTE: A head pressure of 0.5 bar (7.5 psi) is required to create flow through the filter,
       and the recommended pressure of 5 bar (75 psi) should produce the flow rate of 3 to 4 L
       per minute. The maximum operating pressure of 8 bar (120 psi) should not be exceeded.

                             12.3.1.1.4     Allow 2 to 10 L of reagent water to flush the system.
                                            Adjust the pump speed as necessary during this period.
                                            Turn off the pump when the flow rate has been adjusted.
               12.3.1.2      Place filter module into the filter housing bolt head down and secure lid,
                             hand tighten housings, apply gentle pressure to create the seal between
                             the module and the 'O' rings in the base and the lid of the housing.
                             Excessive tightening is not necessary, and may shorten the life of the 'O'
                             rings. Tools may be used to tighten housing to the alignment marks (refer
                             to manufacturer's instructions). 'O' rings should be lightly greased before
                             use (refer to manufacturer's instructions).
               12.3.1.3      Install the filter housing in the line, securing the inlet and outlet ends with
                             the appropriate clamps/fittings. Verify that the filter housing is installed
                             so that the end closest to the screw top cap is the inlet and the opposite
                             end is the outlet.
               12.3.1.4      Record the sample number, sample turbidity (if not provided with the
                             field sample), and the name of the analyst filtering the sample on a bench
                             sheet.
               12.3.1.5      Filtration
                             12.3.1.5.1     Connect the sampling system to the field carboy of
                                            sample water, or transfer the sample water to the
                                            laboratory carboy used in Section 12.3.1.1.1. If the
                                            sample will be filtered from a field carboy, a spigot can
                                            be used with the carboy to facilitate sample  filtration.
       NOTE: If the bulk field sample is transferred to a laboratory carboy, the laboratory
       carboy must be cleaned and disinfected before it is used with another field sample.

                             12.3.1.5.2     Place the drain end of the sampling system tubing into an
                                            empty graduated container with a capacity greater than or
                                            equal to the volume to be filtered. This container will be
                                            used to determine the sample volume filtered.
                                            Alternately, connect a flow meter downstream of the
                                            filter, and record the initial meter reading.
                             12.3.1.5.3     Allow the carboy discharge tube and filter housing to fill
                                            with sample water. Turn on the pump to start water
                                            flowing through the filter. Verify that the flow rate is
                                            between 1 and 4 L per min.
                                               39                                December 2005

-------
 Method 1622 - Cryptosporidium
                              12.3.1.5.4    After all of the sample has passed through the filter, turn
                                            off the pump. Allow the pressure to decrease until flow
                                            stops.
                12.3.1.6      Disassembly
                              12.3.1.6.1     Disconnect the inlet end of the filter housing assembly
                                            while maintaining the level of the inlet fitting above the
                                            level of the outlet fitting to prevent backwashing and the
                                            loss  of oocysts from the filter. Restart the pump and
                                            allow as much water to drain as possible. Turn off the
                                            pump.
                              12.3.1.6.2     Based on the water level in the graduated container or the
                                            meter reading, record the volume filtered on a bench
                                            sheet to the nearest quarter liter.
                              12.3.1.6.3     Loosen the outlet fitting, the filter housing should be
                                            sealed with rubber plugs.
        NOTE:  Fillers should be prevented from drying out, as this can impair their ability to
        expand when decompressed.

        12.3.2  Elution
                12.3.2.1      The filter is eluted to wash the oocysts from the filter. This can be
                              accomplished using the Filta-Max® wash station, which moves a plunger
                              up and down a tube containing the filter and eluting solution (Section
                              12.3.2.2), or a stomacher, which uses paddles to agitate the stomacher bag
                              containing the foam filter in the eluting solution (Section 12.3.2.3). If the
                              Filta-Max® automatic wash station is used please see the manufacturer's
                              operator's guide for instructions on its use. If Filta-Max® Quick Connect
                              kit is used please follow manufacturer's instructions.
                12.3.2.2      Filta-Max® wash station elution procedure
                              12.3.2.2.1     First wash

                                            (a) Detach the removable plunger head using the tool
                                               provided, and remove the splash guard.
                                            (b) Place the filter membrane  flat in the concentrator
                                               base with the rough side up. Locate the concentrator
                                               base in the jaws of the wash station and screw on the
                                               concentrator tube (the longer of the two tubes),
                                               creating a tight seal at the membrane. Take the
                                               assembled concentrator out of the jaws and place on
                                               the bench.
                                            (c) Replace the splash guard and temporarily secure it at
                                               least 15 cm above the end  of the rack. Secure the
                                               plunger head with the tool provided ensuring that the
                                               lever is fully locked down.
                                            (d) Remove the filter module from the filter housing or
                                               transportation container. Pour excess liquid into  the
                                               assembled concentrator, then rinse the housing or
                                               container with PBST and add the rinse to the
                                               concentrator tube.  Screw the filter module onto the
                                               base  of the plunger. Locate the elution tube base in
December 2005                                 40

-------
                                                            Method 1622 - Cryptosporidium
                                        the jaws of the wash station and screw the elution
                                        tube (the shorter of the two tubes) firmly in place.
                                    (c)  Pull the plunger down until the filter module sits at
                                        the bottom of the elution tube; the locking pin (at the
                                        top left of the wash station) should "click" to lock the
                                        plunger in position.
                                    (f)  Remove the filter module bolt by turning the adapted
                                        alien key (provided)  in a clockwise direction (as seen
                                        from above). Attach  the steel tube to the elution tube
                                        base.
                                    (g)  Add 600 mL of PBST to the assembled concentrator.
                                        If more than  50 mL of liquid has been recovered from
                                        the shipped filter module, reduce the volume of
                                        PBST accordingly. Screw the concentrator tube onto
                                        the base beneath the  elution tube. Release the locking
                                        pin.
NOTE: Gentle pressure on the lever, coupled with a pulling action on the locking pin
should enable the pin to be easily released.


                                    (h)  Wash the foam disks by moving the plunger up and
                                        down 20 times. Gentle movements of the plunger are
                                        recommended to avoid generating excess foam.

NOTE:  The plunger has an upper movement limit during the wash process to prevent it
popping out of the top of the chamber.

                                    (i)  Detach the concentrator and hold it such that the
                                        stainless steel tube is just above the level of the
                                        liquid. Purge the remaining liquid from the elution
                                        tube by moving the plunger up and down 5 times,
                                        then lock the plunger in place. To prevent drips,
                                        place the plug provided  in the end of the steel tube.
                                    (j)  Prior to the second wash the eluate from the first
                                        wash can be concentrated using the Filta-Max®
                                        apparatus according to Section 12.3.3.2.1 or the
                                        eluate can be decanted into a 2-L pooling beaker and
                                        set aside.
                      12.3.2.2.2    Second wash

                                    (a)  Add an additional 600 mL of PBST to the
                                        concentrator module, remove the plug from the end
                                        of the steel tube and  screw the concentrator tube back
                                        onto the elution module base. Release the locking
                                        pin.
                                    (b)  Wash the foam disks by moving the plunger up and
                                        down 10 times. Gentle movements of the plunger are
                                        recommended to avoid generating excess.foam.
                                    (c)  The eluate can be concentrated using the Filta-Max®
                                        apparatus according to Section 12.3.3.2.2 or the
                                        eluate can be decanted into the 2-L pooling beaker
                                        containing the eluate from the first wash and

                                       41                                 December 2005

-------
 Method 1622 - Cryptosporidium
                                                concentrated using centrifugation, as described in
                                                Section 12.3.3.3.
                12.3.2.3     Stomacher elution procedure
                             12.3.2.3.1     First wash

                                            (a)  Place the filter module in the stomacher bag then use
                                                the alien key to remove the bolt from the filter
                                                module, allowing the rings to expand. Remove the
                                                end caps from the stomacher bag and rinse with
                                                PBST into the stomacher bag.
                                            (b)  Add 600 mL of PBST to stomacher bag containing
                                                the filter pads. Place bag in stomacher and wash for 5
                                                minutes on a normal setting.
                                            (c)  Remove the bag from the stomacher and decant the
                                                eluate into a 2-L pooling beaker.
                             12.3.2.3.2     Second wash

                                            (a)  Add a second 600-mL aliquot of PBST to the
                                                stomacher bag. Place bag in stomacher and wash for
                                                5 minutes on a normal setting. Remove the bag from
                                                the stomacher and decant the eluate from the
                                                stomacher bag into the 2-L pooling beaker. Wring the
                                                stomacher bag by hand to remove eluate from the
                                                foam filter and add to the pooling beaker. Remove
                                                the foam filter from the bag and using a squirt bottle,
                                                rinse the stomacher bag with reagent water and add
                                                the rinse to the pooling beaker.
                                            (b)  Proceed to concentration (Section 12.3.3).
        12.3.3  Concentration
                12.3.3.1      The eluate can be concentrated using the Filta-Max® concentrator
                             apparatus, which pulls most of the eluate through a membrane filter
                             leaving the oocysts concentrated in a small volume of the remaining
                             eluting solution (Section 12.3..2), or by directly centrifuging all of the
                             eluting solution used to wash the filter (Section 12.3.2.3).
                12.3.3.2     The Filta-Max® concentrator procedure
                             12.3.3.2.1     Concentration of first wash
                                            (a)  If the stomacher was used to elute the sample
                                                (Section 12.3.2.3), transfer 600 mL of eluate from the
                                                pooling beaker to the concentrator tube. Otherwise
                                                proceed to Step (b).
                                            (b)  Stand the concentrator tube on a magnetic stirring
                                                plate and attach the lid (with magnetic stirrer bar).
                                                Connect the waste bottle trap and hand or electric
                                                vacuum pump to the valve on the concentrator base.
                                                Begin stirring and open the tap. Increase the vacuum
                                                using the hand pump.
        NOTE:  The force of the vacuum should not exceed 30 cmHg.


                                            (c)  Allow the liquid to drain until it is approximately
                                                level with the middle of the stirrer bar then close the
                                                valve. Remove the magnetic stirrer, and rinse it with

December 2005                                42

-------
                                                           Method 1622 - Cryptosporidium
                                       PBST or distilled water to recover all oocysts. Decant
                                       the concentrate into a 50-mL tube, then rinse the
                                       sides of the concentration tube and add the rinsate to
                                       the 50-mL tube.
                     12.3.3.2.2     Concentration of second wash

                                    (a) If the stomacher was used to elute the sample
                                       (Section 12.3.2.3), transfer the remaining 600 mL of
                                       eluate from the pooling beaker to the concentrator
                                       tube. Otherwise proceed to Step (b).
                                    (b) Add the concentrate, in the 50-mL tube, retained
                                       from the first concentration (Section 12.3.3.2.1 (c)) to
                                       the 600 mL of eluate from the second wash, then
                                       repeat concentration steps from Sections 12.3.3.2.1
                                       (b) and 12.3.3.2.1 (c). The final sample can be poured
                                       into the same 50-mL tube used to retain the first
                                       concentrate. Rinse the sides of the concentrator tube
                                       with PBST and add the rinse to the 50-mL tube.
                                    (c) Remove the magnetic stirrer. Insert the empty
                                       concentrator module into the jaws of the wash station
                                       and twist off the concentrator tube.
                                    (d) Transfer the membrane from the concentrator base to
                                       the bag provided using membrane forceps.
                     12.3.3.2.3     Membrane elution. The membrane can be washed
                                    manually or using a stomacher:
                                    •   Manual wash. Add 5 mL of PBST to the bag
                                       containing the membrane. Rub the surface of the
                                       membrane through the bag until the membrane
                                       appears clean. Using a pipette, transfer the eluate to a
                                       50-mL tube. Repeat the membrane wash with another
                                       5 mL of PBST and transfer the eluate to the 50-mL
                                       tube. (Optional: Perform a third wash using another 5
                                       mL of PBST, by hand-kneading an additional minute
                                       or placing the bag on a flat-headed vortexer and
                                       vortexing for one minute. Transfer the eluate to the
                                       50-mL tube.)
NOTE: Mark the bag with an "X" to note which side of the membrane has the oocysts to
encourage the hand-kneading to focus on the appropriate side of the membrane.


                                    •   Stomacher wash. Add 5 mL of PBST to the bag
                                       containing the membrane. Place the bag containing
                                       the membrane into a small stomacher and stomach
                                       for 3 minutes. Using a pipette transfer the eluate to a
                                       50-mL tube. Repeat the wash two times using the
                                       stomacher and 5-mL aliquots of PBST. (Optional:
                                       Perform a fourth wash using another 5 mL of PBST,
                                       by hand-kneading an additional minute or placing the
                                       bag on a flat-headed vortexer and vortexing for one
                                       minute. Transfer the eluate to the 50-mL tube.)
                     12.3.3.2.4     If the membrane filter clogs before concentration is
                                    complete, there are two possible options for completion

                                      43                               December 2005

-------
 Method 1622 - Cryptosporidium
                                            of concentration. One option is replacing the membrane
                                            as often as necessary. Filter membranes may be placed
                                            smooth side up during the second concentration step.
                                            Another option is concentrating the remaining eluate
                                            using centrifugation. Both options are provided below.
                                            •   Using multiple membranes. Disassemble the
                                                concentrator tube and pour any remaining eluate back
                                                into the pooling beaker. Remove the membrane using
                                                membrane forceps, placing it in the bag provided.
                                                Place a new membrane in the concentrator tube
                                                smooth side up, reassemble, return the eluate to the
                                                concentrator tube, rinse the pooling beaker and add
                                                rinse to the eluate, and continue the concentration.
                                                Replace the membrane as often as necessary.
                                                Centrifuging remaining volume. Decant the
                                                remaining eluate into a 2-L pooling beaker. Rinse the
                                                sides of the concentrator  tube and add to the pooling
                                                beaker. Remove the filter membrane and place it in
                                                the bag provided. Wash the membrane as described
                                                in Section 12.3.3.2.3, then concentrate the sample as
                                                described in Section 12.3.3.3.1.
                12.3.3.3     If the Filta-Max® concentrator is not used for sample concentration, or if
                             the membrane filter clogs before sample concentration is complete, then
                             the procedures described in Section 12.3.3.3.1 should be used to
                             concentrate the sample. If less than 50 mL of concentrate has been
                             generated, the sample can be further concentrated, as described in Section
                             12.3.3.3.2, to reduce the volume of sample to be processed through IMS.
        NOTE:  The volume must not be reduced to less than 5 mL above the packed pellet. The
        maximum amount of pellet that should be processed through IMS is 0.5 mL. If the packed
       pellet is greater than 0.5 mL then the pellet may be subsampled as described in Section
        13.2.4.

                             12.3.3.3.1     Centrifugation of greater than 50 mL of eluate

                                            (a)  Decant the eluate from the 2-L pooling beaker into
                                                250-mL conical centrifuge tubes. Make sure that the
                                                centrifuge tubes are balanced.
                                            (b)  Centrifuge the 250-mL centrifuge tubes containing
                                                the eluate at 1500 x G for 15 minutes. Allow the
                                                centrifuge to coast to a stop.
                                            (c)  Using a Pasteur pipette, carefully aspirate off the
                                                supernatant to 5 mL above the pellet. If the sample is
                                                reagent water (e.g. initial or ongoing precision and
                                                recovery sample) extra care must be taken to avoid
                                                aspirating oocysts during this step.

                                            (d)  Vortex each 250-mL tube vigorously until pellet is
                                                completely resuspended. Swirl the centrifuge  tube
                                                gently to reduce any foaming after vortexing.
                                                Combine the contents of each 250-mL centrifuge
                                                tube into a 50-mL centrifuge tube. Rinse each of the
December 2005                                 44

-------
                                                                   Method 1622 - Cryptosporidium
                                               250-mL centrifuge tubes with PBST and add the
                                               rinse to the 50-mL tube.
                                           (e) Proceed to Section 12.3.3.3.2.
                             12.3.3.3.2    Centrifugation of less than 50 mL of eluate

                                           (a) Centrifuge the 50-mL centrifuge tube containing the
                                               combined concentrate at 1500 x G for 15 minutes.
                                               Allow the centrifuge to coast to a stop. Record the
                                               initial pellet volume (volume of solids) and the date
                                               and time that concentration was completed on a
                                               bench sheet.
                                           (b) Proceed to Section 13.0 for concentration and
                                               separation (purification).
       12.3.4  Maintenance and cleaning
               12.3.4.1      Maintenance of O-rings
                             12.3.4.1.1    Check all rubber O-rings for wear or deterioration prior
                                           to each use and replace as necessary.
                             12.3.4.1.2    Lubricate the plunger head O-ring inside and out with
                                           silicon before each use.
                             12.3.4.1.3    Lubricate all other O-rings (concentrator tube set, filter
                                           housing) regularly in order to preserve their condition.
               12.3.4.2      Cleaning
                             12.3.4.2.1    All components of the Filta-Max® system can be cleaned
                                           using warm water and laboratory detergent.  After
                                           washing, rinse all components with oocyst and cyst free
                                           reagent water and dry them. All O-rings should be re-
                                           lubricated. Alternatively a mild (40°C) dishwasher cycle
                                           without bleach or rinse aid can be used.
                             12.3.4.2.2    To wash the detachable plunger head slide the locking
                                           pin out and wash the plunger head  and locking pin in
                                           warm water and laboratory detergent. Rinse the plunger
                                           head and locking pin with oocyst and cyst free reagent
                                           water and dry. Lightly lubricate the locking  pin and re-
                                           assemble the plunger head.
124   Sample collection (filtration and concentration) using portable continuous-flow centrifugation.
       Please follow manufacturer's instructions. This procedure was validated for the detection of
       Cryptosporidium using 50-L sample volumes. Alternate sample volumes maybe used, provided
       the laboratory demonstrates acceptable performance on initial and ongoing spiked reagent water
       and source water samples (Section 9.1.2).

13.0  Sample Concentration and  Separation (Purification)
•J3 -j   During concentration and separation, the filter eluate is concentrated through centrifugation, and
       the oocysts in the sample are separated from other particulates through immunomagnetic
       separation (IMS). Alternate procedures and products may be  used if the laboratory first
       demonstrates equivalent or superior performance as per Section 9.1.2.
132   Adj ustment o f pel let volume
       13.2.1  Centrifuge the 250-mL centrifuge tube containing the capsule filter eluate at 1500 x G
               for 15 minutes. Allow the centrifuge to coast to a stop—do not use the  brake. Record the
               pellet volume (volume of solids) on the bench sheet.

                                              45                                 December 2005

-------
 Method 1622 - Cryptosporidium
        NOTE:  Recoveries may be improved if centrifugation force is increased to 2000 x G.
        However, do not use this higher force if the sample contains sand or other gritty material
        that may degrade the condition of any oocysts in the sample.

        13.2.2  Using a Pasteur pipette, carefully aspirate the supernatant to 5 mL above the pellet. Extra
                care must be taken to avoid aspirating oocysts during this step, particularly if the sample
                is reagent water (e.g. initial or ongoing precision and recovery sample).
        13.2.3  If the packed pellet volume is < 0.5 mL, vortex the tube vigorously until pellet is
                completely resuspended. Swirl the centrifuge tube gently to reduce any foaming after
                vortexing. Record the resuspended pellet volume on the bench sheet. Proceed to Section
                13.3.
        NOTE:  Extra care must be taken with samples containing sand or other gritty material
        when vortexing to ensure that the condition of any oocysts in the sample is not
        compromised.

        13.2.4  Jf the packed pellet volume is > 0.5 mL, the concentrate must be separated into
                multiple subsamples (a subsample is equivalent to no greater than 0.5 mL of packed
                pellet material, the recommended maximum amount of particulate material to process
                through the subsequent purification and examination steps in the method). Use the
                following formula to determine the total volume required in the centrifuge tube before
                separating the concentrate into two or more subsamples:

                                                           pellet volume
                               total volume (mL) required =                      x 5 mL
                                                              O.SmL

                (For example, if the packed pellet volume is  1.2 mL, the total volume required is 12 mL.)
                Add reagent water to the centrifuge tube to bring the total volume to the level calculated
	above.	
        NOTE:  Extra care must be taken with samples containing sand or other gritty material
        when vortexing to ensure that the condition of any oocysts in the sample is not
        compromised.

                13.2.4.1      Analysis of entire sample. If analysis of the entire sample is required,
                             determine the number of subsamples to be processed independently
                             through the remainder of the method:
                             13.2.4.1.1     Calculate number of subsamples: Divide the total
                                           volume in the centrifuge tube by 5 mL and round up to
                                           the nearest integer (for example, if the resuspended
                                           volume in Section  13.2.4 is 12 mL, then the number of
                                           subsamples would  be 12 mL / 5 mL = 2.4, rounded = 3
                                           subsamples).
                             13.2.4.1.2     Determine volume of resuspended concentrate per
                                           subsample. Divide the total volume in the centrifuge
                                           tube by the calculated number of subsamples (for
                                           example, if the resuspended volume in Section 13.2.4 is
                                           12 mL, then the volume to use for each  subsample =12
                                           mL / 3 subsamples = 4 mL).
                             13.2.4.1.3     Process subsamples through IMS. Vortex the tube
                                           vigorously for 10 to 15 seconds to completely resuspend
                                           the pellet. Record the resuspended pellet volume on the

December 2005                                46

-------
                                                                   Method 1622 - Cryptosporidium
                                            bench sheet. Proceed immediately to Section 13.3, and
                                            transfer aliquots of the resuspended concentrate
                                            equivalent to the volume in the previous step to multiple,
                                            flat-sided sample tubes in Section 13.3.2.1. Process the
                                            sample as multiple, independent subsamples from Section
                                            13.3 onward, including the preparation and examination
                                            of separate slides for each aliquot. Record the volume of
                                            resuspended concentrate transferred to IMS on the bench
                                            sheet (this will be equal to the volume recorded in
                                            Section 13.2.4).  Also record the number of subsamples
                                            processed independently through the method on the
                                            bench sheet.
                13.2.4.2      Analysis of partial sample. If not all of the concentrate will be
                             examined, vortex the tube vigorously for 10 to 15 seconds to completely
                             resuspend the pellet. Record the  resuspended pellet volume on the bench
                             sheet. Proceed immediately to Section 13.3, and transfer one or more 5-
                             mL aliquots of the resuspended concentrate to one or more flat-sided
                             sample tubes in Section 13.3.2.1. Record the volume of resuspended
                             concentrate transferred to IMS on the bench sheet. To determine the
                             volume analyzed, calculate the percent of the concentrate examined using
                             the following formula:

                                    total volume of resuspended concentrate transferred to IMS
                percent examined =                                                        x 100%
                                    total volume of resuspended concentrate in Section 13.2.4
                             Then multiply the volume filtered (Section 12.2.5.2) by this percentage to
                             determine the volume analyzed.
133   IMS procedure (adapted from Reference 20.13)

       NOTE:  The IMS procedure should be performed on a bench top with all materials at
       room temperature,  ranging from 15°C to 25°C.

       13.3.1   Preparation and addition of reagents
                13.3.1.1      Prepare a IX dilution of SL-buffer-A from the 10X SL-buffer-A (clear,
                             colorless solution) supplied. Use reagent water (demineralized; Section
                             7.3) as the diluent. For every 1 mL of IX SL-buffer-A required, mix 100
                             uL of 10X SL-buffer-A and 0.9 mL diluent water. A volume of 1.5 mL of
                             IX SL-buffer-A will be required per sample or subsample on which the
                             Dynal IMS procedure is performed.
                13.3.1.2      For each lOmL sample or subsample (Section 13.2) to be processed
                             through IMS, add 1 mL of the 10X SL-buffer-A (supplied—not the
                             diluted IX SL-buffer-A) to a flat-sided tube (Section 6.5.4).
                13.3.1.3      For each subsample, add 1 mL of the 10X SL-buffer-B (supplied—
                             magenta solution) to the flat-sided tube containing the 10X SL-buffer-A.
       13.3.2   Oocyst and cyst capture
                13.3.2.1      Use a graduated, 10-mL pipette that has been pre-rinsed with elution
                             buffer to transfer the water sample concentrate from Section 13.2 to the
                             flat-sided tube(s) containing the SL-buffers. If all of the concentrate is
                             used, rinse the centrifuge tube twice with reagent water and add the
                             rinsate to the flat-sided tube containing the concentrate (or to the tube

                                               47                                 December 2005

-------
Method 1622 - Cryptosporidium
                             containing the first subsample, if multiple subsamples will be processed).
                             Each of the two rinses should be half the volume needed to bring the total
                             volume in the flat-sided sample tube to 12 mL (including the buffers
                             added in Sections 13.3.1.2 and 13.3.1.3). (For example, if the tube
                             contained 1 mL of SL-buffer-A and 1 mL of SL-buffer-B, and 5 mL of
                             sample was transferred after resuspension of the pellet, fora total of 7
                             mL, the centrifuge tube would be rinsed twice with 2.5 mL of reagent
                             water to bring the total volume in the flat-sided tube to 12 mL.) Visually
                             inspect the centrifuge tube after completing the transfer to ensure that no
                             concentrate remains. If multiple subsamples will be processed, bring the
                             volume in the remaining flat-sided tubes to  12 mL with reagent water.
                             Label the flat-sided tube(s) with the sample number (and subsample
                             letters).
                13.3.2.2     Vortex the Dynabeads® anti-Cryptosporidium beads vial from the IMS
                             kit for approximately 10  seconds to suspend the beads. Ensure that the
                             beads are fully resuspended by inverting the sample tube and making sure
                             that there is no residual pellet at the bottom.
                13.3.2.3     Add 100 uL of the resuspended Dynabeads® anti-Cryptosporidium beads
                             (Section 13.3.2.2) to the sample tube(s) containing the water sample
                             concentrate and SL-buffers.
                13.3.2.4     Affix the sample tube(s)  to a rotating mixer and rotate at approximately
                             18 rpm for 1 hour at room temperature.
                13.3.2.5     After rotating for 1 hour, remove each sample tube from the mixer and
                             place the tube in the magnetic particle concentrator (MPC®-1 or MPC®-
                             6) with flat side of the tube toward the magnet.
                13.3.2.6     Without removing the sample tube from the MPC®-1, place the magnet
                             side  of the MPC®-1 downwards, so the tube is horizontal and the flat side
                             of the tube is facing down.
                13.3.2.7     Gently rock the sample tube by hand end-to-end through approximately
                             90°,  tilting the cap-end and base-end of the tube up and down in turn.
                             Continue the tilting action for 2 minutes with approximately one tilt per
                             second.
                13.3.2.8     Ensure that the tilting action is continued throughout this period to
                             prevent binding of low-mass, magnetic or magnetizable material. If the
                             sample in the MPC®-1 is allowed to stand motionless for more than 10
                             seconds, remove the flat-sided tube from the MPC®-1, shake the tube to
                             resuspend all material, replace the sample tube in the MPC®-1 and repeat
                             Section 13.3.2.9 before continuing to Section 13.3.2.11.
                13.3.2.9     Return the MPC®-1 to the upright position, sample tube vertical, with
                             cap at top. Immediately remove the cap and, keeping the flat  side of the
                             tube on top, pour off all of the supernatant from the tube held in the
                             MPC®-1 into a suitable container.  Do not shake the tube and do not
                             remove the tube from MPC®-1 during this step. Allow more supernatant
                             to settle; aspirate additional supernatant with pipette.
                13.3.2.10    Remove the sample tube from the MPC®-1  and resuspend the sample in
                             0.5 mL IX SL-buffer-A (prepared from 10X SL-buffer-A
                             stock—supplied). Mix very gently to resuspend all material in the tube.
                             Do not vortex.
                13.3.2.11     Quantitatively transfer (transfer followed by two rinses) all the liquid
                             from the sample tube to a labeled, 1.5-mL microcentrifuge tube. Use 0.5

December 2005                                48

-------
                                                           Method 1622 - Cryptosporidium
                      mL of IX SL-buffcr-A to perform the first rinse and 0.5 mL of IX SL-
                      buffer-A for the second rinse. Allow the flat-sided sample tube to sit for
                      a minimum of I minute after transfer of the second rinse volume, then use
                      a pipette to collect any residual volume that drips down to the bottom of
                      the tube to ensure that as much sample volume is recovered as possible.
                      Ensure that all of the liquid and beads are transferred.
        13.3.2.12     Place the microcentrifuge tube into the second magnetic particle
                      concentrator (MPC®-M or MPC®-S), with its magnetic strip in place.
        13.3.2.13     Without removing the microcentrifuge tube from MPC®-M, gently
                      rock/roll the tube through 180° by hand.  Continue for approximately 1
                      minute with approximately one 180° roll/rock per second. At the end of
                      this step, the beads should produce a distinct brown dot at the back of the
                      tube.
        13.3.2.14     Immediately aspirate the supernatant from the tube and cap held in the
                      MPC®-M. If more than one sample is being processed, conduct three 90°
                      rock/roll actions before removing the supernatant from each tube. Take
                      care not to disturb the material attached  to the wall of the tube adjacent to
                      the magnet. Do not shake the tube. Do not remove the tube from MPC®-
                      M while conducting these steps.
13.3.3  Dissociation of beads/oocyst complex

NOTE: Two acid dissociations are required.

        13.3.3.1      Remove the magnetic strip from the MPC®-M.
        13.3.3.2      Add 50 uL of 0.1  N HC1, then vortex at  the highest setting for
                      approximately 50 seconds.
NOTE: The laboratory must use 0.1-N standards purchased directly from a vendor,
rather than adjusting the normality in-house.

        13.3.3.3      Place the tube in the MPC®-M without the magnetic strip in place and
                      allow to stand in a vertical position for at least 10 minutes at room
                      temperature.
        13.3.3.4      Vortex vigorously for approximately 30 seconds.
        13.3.3.5      Ensure that all of the sample is at the base of the tube. Place the
                      microcentrifuge tube in the MPC®-M.
        13.3.3.6      Replace magnetic strip in MPC®-M  and allow the tube to stand
                      undisturbed for a minimum of 10 seconds.
        13.3.3.7      Prepare a well slide for sample screening and label the slide.
        13.3.3.8      Add 5 uL of 1.0 N NaOH to  the sample  wells of two well slides (add 10
                      uL to the sample well of one well slide if the volume from the two
                      required dissociations will be added  to the same slide).
NOTE: The laboratory must use 1.0-Nstandards purchased directly from a vendor
rather than adjusting the normality in-house.

        13.3.3.9      Without removing the microcentrifuge tube from the MPC®-M, transfer
                      all of the sample from the microcentrifuge tube in the MPC®-M to the
                      sample well with the NaOH. Do not disturb the beads at the back wall of
                      the tube. Ensure that all of the fluid is transferred.
                                       49                                December 2005

-------
 Method 1622 - Cryptosporidium
                13.3.3.10     Do not discard the beads or microcentrifuge tube after transferring the
                              volume from the first acid dissociation to the well slide. Perform the steps
                              in Sections 13.3.3.1 through 13.3.3.9 a second time. The volume from the
                              second dissociation can be added to the slide containing the volume from
                              the first dissociation, or can be applied to a second slide.
        NOTE:  The wells on Dynal Spot-On slides are likely to be too small to accommodate the
        volumes from both dissociations.

                13.3.3.11     Record the date and time the purified sample was applied to the slide(s).
                13.3.3.12     Air-dry the sample on the well slide(s). Because temperature and
                              humidity vary from laboratory to laboratory, no minimum time is
                              specified. However, the laboratory must take care to ensure that the
                              sample has dried completely before staining to prevent losses during the
                              rinse steps. A slide warmer set at 35°C to 42°C also can be used.
        13.3.4  Tips for minimizing carry-over of debris onto microscope slides after IMS

                •   Make sure the resuspended pellet is fully homogenized before placing the tube in the
                   MPC®-1 or MPC®-M to avoid trapping "clumps" or a dirty layer between the beads
                   and the side of the tube.

                •   When using the MPC®-1 magnet, make sure that the tube is snugged flat against the
                   magnet. Push the tube flat if necessary. Sometimes the magnet is not flush with the
                   outside of the holder and, therefore, the attraction between the beads and the magnet
                   is not as strong as it should be. However, it can be difficult to determine this if you do
                   not have more than one MPC®-1 to make comparisons.

                •   After the supernatant has been poured off at Section 13.3.2.11,  leave the tube in the
                   MPC®-1 and allow time for any supernatant remaining in the tube to settle down to
                   the bottom. Then aspirate the settled supernatant and associated particles from the
                   bottom of the tube. The same can be done at Section  13.3.2.16 with the
                   microcentrifuge tube.

                •   An additional rinse can also be performed at Section  13.3.2.11. After the supernatant
                   has been poured off and any settled material is aspirated off the bottom, leave the tube
                   in the MPC®-1 and add an additional 10 mL of reagent water or PBS to the tube and
                   repeat Sections 13.3.2.9 and 13.3.2.11. Although labs have reported successfully
                   using this technique to  reduce carryover, because the attraction  between the MPC®-1
                   and the beads is not as great as the attraction between the MPC®-M and the beads,
                   the chances would be greater for loss of oocysts doing the rinse at this step instead of
                   at Section 13.3.2.16.
                •   After the supernatant has been aspirated from the tube at Section 13.3.2.16, add 0.1
                   mL of PBS, remove the tube from the MPC®-M, and resuspend. Repeat Sections
                   13.3.2.15 and 13.3.2.16.
                •   Use a slide with the largest diameter well available to spread out the sample as much
                   as possible.
December 2005                                 50

-------
                                                                    Method 1622 - Cryptosporidium
14.0  Sample Staining
       NOTE:  The sample must be stained within 72 hours of application of the purified sample
       to the slide.

•J4 -j   Prepare positive and negative controls.
       14.1 .1   For the positive control, pipette 10 uL of positive antigen or 200 to 400 intact oocysts to
                the center of a well.
       14.1.2   For the negative control, pipette 50 uL of PBS (Section 7.4.2.1) into the center of a well
                and spread it over the well area with a pipette tip.
       14.1.3   Air-dry the control slides (see Section 13.3.3.12 for guidance).

       NOTE:  If the laboratory has a large batch of slides that will be examined over several
       days, and is concerned that a single positive control may fade, due to multiple
       examinations, the laboratory should prepare multiple control slides with the batch of field
       slides and alternate between the positive controls when performing the positive control
       check.

142   Follow manufacturer's instructions in applying stain to slides.
•j 4 3   Place the slides in a humid chamber in the dark and incubate at room temperature for
       approximately 30 minutes. The humid chamber consists of a tightly sealed plastic container
       containing damp paper towels on top of which the slides are placed.
14 4   Remove slides from humid chamber and allow condensation to evaporate, if present.
145   Apply one drop of wash buffer (prepared according to the manufacturer's instructions [Section
       7.6]) to each well. Tilt each slide on a clean paper towel, long edge down. Gently aspirate the
       excess detection reagent from below the well using a clean Pasteur pipette or absorb with paper
       towel or other absorbent material placed at edge of slide. Avoid disturbing the sample.
14.6   Apply 5® ^L of 4',6-diamidino-2-phenylindole (DAPI) staining solution (Section 7.7.2) to each
       well. Allow to stand at room temperature for a minimum of 1 minute. (The solution concentration
       may be increased up to  1  fig/mL if fading/diffusion of DAPI staining is encountered, but the
       staining solution must be tested first on expendable environmental samples to confirm that
       staining intensity is appropriate.)
147   Apply one drop of wash buffer (prepared according to the manufacturer's instructions [Section
       7.6]) to each well. Tilt each slide on a clean paper towel, long edge down. Gently aspirate the
       excess DAPI staining solution from below the well using a clean Pasteur pipette or absorb with
       paper towel or other absorbent material placed at edge of slide. Avoid disturbing the sample.

       NOTE:  If using the MeriFluor® Cryptosporidium/Giardia (Section 7.6.1), do not allow
       slides to dry completely.
14.8   ^d mounting medium (Section 7.8) to each well.
149   Apply a cover slip. Use a tissue to remove excess mounting fluid from the edges of the coverslip.
       Seal the edges of the coverslip onto the slide using clear nail polish.
1410  Record the date and time that staining was completed on the bench sheet. If slides will not be read
       immediately, store in a humid chamber in the dark between 1°C and 10°C until ready for
       examination.
                                               51                                 December 2005

-------
 Method 1622 - Cryptosporidium
 15.0  Examination
        NOTE:  Although immunofluorescence assay (FA) and 4',6-diamidino-2-phenylindole
        (DAPI) and differential interference contrast (DIC) microscopy examination should be
        performed immediately after staining is complete, laboratories have up to 168 hours (7
        days) from completion of sample staining to complete the examination and verification of
        samples. However, if fading/diffusion of'FITC or DAPI fluorescence is noticed, the
        laboratory must reduce this holding time. In addition the laboratory may adjust the
        concentration of the DAPI staining solution (Sections 7.7.2) so that fading/diffusion does
        not occur.

 •j g -|    Scanning technique: Scan each well in a systematic fashion. An up-and-down or a side-to-side
        scanning pattern may be used (Figure 4).
 1 5.2    Examination using immunofluorescence assay (FA), 4',6-diamidino-2-phenylindole (DAPI)
        staining characteristics, and differential interference contrast (DIC) microscopy. The minimum
        magnification requirements for each type of examination are noted below.
        NOTE:  All characterization (DAPI and DIC) and size measurements must be determined
        using 1000X magnification and reported to the nearest 0.5
        Record examination results for Cryptosporidium oocysts on a Cryptosporidium examination form.
        All organisms that meet the criteria specified in Sections 15.2.2 and 15.2.3, less atypical
        organisms specifically identified as non-target organisms by DIC or DAPI (e.g. possessing spikes,
        stalks, appendages, pores, one or two large nuclei filling the cell, red fluorescing chloroplasts,
        crystals, spores, etc), must be reported.
        1 5.2.1   Positive and negative staining control. Positive and negative staining controls must be
                acceptable before proceeding with examination of field sample slides.
                15.2.1.1      Each analyst must characterize a minimum of three Cryptosporidium
                              oocysts on the positive staining control slide before examining field
                              sample slides. This characterization must be performed by each analyst
                              during each microscope examination session. FITC examination must be
                              conducted at a minimum of 200X total magnification, DAPI examination
                              must be conducted at a minimum of 400X, and DIC examination and size
                              measurements must be conducted at a minimum of 1000X. Size, shape,
                              and DIC and DAPI characteristics of three Cryptosporidium oocysts must
                              be recorded by the analyst on a microscope log. The analyst also must
                              indicate on each sample examination form whether the positive staining
                              control was  acceptable.
                1 5.2.1.2      Examine the negative staining control to confirm that it does not contain
                              any oocysts (Section 14.1). Indicate on each sample examination form
                              whether the negative staining control was acceptable.
                15.2.1.3      If the positive staining control contains oocysts within the expected range
                              and at the  appropriate  fluorescence for both FA and DAPI, and the
                              negative staining control does not contain any oocysts (Section 14.1),
                              proceed to Sections  15.2.2 and 15.2.3.
        15.2.2   Sample examination — Cryptosporidium
                15.2.2.1      FITC examination (the analyst must use a minimum of 200X total
                              magnification). Use epifluorescence to scan the entire well for apple-
                              green fluorescence of oocyst and cyst shapes. When brilliant apple-green
                              fluorescing ovoid  or spherical objects 4 to 6 urn in diameter are observed
                              with brightly highlighted edges, increase magnification to 400X and
December 2005                                 52

-------
                                                                   Method 1622 - Cryptosporidium
                             switch the microscope to the UV filter block for DAPI (Section 15.2.2.2),
                             then to DIG (Section 15.2.2.3) at 1000X.
               15.2.2.2      DAPI fluorescence examination (the analyst must use a minimum of
                             400X total magnification). Using the UV filter block for DAPI, the
                             object will exhibit one of the following characteristics:
                             (a)  Light blue internal staining (no distinct nuclei) with a green rim
                             (b)  Intense blue internal staining
                             (c)  Up to four distinct, sky-blue nuclei

                             Look for atypical DAPI fluorescence, e.g., more than four stained nuclei,
                             size of stained nuclei, and wall structure and color.  Record oocysts in
                             category (a) as DAPI-negative; record oocysts in categories (b) and (c) as
                             DAPI-positive.
               15.2.2.3      DIG examination (the analyst must use a minimum of 1000X total
                             magnification [oil immersion lens]). Using DIG, look for external or
                             internal morphological characteristics atypical of Cryptosporidium
                             oocysts  (e.g., spikes, stalks, appendages, pores, one or two large nuclei
                             filling the cell, red fluorescing chloroplasts, crystals, spores, etc.)
                             (adapted from Reference 20.10). If atypical structures are not observed,
                             then categorize each apple-green fluorescing object as:
                             (a)  An empty Cryptosporidium oocyst
                             (b)  A Cryptosporidium oocyst with amorphous structure
                             (c)  A Cryptosporidium oocyst with internal structure (one to four
                                 sporozoites/oocyst)
                             Using 1OOOX total magnification,  record the shape, measurements (to the
                             nearest 0.5 urn), and number of sporozoites (if applicable) for each apple-
                             green fluorescing object meeting the size and shape characteristics.
                             Although not a defining characteristic, surface oocyst folds may be
                             observed in some specimens.
               15.2.2.4      A positive result is a Cryptosporidium oocyst which exhibits typical IFA
                             fluorescence, typical size and shape and exhibits nothing atypical on IFA,
                             DAPI fluorescence, or DIC microscopy. A positive result must be
                             characterized and assigned to one of the DAPI and DIC categories in
                             Sections 15.2.2.2 and 15.2.2.3.

       15.2.3  Record the date and time that sample examination was completed on the examination
               form.
       15.2.4  Report Cryptosporidium concentrations as oocysts/L.
       15.2.5  Record analyst name.


16.0  Analysis of Complex Samples
•jg 1   Some samples may contain high levels (>1000/L) of oocysts and/or interfering organisms,
       substances, or materials. Some samples may clog the filter (Section 12.0); others will not allow
       separation of the oocysts from the retentate or eluate; and others may contain materials that
       preclude or confuse microscopic examination.
•jg 2   If the sample holding time has not been exceeded and a full-volume sample cannot be filtered,
       dilute an aliquot of sample with  reagent water and filter this smaller aliquot (Section 12.0). This
       dilution must be recorded and reported with the results.
                                               53                                December 2005

-------
Method 1622 - Cryptosporidium
 163  If the holding times for the sample and for microscopic examination of the cleaned up
       rctentate/eluate have been exceeded, the site should be re-sampled. If this is not possible, the
       results should be qualified accordingly.
 164  Some samples may adhere to the centrifuge tube walls. The use of siliconized or low-adhesion
       centrifuge tubes (Fisherbrand siliconized/low retention microcentrifuge tubes, 02-681-320 or
       equivalent) may reduce adhesion. Alternately, rinse centrifuge tubes with PBST elution buffer or
       Sigmacote® prior to use.

 17.0 Method Performance
 •jy •]   Method acceptance criteria are shown in Tables 3 and 4 in Section 21.0. The initial and ongoing
       precision and recovery criteria are based on the results of spiked reagent water samples analyzed
       during the Information Collection Rule Supplemental Surveys (Reference 20.11). The matrix
       spike and matrix spike duplicate criteria are based on spiked source water data generated during
       the interlaboratory validation study of Method 1622  involving 11 laboratories and 11 raw surface
       water matrices across the U.S. (Reference 20.14).
       NOTE: Some sample matrices  may prevent the MS acceptance criteria in Tables 3 and 4
       to be met. An assessment of the distribution of MS recoveries across 430 MS samples
       from 87 sites during the ICR Supplemental Surveys is provided in Table 5.


 18.0 Pollution Prevention
 •|g -j   The solutions and reagents used in this method pose  little threat to the environment when recycled
       and managed properly.
 182  Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
       the volume of expired materials that need to be discarded.

 19.0 Waste Management
 19.1   ^ is ti16 laboratory's responsibility to comply with all federal, state, and local regulations govern-
       ing waste management, particularly the biohazard and hazardous waste identification rules and
       land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all
       releases from fume hoods and bench operations. Compliance with all sewage discharge permits
       and regulations is also required. An overview of these requirements can be found in the
       Environmental Management Guide for Small Laboratories (EPA 233-B-98-001).
 19.2  Samples, reference materials, and equipment known  or suspected to have viable oocysts attached
       or contained must be sterilized prior to disposal.
 193  For further information on waste management, consult The  Waste Management Manual for
       Laboratory Personnel and Less  is Better: Laboratory Chemical Management for Waste
       Reduction, both available from the American Chemical Society's Department of Government
       Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.

 20.0 References
 20 1   Morgan-Ryan, UM, A. Fall, L.A. Ward, N. Hijjawi, I. Sulaiman, R. Payer, R.C.Thompson, M.
       Olson, A. Lai, L. Xiao. 2002. Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae
       from Homo sapiens). Journal Eukaryot Microbiol 49(6):433^450.

 20.2  Rodgers, Mark R., Flanigan, Debbie J., and Jakubowski, Walter, 1995. Applied and
       Environmental Microbiology 6J. (10), 3759-3763.
 20.3  Fleming, Diane O., et al.(eds.), Laboratory Safety: Principles and Practices, 2nd edition. 1995.
       ASM Press, Washington, DC

December 2005                                54

-------
                                                                 Method 1622 - Cryptosporidium
20.4   "Working with Carcinogens," DHEW, PHS, CDC, NIOSH, Publication 77-206, (1977).
20.5   "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910 (1976).
20.6   "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety (1979).
20.7   APHA, AWWA, and WEF. 2005. Standard Methods for the Examination of Water and
       Wastewater; 21lh Edition. American Public Health Association, American Water Works
       Association, Washington, D.C.
20 8   USEPA 2005. Manual for the Certification of Laboratories Analyzing Drinking Water; Criteria
       and Procedures; Quality Assurance. Fifth Edition. EPA 815-R-05-004. Office of Ground Water
       and Drinking Water, U.S. Environmental Protection Agency, 26 West Martin Luther King Drive,
       Cincinnati, OH 45268.
20 9   ICR Microbial Laboratory Manual, EPA/600/R-95/178, National Exposure Research Laboratory,
       Office of Research and Development, U.S. Environmental Protection Agency, 26 Martin Luther
       King Drive, Cincinnati, OH 45268 (1996).
20.10  Connell, K., C.C. Rodgers, H.L. Shank-Givens, J Scheller, M.L Pope, and K. Miller, 2000.
       Building a Better Protozoa Data Set. Journal AWWA, 92:10:30.
20.11  "Envirochek™ Sampling Capsule," PN 32915, German Sciences, 600 South Wagner Road, Ann
       Arbor, MI 48103-9019 (1996).
20.12  "Dynabeads® GC-Combo," Dynal Microbiology R&D, P.O.  Box 8146 Dep., 0212 Oslo, Norway
       (September 1998, Revision no. 01).
20 13  USEPA. Results of the Interlaboratory Method Validation Study for Determination of
       Cryptosporidium and Giardia Using USEPA Method 1623, EPA-821-R-01-028. Office of Water,
       Office of Science and Technology, Engineering and Analysis Division, Washington, DC (2001).
20 14  USEPA. Implementation and Results of the Information Collection Rule Supplemental Surveys.
       EPA-815-R-01-003. Office of Water, Office of Ground Water and Drinking Water, Standards and
       Risk Management Division, Washington, DC (2001).
20 15  Connell, K., J. Scheller, K. Miller, and C.C. Rodgers, 2000. Performance of Methods 1622 and
       1623 in the ICR  Supplemental Surveys. Proceedings, American Water Works Association Water
       Quality Technology Conference, November 5-9, 2000, Salt Lake City, UT.
                                             55                               December 2005

-------
 Method 1622 - Cryptosporidium
 21.0  Tables and Figures

 Table 1. Method Holding Times (See Section 8.2 for details)
           Sample Processing Step
    Maximum Allowable Time between Breaks
(Samples should be processed as soon as possible)
      Collection
      Filtration
      >• Up to 96 hours are permitted between sample collection (if shipped to the laboratory as a bulk
      sample) or filtration (if filtered in the field) and initiation of elution
      Elution
      Concentration
      Purification
      Application of purified sample to slide
                                            These steps must be completed in 1 working day
      Drying of sample
         Up to 72 hours are permitted from application of the purified sample to the slide to staining
      Staining
         Up to 7 days are permitted between sample staining and examination
      Examination
December 2005
56

-------
                                                                       Method 1622 - Cryptosporidium
Table 2.    Tier 1 and Tier 2 Validation/Equivalency Demonstration Requirements
Test
IPR
(Section 9.4)
Method blank
(Section 9.6)
MS
(Section 9.5.1)
MS/MSD
(Section 9.5)
Description
4 replicates of spiked
reagent water
Unspiked reagent
water
Spiked matrix water
2 replicates of spiked
matrix water
Tier 1 modification'11
Required. Must be accompanied by a
method blank.
Required
Required on each water to which the
modification will be applied and on every
20th sample of that water thereafter. Must be
accompanied by an unspiked field sample
collected at the same time as the MS sample
Recommended, but not required. Must be
accompanied by an unspiked field sample
collected at the same time as the MS sample
Tier 2 modification12'
Required per laboratory
Required per laboratory
Not required
Required per laboratory.
Each laboratory must
analyze a different water.
(1)   If a modification will be used only in one laboratory, these tests must be performed and the results must meet all
     of the QC acceptance criteria in the method (these tests also are required the first time a laboratory uses the
     validated version of the method)
(2)   If nationwide approval of a modification is sought for one type of water matrix (such as surface water), a
     minimum of 3 laboratories must perform the tests and the results from  each lab individually must meet all QC
     acceptance criteria in the method. If more than 3 laboratories are used in a study, a minimum of 75% of the
     laboratories must meet all QC acceptance criteria.

        NOTE:   The initial precision and recovery and ongoing precision and recovery (OPR)

        acceptance criteria listed in Table 3 are based on results from 293 Cryptosporidium OPR
        samples analyzed by six laboratories during the Information Collection Rule
        Supplemental Surveys (Reference 20.15). The matrix spike acceptance criteria are based
        on data generated through interlaboratory validation of Method 1623 (Reference 20.14).
                                                 57
December 2005

-------
Method 1622 - Cryptosporidium
Table 3. Quality Control Acceptance Criteria for Cryptosporidium
Performance test
Initial precision and recovery
Mean recovery (percent)
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (percent)
Matrix spike/matrix spike duplicate (for method modifications)
Mean recovery1'2 (as percent)
Precision (as maximum relative percent difference)
Section
9.4
9.4.3
9.4.3
9.7
9.5
9.5.2.2
9.5.2.3
Acceptance criteria

24-100
55
11-100

13-143
67
(1) The acceptance criteria for mean MS/MSD recovery serves as the acceptance criteria for MS recovery during
    routine use of the method (Section 9.5.1).
(2) Some sample matrices may prevent the acceptance criteria from being met. An assessment of the distribution of
    MS recoveries from multiple MS samples from 87 sites during the ICR Supplemental Surveys is provided in Table
    4.
December 2005
58

-------
                                                                   Method 1622 - Cryptosporidium
Table 4. Distribution of Matrix Spike Recoveries from Multiple Samples Collected from 87 Source Waters
       During the ICR Supplemental Surveys (Adapted from Reference 20.16)
MS Recovery Range
<10%
>10%-20%
>20% - 30%
>30% - 40%
>40% - 50%
>50% - 60%
>60% - 70%
>70% - 80%
>80% - 90%
>90%
Percent of 430 Cryptosporidium MS Samples in
Recovery Range
6.7%
6.3%
14.9%
14.2%
18.4%
17.4%
1 1 .2%
8.4%
2.3%
0.2%
                                              59
December 2005

-------
Method 1622 - Cryptosporidium
                                1 mm
                        A
          1/5 mm
                        D
         B
         C
  Figure 1. Hemacytometer Platform Ruling. Squares 1, 2, 3, and 4
           are used to count stock suspensions of Cryptosporidium
           oocysts (after Miale, 1967)
December 2005
60

-------
                                      Method 1622 - Cryptosporidium
              -
                                  o

                         -•-
                      0
n
                                               o
                                               0
                                             o
                                              G
                                              o
                                               o
                                       o
Figure 2. Manner of Counting Oocysts in 1 Square mm.
          Dark organisms are counted and light
          organisms are omitted (after Miale, 1967).
                        61
                     December 2005

-------
                                                   Method 1622 - Cryptosporidium
   Sample
                                DIRECTION OF HOW
                                   Oirteltuttng
                                              Flow rate
                                                wilh valve
                                                              Effluent tubing
Influent
tubing
           Pressure   Met tubing
                             capsule
                                           Flow tola! izer
Figure 3a. Filtration Systems for Envirochek™ or
           Envirochek™HV Capsule (unpressurized source - top,
           pressurized source - bottom)
                                  62
                                                            December 2005

-------
Method 1622 - Cryptosporidium
                                              with valve
                                Ftowrate
                                 meter   /jOfc=ftlO||
                                 itti valve Ji  •—-——U^H
                                                     \
                  Fto.JWauwltlter housing
     Sample
Influent tubir^f
                             Inlet tubing
                                                \
                                   Outiel tubing
                                    ClUECf ION Of FLOIA)
                                                                        Effluent tub ing
                                          Outlet ti*ng
                                                       Ftow rate meter*-
                                                         with valve
                                                          Effluent tubir^
    Influent
Pressure   Inlet tubing
                                  FKte-Max™
                                 filter housing
                                    Row totalizer
                                         DIRECTION OF fW*V
                                                          Flow controi:
                                                          vate fray b«
                                                          used in plsc* of
                                                          flov< rale iwter
  Figure 3b. Filtration Systems for Filta-Max® filters (unpressurized
               source - top,  pressurized source - bottom)
December 2005
                            63

-------
Method 1622 - Cryptosporidium
Figure 4.  Methods for Scanning a Well Slide
December 2005
64

-------
                                                                  Method 1622 - Cryptosporidium
22.0  Glossary of Definitions and Purposes

       These definitions and purposes are specific to this method but have been conformed to common
       usage as much as possible.

22.1   Units of weight and measure and their abbreviations
       22.1.1 Symbols
              °C     degrees Celsius
              uL     microliter
              <      less than
              >      greater than
              %     percent
       22.1.2 Alphabetical characters
              cm     centimeter
              g      gram
              G     acceleration due to gravity
              hr     hour
              ID     inside diameter
              in.     inch
              L      liter
              m     meter
              MCS  microscope cleaning solution
              mg     milligram
              mL    milliliter
              mm    millimeter
              mM    millimolar
              N     normal; gram molecular weight of solute divided by hydrogen equivalent of
                     solute, per liter of solution
              RSD  relative standard deviation
              sr      standard deviation of recovery
              X     mean percent recovery
22.2   Definitions, acronyms, and abbreviations (in alphabetical order)

       Analyst—The analyst should have at least 2 years of college in microbiology or equivalent or
       closely related field. The analyst also should have a minimum of 6 months of continuous bench
       experience with Cryptosporidium and IFA microscopy. The analyst should have a minimum of 3
       months experience using EPA Method 1622 and/or EPA Method 1623 and should have
       successfully analyzed a minimum of 50 samples using EPA Method 1622 and/or EPA Method
       1623.

       Analyte—A protozoan parasite tested for by this method. The analyte in this method is
       Cryptosporidium.

       Cyst—A phase or a form of an organism produced either in response to environmental conditions
       or as a normal part of the life cycle of the organism. It is characterized by a thick and
       environmentally resistant cell wall.

       Flow cytometer—A particle-sorting instrument capable of counting protozoa.
                                              65                               December 2005

-------
Method 1622 - Cryptosporidium
        Immunomagnetic separation (IMS)—A purification procedure that uses microscopic, magnetically
        responsive particles coated with an antibodies targeted to react with a specific pathogen in a fluid
        stream. Pathogens are selectively removed from other debris using a magnetic field.

        Initial precision and recovery (IPR)—Four aliquots of spiking suspension analyzed to establish
        the ability to generate acceptable precision and accuracy. An IPR is performed prior to the first
        time this method is used and any time the method or instrumentation is modified.

        Laboratory blank—See Method blank

        Laboratory control sample (LCS)—See Ongoing precision and recovery (OPR) standard

        Matrix spike (MS)—A sample prepared by adding a known quantity of organisms to a specified
        amount of sample matrix for which an independent estimate of target analyte concentration is
        available. A matrix spike is used to determine the effect of the matrix on a method's recovery
        efficiency.

        May—This action, activity, or procedural step is neither required nor prohibited.

        May not—This action, activity, or procedural step is prohibited.

        Method blank—An aliquot of reagent water that is treated exactly as a sample, including exposure
        to all glassware, equipment, solvents, and procedures that are used with samples. The method
        blank is used to determine if analytes or interferences  are present in the laboratory environment,
        the reagents, or the apparatus.

        Must—This action, activity, or procedural step is  required.

        Negative control—See Method blank

        Nucleus—A membrane-bound organelle containing genetic material. Nuclei are a prominent
        internal structure  seen both in Cryptosporidium oocysts. In Cryptosporidium oocysts, there is one
        nucleus per sporozoite.

        Oocyst—The encysted zygote of some sporozoa; e.g., Cryptosporidium. The oocyst is a phase or
        form of the organism produced as a normal part of the life cycle of the organism. It is
        characterized by a thick and environmentally resistant outer wall.

        Ongoing precision and recovery (OPR) standard—A method blank spiked with  known quantities
        of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results
        produced by the laboratory remain within the limits specified in this method for precision and
        recovery.

        Oocyst and cyst spiking suspension—See Spiking suspension

        Oocyst and cyst stock suspension—See Stock suspension

        Positive control—See Ongoing precision and recovery standard

        Principal analyst—The principal analyst (may not be applicable to all monitoring programs)
        should have a BS/BA in microbiology or closely related field and a minimum of 1 year of
        continuous bench experience with Cryptosporidium and IFA microscopy. The principal analyst
        also should have a minimum of 6 months experience using EPA Method 1622 and/or EPA


December 2005                                 66

-------
                                                             Method 1622 - Cryptosporidium
Method 1623 and should have analyzed a minimum of 100 samples using EPA Method 1622
and/or EPA Method 1623.

PTFE—Polytetrafluoroethylene

Quantitative transfer—The process of transferring a solution from one container to another using
a pipette in which as much solution as possible is transferred, followed by rinsing of the walls of
the source container with a small volume of rinsing solution (e.g., reagent water, buffer, etc.),
followed by transfer of the rinsing solution, followed by a second rinse and transfer.

Reagent water—Water demonstrated to be free from the analytes of interest and potentially
interfering substances at the method detection limit for the analyte.

Reagent water blank—see Method blank

Relative standard deviation (RSD)—The standard deviation divided by the mean times 100.

RSD—See Relative standard deviation

Should—This action, activity, or procedural step is suggested but not required.

Spiking suspension—Diluted stock suspension containing the organism(s) of interest at a
concentration appropriate for spiking samples.

Sporozoite—A motile, infective stage of certain protozoans; e.g., Cryptosporidium. There are four
sporozoites in each Cryptosporidium oocyst, and they are generally banana-shaped.

Stock suspension—A concentrated suspension containing the organism(s) of interest that is
obtained from a source that will attest to the host source, purity, authenticity, and viability of the
organism(s).

Technician—The technician filters samples, performs centrifugation, elution, concentration, and
purification using IMS, and places purified samples on slides for microscopic examination, but
does not perform microscopic protozoan detection and identification. No minimum education or
experience requirements with Cryptosporidium and IFA microscopy apply to the technician.  The
technician should have at least 3 months of experience in filter extraction and processing of
protozoa samples by EPA Method 1622/1623 and should have successfully processed a minimum
of 50 samples using EPA Method 1622/1623.
                                        67                                 December 2005

-------
                 Appendix C

Method 1623: Cryptosporidium and Giardia in Water by
                Filtration/IMS/FA

-------
This page intentionally left blank

-------
&EPA
   United States
   Environmental Protection
   Agency
   Method 1623: Cryptosporidium and
   Giardia in Water by Filtration/IMS/FA

   December 2005

-------
Office of Water (4607)
EPA815-R-05-002
http://www.epa.gov/microbes/
December 2005
                                                   Printed on Recycled Paper

-------
                                  Acknowledgments

This method was prepared under the direction of William A. Telliard of the Engineering and Analysis
Division within the U.S. Environmental Protection Agency (U.S. EPA) Office of Water. This document
was prepared by CSC under a U.S. EPA contract, with assistance from its subcontractor, Interface, Inc.

The U.S. EPA Office of Water gratefully acknowledges the contributions of the following persons and
organizations to the development of this method:

Mike Arrowood, Centers for Disease Control and Prevention, Division of Parasitic Diseases (MS-F13),
       4770 Buford Highway, N.E., Atlanta, GA 30341-3724, USA

Phil Berger, Office of Groundwater and Drinking Water, U.S. Environmental Protection Agency, 401 M
       Street, S.W., Washington, DC 20460, USA

Jennifer Clancy, Clancy Environmental Consultants, Inc., P.O. Box 314, St. Albans, VT 05478, USA

Kevin Connell, CSC, 6101 Stevenson Avenue, Alexandria, VA 22314, USA
Ricardo DeLeon, Metropolitan Water District of Southern California, 700 Moreno Avenue, LaVerne, CA
       91760, USA
Shirley Dzogan, EnviroTest Laboratories, 745 Logan Avenue; Winnipeg, Manitoba R3E 3L5, Canada

Mary Ann Feige (retired), Technical Support Center, Office of Ground Water and Drinking Water, U.S.
       Environmental Protection Agency, 26 W. Martin Luther King Drive, Cincinnati, OH 45268-1320,
       USA

Colin Fricker, Thames Water Utilities, Manor Farm Road, Reading, Berkshire, RG2 OJN, England
Carrie Moulton (Hancock), Technical Support Center, Office of Ground Water and Drinking Water, U.S.
       Environmental Protection Agency, 26 W. Martin Luther King Drive, Cincinnati, OH 45268-1320,
       USA
Stephanie Harris,Manchester Laboratory, U.S. Environmental Protection Agency, Region 10, 7411
       Beach Drive East, Port Orchard, WA 98366, USA

Dale Rushneck, Interface, Inc., 3194 Worthington Avenue, Fort Collins, CO 80526, USA
Frank Schaefer HI, National Exposure Research  Laboratory, U.S. Environmental Protection Agency, 26
       W. Martin Luther King Drive, Cincinnati, OH 45268-1320, USA
Steve Schaub, Health and Ecological Criteria Division (4304), Office of Science and Technology, U.S.
       Environmental Protection Agency, 401 M Street, S.W., Washington, DC 20460, USA
Ajaib Singh, City of Milwaukee Health Department, 841 North Broadway, Milwaukee, WI 53202, USA
Huw Smith, Department of Bacteriology,  Scottish Parasite Diagnostic Laboratory, Stobhill NHS Trust,
       Springburn, Glasgow, G21 3UW, Scotland

Timothy Straub, Lockheed Martin, 7411 Beach Drive East, Port Orchard, WA 98366, USA

William A. Telliard, Office of Science and Technology, U.S. Environmental Protection Agency, 401 M
       Street, S.W., Washington, DC 20460, USA

Cryptosporidium cover photo  courtesy of the U.S. Centers for Disease Control
Giardia cover photo courtesy of CH Diagnostic & Consulting Service, Inc.

-------
                                       Disclaimer

This method has been reviewed by the U.S. EPA Office of Water and approved for publication. Mention
of trade names or commercial products does not constitute endorsement or recommendation for use.
Questions regarding this method or its application should be addressed to:

       Carrie Moulton
       Coordinator, Laboratory Quality Assurance Program for the Analysis of Cryptosporidium
       U.S. Environmental Protection Agency
       Office of Ground Water and Drinking Water
       Technical Support Center, MCI40
       26 West Martin Luther King Drive
       Cincinnati, OH 45268-1320
       (513)569-7919
       (513)569-7191 (fax)
       moulton.carrie@epa.gov

-------
                                       Introduction

To support future regulation of protozoa in drinking water, the Safe Drinking Water Act Amendments of
1996 require the U.S. Environmental Protection Agency (EPA) to evaluate the risk to public health posed
by drinking water contaminants, including waterborne parasites, such as Cryptosporidium and Giardia. To
implement these requirements, EPA must assess Cryptosporidium and Giardia occurrence in raw surface
waters used as source waters for drinking water treatment plants. EPA Method 1623 was developed to
support this assessment.

Method Development and Validation

EPA initiated an effort in 1996 to identify new and innovative technologies for protozoan monitoring and
analysis. After evaluating potential alternatives to the then-current method through literature searches,
discussions with research and commercial laboratories, and meetings with experts in the field, the
Engineering and Analysis Division within the Office of Science and Technology within EPA's Office of
Water developed draft Method 1622 for Cryptosporidium detection in December 1996. This
Cryptosporidium-only method was validated through an interlaboratory study in August 1998, and was
revised as a final, valid method for detecting Cryptosporidium in water in January 1999.

Although development of an acceptable immunomagnetic separation system for Giardia lagged behind
development of an acceptable system for Cryptosporidium, an acceptable system was identified in
October 1998, and EPA validated a method for simultaneous detection of Cryptosporidium and  Giardia in
February 1999 and developed quality control (QC) acceptance criteria for the method based on this
validation study. To avoid confusion with Method 1622, which already had been validated and was in use
both domestically and internationally as a stand-alone Cryptosporidium-only detection method,  EPA
designated the new combined procedure EPA Method 1623.

The interlaboratory validated versions of Method 1622 (January 1999; EPA-821-R-99-001) and Method
1623 (April 1999; EPA-821-R-99-006) were used to analyze approximately 3,000 field and QC  samples
during the Information Collection Rule Supplemental Surveys (ICRSS) between March  1999 and
February 2000. Method 1622 was used to analyze samples from March 1999 to mid-July 1999; Method
1623 was used from mid-July 1999 to February 2000.

Changes in the April 2001  Versions of the Methods
Both methods were revised in April 2001, after completion of the ICRSS and multiple meetings with
researchers and experienced laboratory staff to discuss potential method updates. Changes incorporated in
the April 2001 revisions of the methods (EPA-821-R-01-025 and EPA-821-R-01-026) included the
following:
•       Nationwide approval of modified versions of the methods using the following components:
       (a)    Whatman Nuclepore CrypTest™ filter
       (b)    IDEXX Filta-Max® filter
       (c)    Waterborne Aqua-Glo™ G/C Direct FL antibody stain
       (d)    Waterborne Crypt-a-Glo™ and Giardi-a-Glo™ antibody stains
•       Clarified sample acceptance criteria

•       Modified capsule filter elution procedure

•       Modified concentrate aspiration procedure

-------
•      Modified IMS acid dissociation procedure
•      Updated QC acceptance criteria for IPR and OPR tests
•      Addition of a troubleshooting section for QC failures
•      Modified holding times
•      Inclusion of flow cytometry-sorted spiking suspensions
Changes in the June 2003 Versions of the Methods
Both methods were revised again in June 2003 to support proposal of EPA's Long Term 2 Enhanced
Surface Water Treatment Rule. Changes incorporated into the December 2002 versions include:
       Nationwide approval of a modified version of the methods using the Pall Gelman Envirochek™
       HV filter
•      Removal of Whatman Nuclepore CrypTest™  filter from the methods as a result of discontinuation
       of the product by the manufacturer
•      Nationwide approval of the use of BTF EasySeed™ irradiated oocysts and cysts for use in routine
       quality control (QC) samples
•      Minor clarifications and corrections
•      Rejection criteria for sample condition upon receipt
•      Guidance on measuring sample temperatures
•      Clarification of QC sample requirements and use of QC sample results
•      Guidance on minimizing carry-over debris onto microscope slides after IMS
Changes in the December 2005 Versions of the Methods
Both methods were revised again in 2005 to support promulgation of EPA's Long Term 2 Enhanced
Surface Water Treatment Rule. Changes incorporated into the June 2003 versions include:
       Nationwide approval of the use of portable continuous-flow centrifugation as a modified version
       of the method. The product met all method acceptance criteria for Cryptosporidium using 50-L
       source water samples (but not Giardia, however, individual laboratories are permitted to
       demonstrate acceptable performance for Giardia in  their laboratory).
•      Addition of BTF EasyStain™ monoclonal antibody stain as an acceptable reagent for staining in
       Methods 1622/1623. The product was validated through an interlaboratory validation study using
       the Pall Envirochek™ HV filter.
       Clarification of the analyst verification procedure
•      Clarification of sample condition criteria upon receipt
Performance-Based Method  Concept and Modifications Approved for Nationwide Use
EPA Method 1623 is a performance-based method applicable to the determination of Cryptosporidium
and Giardia in aqueous matrices.  EPA Method  1623 requires filtration, immunomagnetic separation of the
oocysts and cysts from the material captured, and enumeration of the target organisms based on the results
of immunofluorescence assay, 4',6-diamidino-2-phenylindole (DAPI) staining results, and differential
interference contrast microscopy.
                                              IV

-------
The interlaboratory validation of EPA Method 1623 conducted by EPA used the Pall Gelman capsule
filtration procedure, Dynal immunomagnetic separation (IMS) procedure, and Meridian sample staining
procedure described in this document. Alternate procedures are allowed, provided that required quality
control tests are performed and all quality control acceptance criteria in this method are met.

Since the interlaboratory validation of EPA Method 1623, interlaboratory validation studies have been
performed to demonstrate the equivalency of modified versions of the method using the following
components:
•      Whatman Nuclepore CryptTest™ filter (no longer available)
       IDEXX Filta-Max® filter
       Pall Gelman Envirochek™ HV filter
•      Portable Continuous-Flow Centrifugation (PCFC)
       Waterbome Aqua-Glo™ G/C Direct FL antibody stain
•      Waterborne Crypt-a-Glo™ and Giardi-a-Glo™ antibody stains
•      BTF EasyStain™ antibody stain
•      BTF EasySeed™ irradiated oocysts and cysts for use in routine QC samples

The validation studies for these modified versions of the method met EPA's performance-based
measurement system Tier 2 validation for nationwide use (see Section 9.1.2 for details), and have been
accepted by EPA as equivalent in performance to the original version of the method validated by EPA.
The equipment and reagents used in these modified versions of the method are noted in Sections 6 and 7
of the method.

Because this is a performance-based method, other alternative components not listed  in the method may be
available for evaluation and use by the laboratory. Confirming the acceptable performance of a modified
version of the method using alternate components in a single laboratory does not require that an
interlaboratory validation study be conducted. However, method modifications validated only in a single
laboratory have not undergone sufficient testing to merit inclusion in the method. Only those modified
versions of the method that have been demonstrated as equivalent at multiple laboratories on multiple
water sources through  a Tier 2 interlaboratory study will be cited in the  method.

-------
                                   Table of Contents






1.0    Scope and Application	  1




2.0    Summary of Method	  1




3.0    Definitions 	  2




4.0    Contamination, Interferences, and Organism Degradation	  2




5.0    Safety  	  3




6.0    Equipment and Supplies	4




7.0    Reagents and Standards 	  8




8.0    Sample Collection and Storage	 11




9.0    Quality Control	 13




10.0   Microscope Calibration and Analyst Verification	21




11.0   Oocyst and Cyst Suspension Enumeration and Sample Spiking  	 28




12.0   Sample Filtration and Elution	 36




13.0   Sample Concentration and Separation (Purification)	46




14.0   Sample Staining  	 51




15.0   Examination 	 52




16.0   Analysis of Complex Samples  	 54




17.0   Method Performance	 55




18.0   Pollution Prevention	 55




19.0   Waste Management	55




20.0   References	 55




21.0   Tables and Figures 	57




22.0   Glossary of Definitions and Purposes	66
                                              VI

-------
        Method 1623:  Cryptosporidium and Giardia in  Water
                                by  Filtration/IMS/FA

1.0   Scope and Application
•j 1    This method is for the detection of Cryptosporidium (CAS Registry number 137259-50-8) and
       Giardia (CAS Registry number 137259-49-5) in water by concentration, immunomagnetic
       separation (IMS), and immunofluorescence assay (FA) microscopy. Cryptosporidium and Giardia
       may be verified using 4',6-diamidino-2-phenylindole (DAPI) staining and differential interference
       contrast (DIG) microscopy. The method has been validated in surface water, but may be used in
       other waters, provided the laboratory demonstrates that the method's performance acceptance
       criteria arc met.
-j 2    This method is designed to meet the survey and monitoring requirements of the U.S.
       Environmental Protection Agency (EPA). It is based on laboratory testing of recommendations by
       a panel of experts convened by EPA. The panel was charged with recommending an improved
       protocol for recovery and detection of protozoa that could be tested and implemented with
       minimal additional research.
•j 3    This method identifies the genera, Cryptosporidium or Giardia, but not the species. The method
       cannot determine the host species of origin, nor can it determine the viability or infectivity of
       detected oocysts and cysts.
1 4    This method is for use only by persons experienced in the determination of Cryptosporidium and
       Giardia by filtration, IMS, and FA. Experienced persons are defined in Section 22.2 as analysts or
       principal analysts. Laboratories unfamiliar with analyses of environmental samples by the
       techniques in this method should  gain experience using water filtration techniques, IMS,
       fluorescent antibody staining with monoclonal antibodies,  and microscopic examination of
       biological particulates using bright-field and DIC microscopy.
1 5    Any modification of the method beyond those expressly permitted is subject to the application and
       approval of alternative test procedures under 40 CFR Part 141.27.

2.0   Summary of Method
2i    A water sample is filtered and the oocysts, cysts, and extraneous materials are retained on the
       filter. Although EPA has only validated the method using laboratory filtration of bulk water
       samples shipped from the field, field-filtration also may be used.
2 2    Elution and separation
       2.2.1   Materials on the filter are eluted and the eluate is  centrifuged to pellet the oocysts and
               cysts, and the supernatant fluid is aspirated.
       2.2.2   The oocysts and cysts are magnetized by attachment of magnetic beads conjugated to
               artti-Cryptosporidium and ant\-Giardia antibodies. The magnetized oocysts and cysts are
               separated from the extraneous materials  using a magnet, and the extraneous materials are
               discarded. The magnetic bead complex is then detached from the oocysts and cysts.
23    Enumeration
       2.3.1   The oocysts and cysts are stained on well slides with fluorescently labeled monoclonal
               antibodies and 4',6-diamidino-2-phenylindole (DAPI). The stained sample is examined
               using fluorescence and differential interference contrast (DIC) microscopy.
                                                                               December 2005

-------
Method 1623 - Cryptosporidium and Giardia
        2.3.2    Qualitative analysis is performed by scanning each slide well for objects that meet the
                size, shape, and fluorescence characteristics of Cryptosporidium oocysts or Giardia
                cysts.
        2.3.3    Quantitative analysis is performed by counting the total number of objects on the slide
                confirmed as oocysts or cysts.
2 4     Quality is assured through reproducible calibration and testing of the filtration, immunomagnetic
        separation (IMS), staining, and microscopy systems. Detailed information on these tests is
        provided in Section 9.0.

3.0    Definitions
3.1     Cryptosporidium is a genus of protozoan parasites potentially found in water and other media.
        The recent taxonomy of the genus Cryptosporidium includes the following species and their
        potential hosts: C. hominis (humans; formerly C. parvum genotype I; Reference 20.1); C. parvum
        (bovine and other mammals including humans; formerly genotype II;); C. baileyi and C.
        meleagridis (birds); C.  muris (rodents);  C. canis (dogs); C. felis (cats); C. serpentis (reptiles); and
        C. nasorum (fish). Cryptosporidium oocysts are defined in this method as objects exhibiting
        brilliant apple green fluorescence under UV light (FA-positive), typical size (4 to 6 urn) and shape
        (round to oval), and no atypical characteristics by FA, DAPI fluorescence, or DIC microscopy.
        Examination  and characterization using fluorescence (FITC and DAPI stain) and DIC microscopy
        are required for exclusion of atypical organisms (e.g., those possessing spikes, stalks, appendages,
        pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals, spores, etc.).
3 2     Giardia is a genus of protozoan parasites potentially found in water and other media.  The recent
        taxonomy of the genus  Giardia includes the following species and their potential hosts:  G.
        lamblia (also called G.  intestinalis or G. duodenalis; humans and other mammals); G. muris
        (rodents); G.  agilis (amphibians); G. psittaci and G. ardeae (birds).  Recent molecular studies
        suggest the division of  G. lamblia into multiple genotypes (Reference 20.2). Giardia cysts are
        defined in this method as objects exhibiting brilliant apple green fluorescence under UV light
        (FA-positive), typical size (8 to 18 urn long by 5 to 15 (am wide) and shape (oval to round), and no
        atypical characteristics  by FA, DAPI fluorescence,  or DIC microscopy. Examination and
        characterization by fluorescence (FITC and DAPI stain) and DIC microscopy are required for
        exclusion of atypical organisms (e.g., those possessing spikes, stalks, appendages, pores, one or
        two large nuclei filling  the cell, red fluorescing chloroplasts, crystals, spores, etc.).
3 3     Definitions for other terms used in this method are  given in the glossary (Section 22.0).

4.0    Contamination,  Interferences, and Organism Degradation
4 •]     Turbidity caused by inorganic and organic debris can interfere with the concentration, separation,
        and examination of the  sample for Cryptosporidium oocysts and Giardia cysts. In addition to
        naturally-occurring debris, e.g. clays and algae, chemicals, e.g. iron, alum coagulants and
        polymers added to source waters during the treatment process may result in additional
        interference.
4 2     Organisms and debris that autofluoresce or demonstrate non-specific immunofluorescence, such
        as algal and yeast cells, when examined by epifluorescent microscopy, may interfere with the
        detection of oocysts and cysts and contribute to false positives by immunofluorescence assay (FA)
        (Reference 20.3).
December 2005

-------
                                                          Method 1623 - Cryptosporidium and Giardia
4 3     Solvents, reagents, labware, and other sample-processing hardware may yield artifacts that may
        cause misinterpretation of microscopic examinations for oocysts and cysts. All materials used
        must be demonstrated to be free from interferences under the conditions of analysis by running a
        method blank (negative control sample) initially and a minimum of every week or after changes in
        source  of reagent water. Specific selection of reagents and purification of solvents and other
        materials may be required.
4 4     Freezing samples, filters, eluates, concentrates, or slides may interfere with the detection and/or
        identification of oocysts and cysts.
45     All equipment should be cleaned according to manufacturers' instructions. Disposable supplies
        should  be used wherever possible.

5.0    Safety
g_^     The biohazard associated with, and the risk of infection from, oocysts and cysts is high in this
        method because live organisms are handled. This method does not purport to address all of the
        safety problems associated with its use. It is the responsibility of the laboratory to establish
        appropriate safety and health practices prior to  use of this method. In particular, laboratory staff
        must know and observe the safety procedures required in a microbiology laboratory that handles
        pathogenic organisms while preparing, using, and disposing of sample concentrates,  reagents and
        materials, and while operating sterilization equipment.
5 2     The toxicity or carcinogenicity of each compound or reagent used in this method  has not been
        precisely determined; however, each chemical compound should be treated as a potential health
        hazard. Exposure to these  compounds should be reduced to the lowest possible level. The
        laboratory is responsible for maintaining current knowledge of Occupational Safety and Health
        Administration regulations regarding the safe handling of the chemicals  specified in this method.
        A reference file of material safety data sheets should be made available to all personnel involved
        in these analyses. Additional information on laboratory  safety can be found in References 20.4
        through 20.7.
5 3     Samples may contain high concentrations of biohazards and toxic compounds, and must be
        handled with gloves. Reference materials and standards  containing oocysts and cysts must also be
        handled with gloves and laboratory staff must never place gloves in or near the face after exposure
        to solutions known or suspected to contain oocysts and cysts. Do not mouth-pipette.
5_4     Laboratory personnel must change  gloves after handling filters and other contaminant-prone
        equipment and reagents. Gloves must be removed or changed before touching any other laboratory
        surfaces or equipment.
55     Centers for Disease Control (CDC) regulations (42 CFR 72) prohibit interstate shipment of more
        than 4 L of solution known to contain infectious materials (see
        http://www.cdc.gov/od/ohs/biosftv/shiprcgs.htm for details). State regulations may contain similar
        regulations for intrastate commerce. Unless the sample is known or suspected to contain
        Cryptosporidium, Giardia, or other infectious agents (e.g., during an outbreak), samples should be
        shipped as noninfectious and should not be marked as infectious. If a sample is known or
        suspected to be infectious, and the sample must be shipped to a laboratory by a transportation
        means affected by CDC or state regulations, the sample  should be shipped in accordance with
        these regulations.
                                                                                  December 2005

-------
Method 1623 - Cryptosporidium and Giardia
6.0   Equipment and Supplies
       NOTE:  Brand names, suppliers, and part numbers are for illustrative purposes only. No
       endorsement is implied. Equivalent performance may be achieved using apparatus and
       materials other than those specified here, but demonstration of equivalent performance
       that meets the requirements of this method is the responsibility of the laboratory.
5 -j    Sample collection equipment for shipment of bulk water samples for laboratory filtration.
       Collapsible LDPE cubitainer for collection of 10-L bulk sample(s)—Cole Farmer cat. no. U-
       06100-30 or equivalent. Fill completely to ensure collection of a full 10-L sample. Discard after
       one use.
g 2    Equipment for sample filtration. Four options have been demonstrated to be acceptable for use
       with Method 1623. Other options may be used if their acceptability is demonstrated according to
       the procedures outlined in Section 9.1.2.
       6.2.1   Cubitainer spigot to facilitate laboratory filtration of sample (for use with any filtration
               option)—Cole Farmer cat. no. U-06061-01, or equivalent.
       6.2.2   Original Envirochek™ sampling capsule or Envirochek™ HV sampling capsule
               equipment requirements (for use with the procedure described in Section 12.2). The
               versions of the method using these  filters were validated using 10-L and 50-L sample
               volumes, respectively. Alternate sample volumes maybe used, provided the laboratory
               demonstrates acceptable performance on initial and ongoing spiked reagent water and
               source water samples (Section 9.1.2).
               6.2.2.1       Sampling capsule
                             6.2.2.1.1      Envirochek™, Pall Corporation,  Ann Arbor, MI, part no.
                                            12110 (individual filter) and or part no. 12107 (box of 25
                                           filters) (www.pall.com or (800) 521-1520 ext. 2)
                             6.2.2.1.2      Envirochek™ HV, Pall  Corporation, Ann Arbor, MI, part
                                           no.  12099 (individual filter) or part no. 12098 (box of 25
                                           filters) (www.Dall.com or (800) 521-1520 ext. 2)
               6.2.2.2       Laboratory shaker with arms for agitation of sampling capsules
                             6.2.2.2.1      Laboratory shaker—Lab-Line model 3589 (available
                                           through VWR Scientific cat. no.  57039-055), Pall
                                           Corporation part no. 4821, Fisher cat.  no. 14260-11, or
                                           equivalent
                             6.2.2.2.2      Side arms for laboratory shaker—Lab-Line Model 3587-
                                           4 (available through VWR Scientific cat. no. 57039-045),
                                           Fisher cat. no.  14260-13, or equivalent
       6.2.3   Filta-Max® foam filter equipment requirements  (for use with the procedure described in
               Section 12.3). The version of the method using this filter was validated using 50-L
               sample volumes; alternate sample volumes may be used, provided the laboratory
               demonstrates acceptable performance on initial and ongoing spiked reagent water and
               matrix samples (Section 9.1.2).
December 2005

-------
                                                         Method 1623 - Cryptosporidium and Giardia
                6.2.3.1       Foam filter—Filta-Max®, IDEXX, Westbrook, ME. Filter module cat.
                             no. FMC 10603
        NOTE:  Check at least one filter per batch to ensure that the filters have not been
        affected by improper storage or other factors that could result in brittleness or other
       problems. At a minimum confirm that the test filter expands properly in water before
        using the batch or shipping filters to the field.

                6.2.3.2       Filter processing equipment—Filta-Max® starter kit, IDEXX,
                             Westbrook, ME, cat. no. FMC 11002. Starter kit includes manual wash
                             station with clamp set (FMC 10101 or 10106) including plunger head
                             (FMC 12001), tubing set (FMC 10307), vacuum set (FMC 10401), MKII
                             filter housing with hose-tail fittings (FMC 10504) and green housing
                             tools (FMC 10506). In addition, processing requires magnetic stirrer
                             (FMC 10901) and filter membranes, 100 pk, (FMC 10800).
        6.2.4    Portable Continuous-Flow Centrifuge (PCFC) requirements (for use with procedures
                described in Section 12.4). The version of the method using this technique was validated
                for Cryptosporidium in 50-L sample volumes; alternate sample volumes may be used,
                provided the laboratory demonstrates acceptable performance on initial and ongoing
                spiked reagent water and matrix samples (Section 9.1.2). Individual laboratories are also
                permitted to demonstrate acceptable performance for Giardia in their laboratory. The
                technique is based on technology from Haemonetics Corporation, Braintree, MA.
g 3     Ancillary sampling equipment
        6.3.1    Tubing—Glass, polytetrafluoroethylene (PTFE), high-density polyethylene (HDPE), or
                other tubing to which oocysts and cysts will not easily adhere, Tygon formula R-3603, or
                equivalent. If rigid tubing (glass, PTFE, HDPE) is used and the sampling system uses a
                peristaltic pump, a minimum length of compressible tubing may be used in the pump.
                Before use, the tubing must be autoclaved, thoroughly rinsed with detergent solution,
                followed by repeated rinsing with reagent water to minimize sample contamination.
                Alternately, decontaminate using hypochlorite solution, sodium thiosulfate, and multiple
                reagent water rinses. Dispose of tubing after one use whenever possible or when wear is
                evident.
       6.3.2    Flow control valve—0.5 gpm (0.03 L/s), Bertram Controls, Plast-O-Matic cat. no.
                FC050B!/2-PV, or equivalent; or 0.4- to 4-Lpm flow meter with valve, Alamo Water
                Treatment, San Antonio, TX, cat. no. R5310, or equivalent
       6.3.3    Pump— peristaltic, centrifugal, impeller, or diaphragm pump;  MasterFlex I/P®
                EasyLoad® peristaltic pump (Cole-Parmer cat. No. EW-77963-10) with 77601-10
                pumphead, 77410-00 drive unit, and 06429-73 Tygon LFL tubing; Dayton, model
                number 3YU61 (available through Grainger), Jabsco Flexible Impeller Pump (Cole-
                Parmer cat. No. EW-75202-00); Simer, model number M40;> or equivalent. It is
                recommended that the pump be placed on the effluent side of the filter, when possible, to
                reduce the risk of contamination and the amount of tubing replaced or cleaned.
       6.3.4    Flow meter—SaMeCo cold water totalizer, E. Clark and Associates, Northboro, MA,
                product no. WFU 10.110; Omega flow meter, Stamford, CT, model FTB4105; or
                equivalent. Alternatively, use a graduated carboy(s) (See Section 6.18)
54    Equipment for spiking samples in the laboratory
       6.4.1    Collapsible 10-L LDPE cubitainer with cubitainer spigot—Cole Farmer cat. no. U-
                06100-30 or equivalent and Cole Farmer cat. no. U-06061-01, or equivalent. Discard
                after one use to eliminate possible contamination. Alternatively, use clean, 10-L carboy
                                                                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                with bottom delivery port ('A"), Cole-Palmer cat. no. 06080-42, or equivalent; calibrate
                to 10.0 L and mark level with waterproof marker
       6.4.2    Stir bar—Fisher cat. no. 14-513-66, or equivalent
       6.4.3    Stir plate—Fisher cat. no. 11-510-49S, S50461HP, or equivalent
       6.4.4    Hemacytometer—Neubauer type, Hausser Scientific, Horsham, PA, product no. 3200 or
                1475, or equivalent
       6.4.5    Hemacytometer coverslip—Hausser Scientific, product no. 5000 (for hemacytometer cat.
                no. 3200) or 1461 (for hemacytometer cat. no 1475), or equivalent
       6.4.6    Lens paper without silicone—Fisher cat. no. 11-995, or equivalent
       6.4.7    Polystyrene or polypropylene conical tubes with screw caps—15- and 50-mL
       6.4.8    Equipment required for enumeration of spiking suspensions using membrane filters
                6.4.8.1       Glass microanalysis filter holder—25-mm-diameter, with fritted glass
                             support, Fisher cat. no. 09-753E, or equivalent. Replace stopper with size
                             8, one-hole rubber stopper, Fisher Cat. No. 14-135M, or equivalent.
                6.4.8.2       Three-port vacuum filtration manifold and vacuum source—Fisher Cat.
                             No. 09-753-39A, or equivalent
                6.4.8.3       Cellulose acetate support membrane—1.2-um-pore-size, 25-mm-
                             diameter, Fisher cat. no. A12SP02500, or equivalent
                6.4.8.4       Polycarbonate track-etch hydrophilic membrane filter—1 -um-pore-size,
                             25-mm-diameter, Fisher cat. no. K10CP02500, or equivalent
                6.4.8.5       100 x 15 mm  polystyrene petri dishes (bottoms only)
                6.4.8.6       60 x  15 mm polystyrene petri dishes
                6.4.8.7       Glass microscope slides—1  in. x 3 in or 2 in. x 3 hi.
                6.4.8.8       Coverslips—25 mm2
g 5    Immunomagnetic separation (IMS) apparatus
       6.5.1    Sample mixer—Dynal Inc., Lake Success, NY, cat. no. 947.01, or equivalent
       6.5.2    Magnetic particle concentrator for 10-mL test tubes—Dynal MPC®-1 , cat. no. 120.01 or
                MPC®-6, cat. No 120.02, or  equivalent
       6.5.3    Magnetic particle concentrator for microcentrifuge tubes—Dynal MPC®-M, cat. no.
                120.09 (no longer available); Dynal MPC®-S, cat. no. 120.20, or equivalent
       6.5.4    Flat-sided sample tubes—16  x 125 mm Leighton-type tubes with 60 x 10 mm flat-sided
                magnetic capture area, Dynal L10, cat. no. 740.03, or equivalent
g g    Powder-free latex gloves—Fisher cat no.  113945B, or equivalent
g_7    Graduated cylinders, autoclavable—10-, 100-, and 1000-mL
g g    Centrifuges
       6.8.1    Centrifuge capable of accepting  15- to 250-mL conical centrifuge tubes and achieving
                1500 x G—International Equipment Company, Needham Heights, MA, Centrifuge Size
                2, Model K with swinging bucket,  or equivalent
       6.8.2    Centrifuge tubes—Conical, graduated, 1.5-, 50-, and 250-mL
g g    Microscope
       6.9.1    Epifluorescence/differential interference contrast (DIG) with stage and ocular
                micrometers and 20X (N.A.=0.4) to 100X (N.A.=1.3) objectives—Zeiss™ Axioskop,
                Olympus™ BH, or equivalent. Hoffman Modulation Contrast optics may be equivalent.
December 2005

-------
                                                         Method 1623 - Cryptosporidium and Giardia
6.10
6.11
6.12
6.13
6.14
6.15
6.16

6.17
       6.9.2
        6.9.3
         Excitation/band-pass filters for immunofluorescence assay (FA)—Zeiss™ 487909 or
         equivalent, including, 450- to 490-nm exciter filter, 510-nm dicroic beam-splitting
         mirror, and 515- to 520-nm barrier or suppression filter
         Excitation/band-pass filters for DAPI—Filters cited below (Chroma Technology,
         Brattleboro, VT), or equivalent
Microscope
model
Zeiss™ -
Axioskop
Zeiss™ -IM35
Olympus™ BH
Olympus™ BX
Olympus™
IMT2
Fluoro-chrome
DAPI (UV)
DAPI (UV)
DAPI (UV)
Excitation
filter (nrn)
340-380
340-380
340-380
Dichroic
beam- splitting
mirror (nm)
400
400
400
Barrier or
suppression
filter (nm)
420
420
420
Filter holder
DAPI (UV)
340-380
400
420
Filter holder
DAPI (UV)
340-380
400
420
Filter holder
Chroma
catalog
number
CZ902
CZ702
11000
91002
11000
91008
11000
91003
Ancillary equipment for microscopy
6.10.1   Well slides— Spot-On well slides, Dynal cat. no. 740.04; treated, 12-mm diameter well
         slides, Meridian Diagnostics Inc., Cincinnati, OH, cat. no. R2206; or equivalent
6.10.2   Glass coverslips—22 * 50 mm
6.10.3   Nonfluorescing immersion oil—Type FF, Cargille cat. no. 16212, or equivalent
6.10.4   Micropipette, adjustable:      0- to 10-uL with 0- to 10-uL tips
                                     10- to 100-uL, with 10- to 200-uL tips
                                     100- to 1000-uL with 100- to 1000-uL tips
6.10.5   Forceps—Splinter, fine tip
6.10.6   Forceps—Blunt-end
6.10.7   Desiccant—Drierite™ Absorbent, Fisher cat. no. 07-577-1 A, or equivalent
6.10.8   Humid chamber—A tightly sealed plastic container containing damp paper towels on top
         of which the slides are placed
Pipettes—Glass or plastic
6.11.1   5-, 10-, and 25-mL
6.11.2   Pasteur, disposable
Balances
6.12.1   Analytical—Capable of weighing 0.1 mg
6.12.2   Top  loading—Capable of weighing 10 mg
pH meter
Incubator-'-Fisher Scientific Isotemp™, or equivalent
Vortex mixer—Fisons Whirlmixer,  or equivalent
Vacuum  source—Capable of maintaining 25 in. Hg, equipped with shutoff valve and vacuum
gauge
Miscellaneous labware and supplies
6.17.1   Test tubes and rack
                                                                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
       6.17.2  Flasks—Suction, Erlcnmeyer, and volumetric, various sizes
       6.17.3  Beakers—Glass or plastic, 5-, 10-, 50-, 100-, 500-, 1000-, and 2000-mL
       6.17.4  Lint-free tissues
618   10- to 15-L graduated container—Fisher cat. no. 02-961-50B, or equivalent; calibrate to 9.0, 9.5,
       10.0, 10.5, and 11.0 L and mark levels with waterproof marker
gr-jg   Filters for filter-sterilizing reagents—Sterile Acrodisc, 0.45 ^im, Pall Corporation, cat. no. 4184,
       or equivalent

7.0   Reagents and  Standards
7 •]    Reagents for adjusting pH
       7.1.1   Sodium hydroxide (NaOH)—ACS reagent grade, 6.0 N and 1.0 N in reagent water
       7.1.2   Hydrochloric acid (HC1)—ACS reagent grade, 6.0 N, 1.0 N, and 0.1 N in reagent water

       NOTE: Due to the low volumes oj pH-adjusting reagents used in this method, and the
       impact that changes in pH have on the immunofluorescence assay, the laboratory must
       purchase standards at the required normality directly from a vendor. Normality must not
       be adjusted by the laboratory.

7 2    Solvents—Acetone, glycerol, ethanol, and methanol, ACS reagent grade
7 3    Reagent water—Water in which oocysts and cysts and interfering materials and substances,
       including magnetic minerals, are not detected by this method. See Reference 20.8 (Section 9020)
       for reagent water requirements.
7 4    Reagents for eluting filters

       NOTE: Laboratories should store prepared eluting solution/or no more than 1 week or
       when noticeably turbid, whichever comes sooner.

       7.4.1   Reagents for eluting Envirochek™ and Envirochek™ HV sampling capsules (Section
               6.2.2)
               7.4.1.1       Laureth-12—PPG Industries, Gurnee, IL, cat. no. 06194, or equivalent.
                             Store Laureth-12 as a 10% solution in reagent water. Weigh 10 g of
                             Laureth-12 and dissolve using a microwave or hot plate in 90 mL of
                             reagent water. Dispense 10-mL aliquots into sterile vials and store at
                             room temperature for up to 2 months, or in the freezer for up to a year.
               7.4.1.2       I M Tris, pH 7.4—Dissolve 121.1 g Tris (Fisher cat. no. BP152) in 700
                             mL of reagent water and adjust pH to 7.4 with 1 N HC1 or NaOH. Dilute
                             to a final 1000 mL with reagent water and adjust the final pH. Filter-
                             sterilize through a 0.2-um membrane into a sterile plastic container and
                             store at room temperature. Alternatively, use prepared TRIS, Sigma
                             T6066 or equivalent.
               7.4.1.3       0.5 M EDTA, 2 Na, pH 8.0—Dissolve 186.1 g ethylenediamine
                             tetraacetic acid, disodium salt dihydrate (Fisher cat. no. S311) in 800 mL
                             of reagent water and adjust pH to 8.0 with 6.0 N HC1 or NaOH. Dilute to
                             a final volume of 1000 mL with reagent water and adjust to pH 8.0 with
                             1.0 N HC1 or NaOH. Alternatively, use prepared EDTA, Sigma E5134 or
                             equivalent.
               7.4.1.4       Antifoam A—Sigma Chemical Co. cat. no. A5758, or equivalent
               7.4.1.5       Preparation of elution buffer solution—Add the contents of a pre-
                             prepared Laureth-12 vial (Section 7.4.1.1) to a 1000-mL graduated

December 2005                                8

-------
                                                        Method 1623 - Cryptosporidium and Giardia
                             cylinder. Rinse the vial several times-to ensure the transfer of the
                             detergent to the cylinder. Add 10 mL of Tris solution (Section 7.4.1.2), 2
                             mL of EDTA solution (Section 7.4.1.3), and 150 uL Antifoam A (Section
                             7.4.1.4). Dilute to 1000 mL with reagent water.
       7.4.2   Reagents for eluting Filta-Max® foam filters (Section 6.2.3)
               7.4.2.1       Phosphate buffered saline (PBS), pH 7.4—Sigma Chemical Co. cat. no.
                             P-3813, or equivalent. Alternately, prepare PBS by adding the following
                             to I L of reagent water: 8 g NaCl; 0.2 g KC1;  1.15 g Na,HPO4, anhydrous;
                             and 0.2 g KH2PO4.
               7.4.2.2       Tween® 20 —Sigma Chemical  Co. cat. no. P-7949, or equivalent
               7.4.2.3       High-vacuum grease—BDH/Merck. cat. no. 636082B, or equivalent
               7.4.2.4       Preparation of PBST elution buffer. Add 100 uL of Tween®  20 to
                             prepared PBS (Section 7.4.2.1). Alternatively, add the contents of one
                             packet of PBS to  1.0 L of reagent water. Dissolve by stirring  for 30
                             minutes. Add 100 uL of Tween® 20 . Mix by stirring for 5 minutes.
       7.4.3   Reagents for Portable Continuous-Flow Centrifuge (Section  6.2.4)
               7.4.3.1       Sodium dodecyl sulfate—Sigma Chemical Co. cat. no. 71730 or
                             equivalent
               7.4.3.2       TWEEN 80— Sigma Chemical  Co. cat. no. P1754 or equivalent
               7.4.3.3       Antifoam A—Sigma Chemical Co. cat. no. A5758, or equivalent
               7.4.3.4       Preparation of concentrated elution buffer. Add above reagents to obtain
                             a final concentration of 1% sodium dodecyl sulfate, 0.01% TWEEN 80,
                             and 0.001% Antifoam A in concentrated sample volume of ~250mL
7 5    Reagents for immunomagnetic separation (IMS)—Dynabeads® GC-Combo, Dynal cat. nos.
       730.02/730.12, or equivalent
7 g    Direct antibody labeling reagents for detection of oocysts and cysts. Store reagents between 1°C
       and 10°C and return promptly to this temperature after each use. Do not allow any of the reagents
       to freeze. The reagents should be protected from exposure to light. Diluted, unused working
       reagents should be discarded after 48 hours. Discard reagents after the expiration date is reached.
       The labeling reagents in Sections 7.6.1-7.6.3 have been approved for use with this method.
       7.6.1   MeriFluor® Cryptosporidium/Giardia, Meridian Diagnostics cat. no. 250050,
               Cincinnati, OH, or equivalent
       7.6.2   Aqua-Glo™ G/C Direct FL, Waterborne cat. no. AlOOFLR, New Orleans, LA, or
               equivalent
       7.6.3   Crypt-a-Glo™ and Giardi-a-Glo™, Waterborne cat. nos. A400FLR and A300FLR,
               respectively, New Orleans, LA, or equivalent
       7.6.4   EasyStain™C&G, BTF Pty Limited, Sydney, Australia or equivalent

       NOTE: If a laboratory will use multiple types of labeling reagents, the laboratory must
       demonstrate acceptable performance through an initial precision and recovery test
       (Section 9.4) for each type, and must perform positive and negative staining controls for
       each batch of slides stained using each product. However, the laboratory is not required
       to analyze additional ongoing precision and recovery samples or method blank samples
       for each type. The performance of each labeling reagent used also should be monitored in
       each source water type.

       7.6.5   Diluent for labeling reagents—Phosphate buffered saline (PBS) (Section 7.4.2).
                                                                                December 2005

-------
Method 1623 - Cryptosporidium and Giardia
7 7    4',6-diamidino-2-phenylindole (DAPI) stain—Sigma Chemical Co. cat. no. D9542, or equivalent
       7.7.1   Stock solution—Dissolve 2 mg/mL DAPI in absolute methanol. Prepare volume
               consistent with minimum use. Store between l°C and 10°C in the dark. Do not allow to
               freeze. Discard unused solution when positive staining control fails or after specified
               time determined by laboratory.
       7.7.2   Staining solution—Follow antibody kit manufacturer's instructions. Add 10 uL of 2
               mg/mL DAPI stock solution to 50 mL of PBS for use with Aqua-Glo™ G/C Direct FL or
               MeriFluor® Cryptosporidium/Giardia.  Add 50 uL of 2 mg/mL DAPI stock solution to
               50 mL of PBS for use with EasyStain™. Prepare working solution daily and store
               between 1°C and 10°C (do not allow to freeze). DAPI is light sensitive; therefore, store
               in the dark except when staining. The DAPI concentration may be increased if
               fading/diffusion of DAPI staining is encountered, but the staining solution must be tested
               first on expendable environmental samples to confirm that staining intensity is
               appropriate.
78    Mounting medium
       7.8.1   DABCO/glycerol mounting medium (2%)—Dissolve 2 g of DABCO (Sigma Chemical
               Co. cat no. D-2522, or equivalent) in 95 mL of warm glycerol/PBS (60% glycerol, 40%
               PBS). After the DABCO has dissolved completely, adjust the solution volume to  100 mL
               by adding an appropriate volume of glycerol/PBS solution. Alternately, dissolve the
               DABCO in 40 mL of PBS, then add azide (1 mL of 100X, or 10% solution), then 60 mL
               of glycerol.
       7.8.2   Mounting medium supplied with MeriFluor® Cryptosporidium/Giardia, Meridian
               Diagnostics cat. no. 250050, or equivalent (Section 7.6.1)
       7.8.3   Mounting medium supplied with Aqua-Glo™ G/C Direct FL kit, Waterborne cat. no.
               A100FLR, cat. no. M101, or equivalent (Section 7.6.2)
       7.8.4   Mounting medium supplied with EasyStain™C&G, BTF Pty Limited or equivalent
               (Section 7.6.4)
       7.8.5   Elvanol or equivalent permanent, non-fade archiving  mounting medium

7 g    Clear fingernail polish or clear fixative, PGC Scientifics, Gaithersburg, MD, cat. no.  60-4890-00,
       or equivalent
710   Oocyst and cyst suspensions for spiking
       7.10.1  Enumerated spiking suspensions prepared by flow cytometer—not formalin fixed.
               7.10.1.1      Live, flow cytometer-sorted oocysts and cysts—Wisconsin State
                             Laboratory of Hygiene Flow Cytometry Unit ([608] 224-6260), or
                             equivalent
               7.10.1.2      Irradiated, flow cytometer-sorted oocysts and cysts—flow
                             cytometer-sorted oocysts and cysts—BTF EasySeed™
                             (contact(a),btibio.com). or equivalent
       7.10.2  Materials for manual enumeration of spiking suspensions
               7.10.2.1      Purified Cryptosporidium oocyst stock suspension for manual
                             enumeration—not formalin-fixed: Sterling Parasitology Laboratory,
                             University of Arizona, Tucson, or equivalent
               7.10.2.2      Purified Giardia cyst stock suspension for manual enumeration—not
                             formalin-fixed: Waterborne, Inc., New Orleans, LA; Hyperion Research,
                             Medicine Hat, Alberta, Canada; or equivalent
December 2005                                10

-------
                                                        Method 1623 - Cryptosporidium and Giardia
               7.10.2.3      Tween® 20 , 0.01 %—Dissolve 1.0 mL of a 10% solution of Tween® 20
                             in 1 L of reagent water
       7.10.3  Storage procedure—Store oocyst and cyst suspensions between 1°C and  10°C, until
               ready to use; do not allow to freeze
7_^   Additional reagents for enumeration of spiking suspensions using membrane filtration (Section
       11.3.6)—Sigmacote® Sigma Company Product No. SL-2, or equivalent

8.0   Sample Collection and Storage
8<|    Sample collection, shipment, and receipt
       8.1.1   Sample collection. Samples are collected as bulk samples and shipped to the laboratory
               on ice for processing through the entire method, or are filtered in the field and shipped to
               the laboratory on ice for processing from elution (Section 12.2.6) onward.
       8.1.2   Sample shipment Ambient water samples are dynamic environments and, depending  on
               sample constituents and environmental conditions, Cryptosporidium oocysts or Giardia
               cysts present in a sample can degrade, potentially bias ing analytical results. Samples
               should be chilled to reduce biological activity, and preserve the state of source water
               samples between collection and analysis. Samples analyzed by an off-site laboratory
               should be shipped on ice via overnight service on the day they arc collected.
       NOTE:  See transportation precautions in Section 5.5.

               8.1.2.1        If samples arc collected early in the day, chill samples by storing in a
                             refrigerator between 1°C  and 10°C or pre-icing the sample in a cooler. If
                             the sample is pre-iced before shipping, replace with fresh  ice immediately
                             before shipment.
               8.1.2.2       If samples are collected later in the day, these samples may be chilled
                             overnight in a refrigerator between l°C and 10°C. This should be
                             considered for bulk water samples that will be shipped off-site, as this
                             minimizes the potential for water samples collected during the summer to
                             melt the ice in which they are packed and arrive at the laboratory at
                             >20°C.
               8.1.2.3        If samples are shipped after collection at >20°C with no chilling, the
                             sample will not maintain  the temperature during shipment at <20°C.
               8.1.2.4       Public water systems shipping samples to off-site laboratories for analysis
                             should include in the shipping container a means for monitoring the
                             temperature of the sample during shipping to verify that the sample did
                             not freeze or exceed 20°C. Suggested approaches for monitoring sample
                             temperature during shipping are discussed in Section 8.1.4.
       8.1.3   Sample receipt. Upon receipt, the laboratory must record the sample temperature.
               Samples that were not collected the same day they were received, and that are received at
               >20°C or frozen, or samples that the laboratory has determined exceeded  >20°C or froze
               during shipment, must be rejected. After receipt, samples must be stored at the laboratory
               between 1°C and 10°C, and not frozen, until processed.
       8.1.4   Suggestions on measuring sample temperature. Given the importance of maintaining
               sample temperatures for Cryptosporidium and Giardia determination, laboratories
               performing analyses using this method must establish acceptance criteria  for receipt of
               samples transported to their laboratory. Several options are available to measure sample
               temperature upon receipt at the laboratory and, in some cases, during shipment:
               8.1.4.1       Temperature sample. One option, for filtered samples only (not for 10-L
                            bulk samples), is for the sampler to fill a small, inexpensive sample bottle

                                              11                                December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                             with water and pack this "temperature sample" next to the filtered
                             sample. The temperature of this extra sample volume is measured upon
                             receipt to estimate the temperature of the filter. Temperature sample
                             bottles are not appropriate for use with bulk samples because of the
                             potential effect that the difference in sample volume may have in
                             temperature equilibration in the sample cooler. Example product: Cole
                             Farmer cat. no. U-06252-20.
               8.1.4.2       Thermometer vial. A similar option  is to use a thermometer that is
                             securely housed in a liquid-filled vial. Unlike temperature samples, the
                             laboratory does not need to perform an additional step to monitor the
                             temperature of the vial upon receipt, but instead just needs to read the
                             thermometer. The thermometer vial is appropriate for use with filtered
                             samples not bulk samples. Example product: Eagle-Picher  Sentry
                             Temperature Vial 3TIMOCS-F or 3TR-40CS.
               8.1.4.3       {Button. Measures the sample temperature during shipment and upon
                             receipt. An iButton is a small, waterproof device that contains a
                             computer chip that can be programmed to record temperature at different
                             time intervals.  The information is then downloaded from the iButton
                             onto a computer. The iButton should be placed in a temperature sample,
                             rather than placed loose in the cooler, or attached to the sample container.
                             This option is appropriate for use with both filtered and bulk samples.
                             Information on Thermocron® iButtons is available from
                             http://www.ibutton.com/.  Distributors include http://www.pointsix.com/.
                             http://www.rdsdistributing.com. and http://www.scigiene.com/.
               8.1.4.4       Stick-on temperature strips. Another option is for the laboratory to
                             apply a stick-on temperature strip to the outside of the sample container
                             upon receipt at the laboratory. This option does not measure temperature
                             as precisely as the other options, but provides an indication of sample
                             temperature to verify that the sample temperature is acceptable. This
                             option  is appropriate for use with both filtered and bulk samples. Example
                             product: Cole Farmer cat. no. U-90316-00.
               8.1.4.5       Infrared thermometers. A final option is to measure the temperature of
                             the  surface of the sample container or filter using an infrared
                             thermometer. The thermometer is pointed at the sample, and measures the
                             temperature without coming in contact with the sample volume. This
                             option is appropriate for use with both filtered and bulk samples. Example
                             product: Cole Farmer cat. no. EW-39641-00.
               As with other laboratory equipment, all temperature  measurement devices must be
               calibrated routinely to ensure accurate measurements. See the EPA Manual for the
               Certification of Laboratories Analyzing Drinking Water (Reference 20.9) for more
               information.
8 2    Sample holding times. Samples must be processed or examined within each of the holding times
       specified in Sections 8.2.1 through 8.2.4. Sample processing should be completed as soon as
       possible by the laboratory. The laboratory should complete sample filtration, elution,
       concentration, purification,  and staining the day the sample is received wherever possible.
       However,  the laboratory is permitted to split up the sample processing steps if processing a
       sample completely in one day is not possible. If this is necessary, sample processing can be halted
       after filtration, application of the purified sample onto the slide, or staining. Table  1, in Section
       21.0 provides  a breakdown of the holding times for each set of steps. Sections 8.2.1 through 8.2.4
       provide descriptions of these holding times.
December 2005                                 12

-------
                                                         Method 1623 - Cryptosporidium and Giardia
       8.2.1    Sample collection and filtration. Sample elution must be initiated within 96 hours of
                sample collection (if shipped to the laboratory as a bulk sample) or filtration (if filtered
                in the field).
       8.2.2    Sample elution, concentration, and purification. The laboratory must complete
                elution, concentration, and purification (Sections 12.2.6 through 13.3.3.11) in one work
                day. It is critical that these steps be completed in one work day to minimize the time that
                any target organisms present in the sample sit in eluate or concentrated matrix. This
                process ends with the application of the purified sample on the slide for drying.
       8.2.3    Staining. The sample must be stained within 72 hours of application of the purified
                sample to the slide.
       8.2.4    Examination. Although immunofluorescence assay (FA) and 4',6-diamidino-2-
                phenylindole (DAPI) and differential interference contrast (DIG) microscopy
                examination and characterization should be performed immediately after staining is
                complete, laboratories have up to 168 hours (7 days) from the completion of sample
                staining to perform the examination and verification of samples. However, if
                fading/diffusion of FITC or DAPI staining is noticed, the laboratory must reduce this
                holding time. In addition the laboratory may adjust the concentration of the DAPI
                staining solution (Sections 7.7.2) so that fading/diffusion does not occur.
g 3    Spiking suspension enumeration holding times. Flow-cytometer-sorted spiking suspensions
       (Sections 7.10.1  and 11.2) used for spiked quality control (QC) samples (Section 9) must be used
       within the expiration date noted on  the suspension. Manually enumerated spiking suspensions
       must be used within 24 hours  of enumeration of the spiking suspension if the  hemacytometer
       chamber technique is used (Section 11.3.4); or within 24 hours of application of the spiking
       suspension to the slides if the  well slide or membrane filter enumeration technique is used
       (Sections 11.3.5  and 11.3.6). Oocyst and cyst suspensions must be stored between 1°C and 10°C,
       until ready to use; do not allow to freeze.

9.0   Quality Control
91    Each laboratory  that uses this  method is required to operate a formal quality assurance (QA)
       program that addresses and documents data quality, instrument and equipment maintenance and
       performance, reagent quality and performance, analyst training and certification, and records
       storage and retrieval. General  requirements and recommendations for QA and quality control
       (QC) procedures for microbiology laboratories are provided in References 20.8, 20.9, 20.10. The
       minimum analytical requirements of this program consist of an initial demonstration of laboratory
       capability (IDC) through performance of the initial precision and recovery (IPR) test (Section
       9.4), and ongoing demonstration of laboratory  capability and method performance through the
       matrix spike (MS) test (Section 9.5.1), the method blank test (Section 9.6), the ongoing precision
       and recovery (OPR) test (Section 9.7), staining controls (Section 14.1  and 15.2.1), and analyst
       verification tests (Section 10.6). Laboratory performance is compared to established performance
       criteria to determine if the results of analyses meet the performance characteristics of the method.
       9.1.1    A test of the microscope used for detection of oocysts and cysts is performed prior to
                examination of slides. This test is described in Section  10.0.
       9.1.2    In recognition of advances that are occurring in analytical technology, the laboratory is
                permitted to modify certain method procedures to improve recovery  or lower the costs of
                measurements,  provided that all required quality control (QC) tests are performed and all
                QC acceptance criteria are met. Method procedures that can be modified include front-
                end techniques, such as filtration or immunomagnetic separation (IMS). The laboratory
                is not permitted to use an alternate determinative technique to replace
                immunofluorescence assay in this method (the use of different determinative techniques
                are considered to be different methods, rather than modified version of this method).


                                               13                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
               However, the laboratory is permitted to modify the immunofluorescence assay
               procedure, provided that all required QC tests are performed (Section 9.1.2.1) and all QC
               acceptance criteria are met (see guidance on the use of multiple labeling reagents in
               Section 7.6).
       NOTE: Method modifications should be considered only to improve method
       performance, reduce cost, or reduce sample processing time. Method modifications that
       reduce cost or sample processing time, but that result in poorer method performance
       should not be used.

               9.1.2.1       Method modification validation/equivalency demonstration requirements
                             9.1.2.1.1      Method modifications at a single laboratory. Each
                                           time a modification is made to this method for use in a
                                           single laboratory, the laboratory must, at a minimum,
                                           validate the modification according to Tier 1 of EPA's
                                           performance-based measurement system (PBMS) (Table
                                           2) to demonstrate that the modification produces results
                                           equivalent  or superior to results produced by this method
                                           as written.  Briefly, each time a modification is made to
                                           this method, the  laboratory is required to demonstrate
                                           acceptable  modified method performance through the
                                           IPR test (Section 9.4). IPR results must meet the QC
                                           acceptance criteria in Tables 3 and 4 in Section 21.0, and
                                           should be comparable to previous results using the
                                           unmodified procedure. Although not required, the
                                           laboratory also should perform a matrix spike/matrix
                                           spike duplicate (MS/MSD) test to demonstrate the
                                           performance of the modified method in at least one real-
                                           world matrix before analyzing field samples using the
                                           modified method. The laboratory is required to perform
                                           MS samples using the modified method at the frequency
                                           noted in Section 9.1.8. If the modified method involves
                                           changes that cannot be adequately evaluated through
                                           these tests, additional tests may be required to
                                           demonstrate acceptability.
                             9.1.2.1.2     Method modifications for nationwide approval. If the
                                           laboratory or a manufacturer seeks EPA approval of a
                                           method modification for nationwide use, the laboratory
                                           or manufacturer  must, at a minimum, validate the
                                           modification according to Tier 2 of EPA's PBMS (Table
                                           2). Briefly, at least three laboratories must perform IPR
                                           tests (Section 9.4) and MS/MSD (Section 9.5) tests using
                                           the modified method, and all tests must meet the QC
                                           acceptance criteria specified in Tables 3 and 4 in Section
                                           21.0. Upon nationwide approval, laboratories electing to
                                           use the modified method still must demonstrate
                                           acceptable  performance in their own laboratory according
                                           to the requirements in Section 9.1.2.1.1. If the modified
                                           method involves changes that cannot be adequately
                                           evaluated through these tests, additional tests may be
                                           required to demonstrate acceptability.
               9.1.2.2       The laboratory is required to maintain records of modifications made to
                             this method. These records  include the following, at a minimum:

December 2005                                14

-------
                                                  Method 1623 - Cryptosporidium and Giardia
                       9.1.2.2.1      The names, titles, addresses, and telephone numbers of
                                     the analyst(s) who performed the analyses and
                                     modification, and of the quality control officer who
                                     witnessed and will verify the analyses and modification.
                       9.1.2.2.2      A listing of the analyte(s) measured (Cryptosporidium
                                     and Giardia).
                       9.1.2.2.3      A narrative stating reason(s) for the modification.
                       9.1.2.2.4      Results from all QC tests comparing the modified method
                                     to this method, including:
                                     (a)     IPR (Section 9.4)
                                     (b)     MS/MSD (Section 9.5)
                                     (c)     Analysis of method blanks (Section 9.6)
                       9.1.2.2.5      Data that will allow an independent reviewer to validate
                                     each determination by tracing the following processing
                                     and analysis steps leading to the final result:
                                     (a)     Sample numbers and other identifiers
                                     (b)     Source of spiking suspensions, as well as lot
                                             number and date received (Section 7.10)
                                     (c)     Spike enumeration date and time
                                     (d)     All spiking suspension enumeration counts and
                                             calculations (Section  11.0)
                                     (e)     Sample spiking dates and times
                                     (f)     Volume filtered (Section 12.2.5.2)
                                     (g)     Filtration and elution dates and times
                                     (h)     Pellet volume, resuspended concentrate volume,
                                             resuspendcd concentrate volume transferred to
                                             IMS, and all calculations required to verify the
                                             percent of concentrate examined (Section 13.2)
                                     (i)      Purification completion dates and times (Section
                                             13.3.3.11)
                                     (j)      Staining completion dates and times (Section
                                             14.10)
                                     (k)     Staining control results (Section 15.2.1)
                                     (1)      All required examination information (Section
                                             15.2.2)
                                     (m)     Examination completion dates and times (Section
                                             15.2.4)
                                     (n)     Analysis sequence/run chronology
                                     (o)     Lot numbers of elution, IMS, and staining
                                             reagents
                                     (p)     Copies of bench sheets, logbooks, and other
                                             recordings of raw data
                                     (q)     Data system outputs, and other data to link the
                                             raw data to the results reported
9.1.3   The laboratory shall spike a separate sample aliquot from the same source to monitor
        method performance. The frequency of the MS test is described in Section 9.1.8 and the
        procedures are described in Section 9.5.1.
9.1.4   Analysis of method blanks is required to demonstrate freedom from contamination. The
        frequency of the analysis of method blanks is described in Section 9.1.7 and the
        procedures and criteria for analysis of a method blank are described in Section 9.6.
                                        15                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
        9.1.5   The laboratory shall, on an ongoing basis, demonstrate through analysis of the ongoing
                precision and recovery (OPR) sample that the analysis system is in control. Frequency of
                OPR samples is described in Section 9.1.7 and the procedures are described in Section
                9.7.
        9.1.6   The laboratory shall maintain records to define the quality of data that are generated.
                Development of accuracy statements is described in Sections 9.5.1.4 and  9.7.6.
        9.1.7   The laboratory shall analyze one method blank (Section 9.6) and one OPR sample
                (Section 9.7) each week (7 day or 168 hours time period which begins with processing
                the OPR) in which samples are analyzed if 20 or fewer field samples are analyzed during
                this period. The laboratory shall analyze one laboratory blank and one OPR sample for
                every 20 samples if more than 20 samples are analyzed in a one week (7 day or 168
                hours) period.
        9.1.8   The laboratory shall analyze MS samples (Section 9.5.1) at a minimum frequency of 1
                MS sample per 20 field samples from each source analyzed. The laboratory should
                analyze an MS sample when samples are first received from a PWS for which the
                laboratory has never before analyzed samples to identify potential method performance
                issues with the matrix (Section 9.5.1; Tables 3 and 4). If an MS sample cannot be
                analyzed on the first sampling event, the first MS sample should be analyzed as soon as
                possible to identify potential method performance issues with the matrix.
92     Micropipette calibration
        9.2.1    Micropipettes must be sent to the manufacturer for calibration annually. Alternately, a
                qualified independent technician specializing  in micropipette calibration can be used, or
                the calibration can be performed by the laboratory, provided the laboratory maintains a
                detailed procedure that can be evaluated by an independent auditor. Documentation on
                the precision of the recalibrated micropipette must be obtained  from the manufacturer or
                technician.
        9.2.2   Internal and external calibration records must be kept on file in the laboratory's QA
                logbook.
        9.2.3   If a micropipette calibration problem is suspected, the laboratory shall tare an empty
                weighing boat on the analytical balance and pipette the following volumes of reagent
                water into the weigh boat using the pipette in question: 100% of the maximum
                dispensing capacity of the micropipette, 50% of the capacity, and 10% of the capacity.
                Ten replicates should be performed at each  weight. Record the weight of the water
                (assume that 1.00 mL of reagent water weighs 1.00 g) and calculate the relative standard
                deviation (RSD) for each. If the weight of the reagent water is within 1% of the desired
                weight (mL) and the RSD of the replicates at each weight is within 1%, then the pipette
                remains acceptable for use.
        9.2.4   If the weight of the reagent water is outside the acceptable limits, consult the
                manufacturer's instruction manual troubleshooting section and  repeat steps described in
                Section 9.2.3. If problems with the pipette persist, the laboratory must send the pipette to
                the manufacturer for recalibration.
9 3     Microscope adjustment and calibration —Adjust the microscope as specified in Section 10.0. All
        of the requirements in Section 10.0 must be met prior to analysis of IPRs, method blanks, OPRs,
        field samples, and MS/MSDs.
g 4     Initial precision and recovery (IPR)—To establish the ability to demonstrate control over the
        analytical system and to generate acceptable precision and recovery, the  laboratory shall perform
        the following operations:
        9.4.1    Using the spiking procedure in Section 11.4 and enumerated spiking suspensions
                (Section 7.10.1  or Section 11.3), spike, filter, elute, concentrate, separate (purify), stain,


December 2005                                 16

-------
                                                  Method 1623 - Cryptosporidium and Giardia
        and examine the four reagent water samples spiked with -100-500 oocysts and -100-500
        cysts.
        9.4.1.1       The laboratory is permitted to analyze the four spiked reagent samples on
                      the same day or on as many as four different days (provided that the
                      spiked reagent samples are analyzed consecutively), and also may use
                      different analysts and/or reagent lots for each sample (however, the
                      procedures used for all analyses must be identical). Laboratories should
                      note that the variability of four measurements performed on multiple days
                      or using multiple analysts or reagent lots may be greater than the
                      variability of measurements performed on the same day with the same
                      analysts and reagent lots. As a result, the laboratory is at a greater risk of
                      generating unacceptable IPR results if the test is performed across
                      multiple days, analysts, and /or reagent lots.
        9.4.1.2       If more than one modification will be used for filtration and/or separation
                      of samples, a separate set of IPR samples must be prepared for each
                      modification.
        9.4.1.3       The set of four IPR samples must be accompanied by analysis of an
                      acceptable method blank (Section 9.6).
9.4.2   For each organism, calculate the percent recovery (R) using the following equation:
                         N
           R=   100 x  	
        where:
               R = the percent recovery
               N = the number of oocysts or cysts counted
               T = the number of oocysts or cysts spiked

        This calculation assumes that the total volume spiked was processed and examined.
9.4.3   Using percent recovery (R) of the four analyses, calculate the mean percent recovery and
        the relative standard deviation (RSD) of the recoveries for Cryptosporidium and for
        Giardia. The RSD is the standard deviation divided by the mean, times 100.
9.4.4   Compare the mean and RSD to the corresponding method performance acceptance
        criteria for initial precision and recovery in Tables 3 and 4 in Section 21.0. If the mean
        and RSD for recovery meet the acceptance criteria, system performance is acceptable
        and analysis of blanks and samples may begin. If the mean or RSD falls outside the range
        for recovery, system performance is unacceptable. In this event, trouble-shoot the
        problem by starting at the end of the method (see guidance in Section 9.7.5), correct the
        problem and repeat the IPR test (Section 9.4.1).
9.4.5   Examine and document the IPR slides following the procedure in Section 15.0. The first
        three Cryptosporidium oocysts and first three Giardia cysts identified in each IPR
        sample must be characterized (size, shape, DAPI category, and DIC category) and
        documented on the examination form, as well as any additional comments on organisms
        appearance, if notable.
9.4.6   Using 200X to 400X magnification, more than 50% of the oocysts  or cysts must appear
        undamaged and morphologically intact; otherwise, the organisms in the spiking
        suspension may be of unacceptable quality or the analytical process may be damaging
        the organisms. If the quality of the organisms on the IPR test slides is unacceptable,
        examine the spiking suspension organisms directly (by centrifuging, if possible, to
        concentrate the organisms in a volume that can be applied directly  to a slide). If the

                                       17                                December 2005

-------
 Method 1623 - Cryptosporidium and Giardia
                unprocessed organisms appear undamaged and morphologically intact under DIG,
                determine the step or reagent that is causing damage to the organisms. Correct the
                problem (see Section 9.7.5) and repeat the IPR test.
 9 5    Matrix spike (MS) and matrix spike duplicate (MSD)
        9.5.1   Matrix spike— The laboratory shall spike and analyze a separate field sample aliquot to
                determine the effect of the matrix on the method's oocyst and cyst recovery. The MS and
                field sample must be that was collected from the same sampling location as split samples
                or as samples sequentially collected immediately after one another. The MS sample
                volume analyzed must be within 10% of the field sample volume. The MS shall be
                analyzed according to the frequency in Section 9.1.8.
                9.5.1.1       Analyze an unspiked field sample according to the procedures in Sections
                              12.0 to 15.0. Using the spiking procedure in Section 11.4 and enumerated
                              spiking suspensions (Section 7.10.1 or Section 11.3), spike, filter, elute,
                              concentrate, separate (purify), stain, and examine a second field sample
                              aliquot with a similar number of organisms as that used in the IPR or
                              OPR tests (Sections 9.4 and 9.7).
                9.5.1.2       For each organism, calculate the percent recovery (R) using the following
                              equation.

                                               Nsp - Ns
                                R=   100 x 	
                              where
                                     R is the percent recovery
                                     Nsp is the number of oocysts or cysts counted in the spiked sample
                                     Ns is the number of oocysts or cysts counted in the unspiked
                                     sample
                                     T is the true value of the oocysts or cysts spiked

                9.5.1.3       Compare the recovery for each organism with the acceptance criteria in
                              Tables 3 and 4 in Section 21.0.
        NOTE: Some sample matrices may prevent the acceptance criteria in Tables 3 and 4
       from being met. An assessment of the distribution of MS recoveries across 430 MS
        samples from 87 sites during the ICR Supplemental Surveys is provided in Table 5.

                9.5.1.4       As part of the QA program for the laboratory, method precision for
                              samples should be assessed and records maintained. After the analysis of
                              five samples, the  laboratory should calculate the mean percent recovery
                              (P) and the standard deviation of the percent recovery (sr). Express the
                              precision assessment as a percent recovery interval from P - 2 sr to P + 2
                              sr for each matrix. For example, if P =  80% and sr = 30%, the accuracy
                              interval is expressed as 20% to 140%.  The precision assessment should
                              be updated regularly across all MS samples and stratified by MS samples
                              for each source.
        9.5.2   Matrix spike duplicate—MSD analysis is required as part of Tier 2 or nationwide
                approval of a modified version of this method to demonstrate that the modified version
                of this method produces results equal or superior to results produced by the method as
                written (Section 9.1.2.1.2). At the same time the laboratory spikes and analyzes the
                second field sample aliquot in Section 9.5.1.1, the laboratory shall spike and analyze a
                third, identical field sample aliquot.

.December 2005                                 18

-------
                                                         Method 1623 - Cryptosporidium and Giardia
        NOTE: Matrix spike duplicate samples are only required for Tier 2 validation studies.
        They are recommended for Tier 1 validation, but not required.

                9.5.2.1       For each organism, calculate the percent recovery (R) using the equation
                              in Section 9.5.1.2.
                9.5.2.2       Calculate the mean of the number of oocysts or cysts in the MS and MSD
                              (Xmean) (= [MS+MSD]/2).
                9.5.2.3       Calculate the relative percent difference (RPD) of the recoveries using the
                              following equation:
                                                    N  - N
                                                    '"MS  INMSD
                                RPD =    100 x
                              where
                                     RPD is the relative percent difference
                                     NMS is the number of oocysts or cysts counted in the MS
                                     NMSD is the number of oocysts or cysts counted in the MSD
                                     Xmean is the mean number of oocysts or cysts counted in the MS
                                     and MSD
                9.5.2.4       Compare the mean MS/MSD recovery and RPD with the acceptance
                              criteria in Tables 3 and 4 in Section 21.0 for each organism.
g g     Method blank (negative control sample, laboratory blank)—Reagent water blanks are routinely
        analyzed to demonstrate freedom from contamination. Analyze the blank immediately after
        analysis of the PR test (Section 9.4) and OPR test (Section 9.7) and prior to analysis of samples
        for the week to demonstrate freedom from contamination.
        9.6.1    Filter, elute, concentrate, separate (purify), stain, and examine at least one reagent water
                method blank per week (Section 9.1.7) according to the procedures in Sections 12.0 to
                15.0. A method blank must be analyzed each week (7 day or 168 hours time period that
                begins with processing the OPR) in which samples are analyzed if 20 or fewer field
                samples are analyzed during this period. If more than 20 samples are analyzed in a week
                (7 days or 168 hours), process and analyze one reagent water method blank for every 20
                samples.
        9.6.2    Actions
                9.6.2.1       If Cryptosporidium oocysts, Giardia cysts, or potentially interfering
                             organisms or materials that may be misidentified as oocysts or cysts are
                             not found in the method blank, the method blank test is acceptable and
                             analysis of samples may proceed.
                9.6.2.2       If Cryptosporidium oocysts, Giardia cysts (as defined in Section 3), or
                             any potentially interfering organism or materials that may be
                             misidentified as oocysts or cysts are found in the method blank, the
                             method blank test is unacceptable. Any field sample in a batch associated
                             with an unacceptable method blank is assumed to be contaminated and
                             should be recollected. Analysis of additional samples is halted until the
                             source of contamination is eliminated, the method blank test is performed
                             again, and no evidence of contamination is detected.
97     Ongoing precision and recovery (OPR; positive control sample; laboratory control
        sample)—Using the spiking procedure in Section 11.4 and enumerated spiking suspensions
        (Section 7.10.1 or Section 11.3), filter, elute, concentrate, separate (purify), stain, and examine at
        least one reagent water sample spiked with -100 to 500 oocysts and -100 to 500 cysts each week

                                               19                                December 2005

-------
Method 1623 - Cryptosporidium and Giardia
       to verify all performance criteria. The laboratory must analyze one OPR sample for every 20
       samples if more than 20 samples are analyzed in a week. If multiple method variations are used,
       separate OPR samples must be prepared for each method variation. Adjustment and/or
       recalibration of the analytical system shall be performed until all performance criteria are met.
       Only after all performance criteria are met should samples be analyzed.
       9.7.1   Examine the slide from the OPR prior to analysis of samples from the same batch.
               9.7.1.1       Using 200X to 400X magnification, more than 50% of the oocysts or
                             cysts must appear undamaged and morphologically intact; otherwise, the
                             organisms in the spiking suspension may be of unacceptable quality or
                             the analytical process may be damaging the organisms. Examine the
                             spiking suspension organisms directly (by centrifuging, if possible, to
                             concentrate the organisms in a volume that can be applied directly to a
                             slide). If the organisms  appear undamaged and morphologically intact
                             under DIC, determine the step or reagent that is causing damage to the
                             organisms. Correct the problem and repeat the OPR test.
               9.7.1.2       Identify and enumerate  each organism using epifluorescence microscopy.
                             The first three Cryptosporidium oocysts and three Giardia cysts
                             identified in the OPR sample must be examined using FITC, DAPI, and
                             DIC, as per Section  15.2, and the detailed characteristics (size, shape,
                             DAPI category, and  DIC category) reported on the Cryptosporidium and
                             Giardia report form, as well as any additional comments on organism
                             appearance, if notable.
       9.7.2   For each organism, calculate the percent recovery (R) using the following equation:
                                N
                  R=   100 x  	
               where:
                      R = the percent recovery
                      N = the number of oocysts or cysts detected
                      T = the number of oocysts or cysts spiked
       9.7.3   Compare the recovery with the acceptance criteria for ongoing precision and recovery in
               Tables 3 and 4 in Section 21.0.
       9.7.4   Actions
               9.7.4.1       If the recoveries for Cryptosporidium and Giardia meet the acceptance
                             criteria, system performance is acceptable and analysis of samples may
                             proceed.
               9.7.4.2       If the recovery for Cryptosporidium or Giardia falls outside of the
                             criteria, system performance is unacceptable. Any sample in a batch
                             associated with an unacceptable OPR sample is unacceptable. Analysis of
                             additional samples is halted until the analytical system is brought under
                             control. Troubleshoot the problem using the procedures at Section 9.7.5
                             as a guide. After assessing the issue, perform another OPR test and verify
                             that Cryptosporidium and Giardia recoveries meet the acceptance
                             criteria.
       9.7.5   Troubleshooting. If an OPR sample has failed, and the cause of the failure is not known,
               the laboratory generally should identify the problem working backward in the analytical
               process from the microscopic examination to filtration.
December 2005                                 20

-------
                                                         Method 1623 - Cryptosporidium and Giardia
                9.7.5.1        Quality of spiked organisms. Examine the spiking suspension organisms
                              directly (by ccntrifuging, if possible, to concentrate the organisms in a
                              volume that can be applied directly to a slide). If the organisms appear
                              damaged under DIC, obtain fresh spiking materials. If the organisms
                              appear undamaged and morphologically intact, determined whether the
                              problem is associated with the microscope system or antibody stain
                              (Section 9.7.5.2).
                9.7.5.2        Microscope system and antibody stain:  To determine if the failure of
                              the OPR test is due to changes in the microscope or problems with the
                              antibody stain, re-examine the positive staining control (Section 15.2.1),
                              check Kohler illumination, and check the fluorescence of the fluorescein-
                              labeled monoclonal antibodies (Mabs) and 4',6-diamidino-2-phenylindole
                              (DAPI). If results are unacceptable, re-examine a previously-prepared
                              positive staining control to determine whether the problem is associated
                              with the microscope or the antibody stain.
                9.7.5.3        Separation (purification) system: To determine if the failure of the
                              OPR test is attributable to the separation system, check system
                              performance by spiking a 10-mL volume of reagent water with -100 - 500
                              oocysts and cysts and processing the sample through the IMS, staining,
                              and examination procedures in Sections 13.3 through 15.0. Recoveries
                              should be greater than 70%.
                9.7.5.4        Filtration/elution/concentration system: If the failure of the OPR test is
                              attributable to the filtration/elution/concentration system, check system
                              performance by processing spiked reagent water according to the
                              procedures in Section 12.2 through 13.2.2, and filter, stain, and examine
                              the sample concentrate according to Section 11.3.6.
       9.7.6    The laboratory should add results that pass the specifications in Section 9.7.3 to initial
                and previous ongoing data and update the QC chart to form a graphic representation of
                continued laboratory performance. The laboratory should develop a statement of
                laboratory accuracy (reagent water, raw surface water) by calculating the mean percent
                recovery (R) and the standard deviation of percent recovery (sr). Express the accuracy as
                a recovery interval from R - 2 sr to R + 2 sr. For example, if R = 95% and sr = 25%, the
                accuracy is 45% to 145%.
g g    The laboratory should periodically analyze an external QC sample, such as  a performance
       evaluation or standard reference material, when available. The laboratory also should periodically
       participate in interlaboratory comparison studies using the method.
g g    The specifications  contained in this method can be met if the analytical system is under control.
       The standards used for initial  (Section 9.4) and ongoing (Section 9.7) precision and recovery
       should be identical, so that the most precise results will be obtained. The microscope in particular
       will provide the most reproducible results if dedicated to the settings and conditions required for
       the determination of Cryptosporidium and Giardia by this method.
g •] Q   Depending on specific program requirements, field replicates may  be collected to determine the
       precision of the sampling technique, and duplicate spiked samples  may be required to determine
       the precision of the analysis.

10.0  Microscope Calibration and  Analyst  Verification
•jQ_1   In a room capable of being darkened to near-complete darkness, assemble the microscope, all
       filters, and attachments. The microscope should be placed on a solid surface free from vibration.
       Adequate workspace should be provided on either side of the microscope for taking notes and
       placement of slides and ancillary materials.

                                               21                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
102   Using the manuals provided with the microscope, all analysts must familiarize themselves with
       operation of the microscope.
10.3   Microscope adjustment and calibration (adapted from Reference 20.10)
       10.3.1   Preparations for adjustment
                10.3.1.1      The microscopy portion of this procedure depends upon proper alignment
                             and adjustment of very sophisticated optics. Without proper alignment
                             and adjustment, the microscope will not function at maximal efficiency,
                             and reliable identification and enumeration of oocysts and cysts will not
                             be possible. Consequently, it is imperative that all portions of the
                             microscope from the light sources to the oculars are properly adjusted.
                10.3.1.2      While microscopes from various vendors are configured somewhat
                             differently, they all operate on the same general physical principles.
                             Therefore, slight deviations or adjustments may be required to make the
                             procedures below work for a particular instrument.
                10.3.1.3      The sections below assume that the mercury bulb has not exceeded time
                             limits of operation, that the lamp socket is  connected to the lamp house,
                             and that the condenser is adjusted to produce Kohler illumination.
                10.3.1.4      Persons with astigmatism should always wear contact lenses or glasses
                             when using the microscope.

       CAUTION:    In  the procedures below, do not touch  the quartz portion of the mercury

       bulb with your bare fingers. Finger oils can cause rapid degradation of the  quartz and
       premature failure of the bulb.

       WARNING:   Never look at the ultraviolet (UV) light from the mercury lamp, lamp

       house, or the UV image without a barrier filter in place. UV radiation can cause serious
       eye damage.

       10.3.2   Epifluorescent mercury bulb adjustment: The purpose of this procedure is to ensure even
                field illumination. This procedure must be followed when the microscope is first used,
                when replacing bulbs, and if problems such as diminished fluorescence or uneven field
                illumination are experienced.
                10.3.2.1      Remove the diffuser lens between the lamp and microscope or swing it
                             out of the transmitted light path.
                10.3.2.2      Using a prepared microscope slide, adjust the focus so the image in the
                             oculars is sharply defined.
                10.3.2.3      Replace the slide with a business card or a piece of lens paper.
                10.3.2.4      Close the field diaphragm (iris diaphragm in the microscope base) so only
                             a small point of light is visible on the card. This dot of light indicates the
                             location of the center of the field of view.
                10.3.2.5      Mount the mercury lamp house on the microscope without the UV
                             diffuser lens in place and turn  on the mercury bulb.
                10.3.2.6      Remove the objective in  the light path from the nosepiece. A primary
                             (brighter) and secondary image (dimmer) of the mercury bulb arc should
                             appear on the card after focusing the image with the appropriate
                             adjustment.
December 2005                                 22

-------
                                                  Method 1623 - Cryptosporidium and Giardia
         10.3.2.7      Using the lamp house adjustments, adjust the primary and secondary
                      mercury bulb images so they are side by side (parallel to each other) with
                      the transmitted light dot in between them.
         10.3.2.8      Rcattach the objective to the nosepiece.
         10.3.2.9      Insert the diffuser lens into the light path between the mercury lamp
                      house and the microscope.
         10.3.2.10    Turn off the transmitted light and replace the card with a slide of
                      fluorescent material. Check the field for even fluorescent illumination.
                      Adjustment of the diffuser lens probably will be required.  Additional
                      slight adjustments as in Section 10.3.2.7 above may be required.
         10.3.2.11    Maintain a log of the number of hours the UV bulb has been used. Never
                      use the bulb for longer than it has been rated. Fifty-watt bulbs should not
                      be used longer than 100 hours;  100-watt bulbs should not be used longer
                      than 200 hours.
10.3.3   Transmitted bulb adjustment: The purpose  of this procedure is to center the filament and
         ensure even field illumination. This procedure must be followed when the bulb is
         changed.
         10.3.3.1      Remove the diffuser lens between the lamp and microscope or swing it
                      out of the transmitted light path.
         10.3.3.2      Using a prepared microscope slide and a 40X (or similar) objective,
                      adjust the focus so the image in the oculars is sharply defined.
         10.3.3.3      Without the ocular or Bertrand optics in place, view the pupil and
                      filament image at the bottom of the tube.
         10.3.3.4      Focus the lamp filament image with the appropriate adjustment on the
                      lamp house.
         10.3.3.5      Similarly, center the lamp filament image within the pupil with the
                      appropriate adjustment(s) on the lamp house.
         10.3.3.6      Insert the diffuser lens into the light path between the transmitted lamp
                      house and the microscope.
10.3.4   Adjustment of the interpupillary distance and oculars for each eye: These adjustments
         are necessary so that eye strain is reduced to a minimum, and must be made for each
         individual using the microscope. Section 10.3.4.2 assumes use of a microscope with both
         oculars adjustable; Section 10.3.4.3 assumes use of a microscope with a single adjustable
         ocular. The procedure must be followed each time an analyst uses the microscope.
         10.3.4.1      Interpupillary distance
                      10.3.4.1.1    Place a prepared slide on the microscope stage, turn on
                                    the transmitted light, and focus the specimen image using
                                    the coarse and fine adjustment knobs.
                      10.3.4.1.2    Using both hands, move the oculars closer together or
                                    farther apart until a single circle of light is observed
                                    while looking through the oculars with both eyes. Note
                                    interpupillary distance.
         10.3.4.2      Ocular adjustment for microscopes capable of viewing a photographic
                      frame through the viewing binoculars: This procedure assumes both
                      oculars are adjustable.
                      10.3.4.2.1     Place a card between the right ocular and eye keeping
                                    both eyes open. Adjust the correction (focusing) collar on


                                       23                                December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                                            the left ocular by focusing the left ocular until it reads the
                                            same as the interpupillary distance. Bring an image
                                            located in the center of the field of view into as sharp a
                                            focus as possible.
                             10.3.4.2.2     Transfer the card to between the left eye and ocular.
                                            Again keeping both eyes open, bring the same image into
                                            as sharp a focus for the right eye as possible by adjusting
                                            the ocular correction (focusing) collar at the top of the
                                            right ocular.
                10.3.4.3     Ocular adjustment for microscopes without binocular capability: This
                             procedure assumes a single focusing ocular. The following procedure
                             assumes that only the right ocular is capable of adjustment.
                             10.3.4.3.1     Place a card between the right ocular and eye keeping
                                            both eyes open. Using the fine adjustment, focus the
                                            image for the left eye to its sharpest point.
                             10.3.4.3.2     Transfer the card to between the left eye and ocular.
                                            Keeping both eyes  open, bring the image for the right eye
                                            into sharp focus by adjusting the ocular collar at the top
                                            of the ocular without touching the coarse or fine
                                            adjustment.
       10.3.5  Calibration of an ocular micrometer: This section assumes that a reticle has been
                installed in one of the oculars by a microscopy specialist and that a stage micrometer is
                available for calibrating the ocular micrometer (reticle).  Once installed, the ocular reticle
                should be left in place. The more an ocular is manipulated the greater the probability is
                for it to become contaminated with dust particles. This calibration should be done for
                each objective in use on the microscope. If there is a top lens on the microscope, the
                calibration procedure must be done for the respective objective at each top lens setting.
                The procedure must be followed when the microscope is first used and each time  the
                objective is changed.
                10.3.5.1      Place the stage micrometer on the microscope stage, turn on the
                             transmitted light, and focus the micrometer image using the coarse and
                             fine  adjustment knobs for the objective to be calibrated. Continue
                             adjusting the focus on the stage micrometer so you can distinguish
                             between the large (0.1 mm) and the small (0.01 mm) divisions.
                10.3.5.2     Adjust the stage and ocular with the micrometer so the "0" line on the
                             ocular micrometer is exactly superimposed on the "0" line on the stage
                             micrometer.
                10.3.5.3     Without changing the stage adjustment, find a point as distant as possible
                             from the two 0 lines where two other lines are exactly superimposed.
                10.3.5.4     Determine the number of ocular micrometer spaces as well as the number
                             of millimeters on the stage micrometer between the two points of
                             superimposition. For example: Suppose 48 ocular micrometer spaces
                             equal 0.6 mm.
                10.3.5.5     Calculate the number of mm/ocular micrometer space. For example:


                                            0.6mm                            0.0125mm
                                   48 ocular micrometer spaces             ocular micrometer space
December 2005                                 24

-------
                                                  Method 1623 - Cryptosporidium and Giardia
        10.3.5.6      Because most measurements of microorganisms are given in um rather
                      than mm, the value calculated above must be converted to urn by
                      multiplying it by 1000 urn/mm. For example:
                               0.0125mm
                                1,000 urn
       12.5 um
                          ocular micrometer space
                                  mm
ocular micrometer space
        10.3.5.7
Follow the procedure below for each objective. Record the information as
shown in the example below and keep the information available at the
microscope.
Item
no.
1
2
3
4
Objective
power
10X
20X
40X
100X
Description
N.A.3=
N.A.=
N.A.=
N.A.=
No. of ocular
micrometer
spaces




No. of stage
micrometer
mm1




Mm/ocular
micrometer
space2




                      11000 urn/mm
                      2(Stage micrometer length in mm * (1000 urn/mm)) + no. ocular micrometer
                      spaces
                      3N.A. refers to numerical aperature. The numerical aperature value is engraved
                      on the barrel of the objective.
10.3.6  Kohler illumination: This section assumes that Kohler illumination will be established
        for only the 100X oil DIG objective that will be used to identify internal morphological
        characteristics in Cryptosporidium oocysts and Giardia cysts. If more than one objective
        is to be used for DIC, then each time the objective is changed, Kohler illumination must
        be reestablished for the new objective lens. Previous sections have adjusted oculars and
        light sources. This section aligns and focuses the light going through the condenser
        underneath the stage at the specimen to be observed. If Kohler illumination is not
        properly established, then DIC will not work to its maximal potential. These steps need
        to become second nature and must be practiced regularly until they are  a matter of reflex
        rather than a chore. The procedure must be followed each time an analyst uses the
        microscope and each time the objective is changed.
        10.3.6.1      Place a prepared slide on the microscope stage, place oil on the slide,
                      move the 100X  oil objective into place, turn on the transmitted light, and
                      focus the specimen image using the coarse and fine adjustment knobs.
        10.3.6.2      At this point both the radiant field diaphragm in the microscope base and
                      the  aperture diaphragm in the condenser should be wide open. Now close
                      down the radiant field diaphragm in the microscope base until the lighted
                      field is reduced  to a small opening.
        10.3.6.3      Using the condenser centering screws on the front right  and left of the
                      condenser, move the small lighted portion of the field to the center of the
                      visual field.
        10.3.6.4      Now look to see whether the leaves of the iris field diaphragm are sharply
                      defined (focused) or not. If they are not sharply defined, then they can be
                      focused distinctly by changing the height of the condenser up and down
                      with the condenser focusing knob while you are looking through the
                      binoculars. Once you have accomplished the precise focusing of the
                                       25
                                                    December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                             radiant field diaphragm leaves, open the radiant field diaphragm until the
                             leaves just disappear from view.
                10.3.6.5     The aperture diaphragm of the condenser should now be adjusted to make
                             it compatible with the total numerical aperture of the optical system. This
                             is done by removing an ocular, looking into the tube at the rear focal
                             plane of the objective, and stopping down the aperture diaphragm iris
                             leaves until they are visible just inside the rear plane of the objective.
                10.3.6.6     After completing the adjustment of the aperture diaphragm in the
                             condenser, return the ocular to its tube and proceed with the adjustments
                             required to establish DIG.
104   Microscope cleaning procedure
       10.4.1   Use canned air to remove dust from the lenses,  filters, and microscope body.
       10.4.2   Use a Kimwipe-dampened with a microscope cleaning solution (MCS) (consisting of 2
                parts 90% isoproponal and 1 part acetone) to wipe down all surfaces of the microscope
                body. Dry off with a clean, dry Kimwipe.
       10.4.3   Protocol for cleaning oculars and condenser
                10.4.3.1      Use a new, clean Q-tip dampened with MCS to clean each lense. Start at
                             the center of the lens and spiral the Q-tip outward using little to no
                             pressure. Rotate the Q-tip head while spiraling to ensure a clean surface is
                             always contacting the lens.
                10.4.3.2     Repeat the procedure using a new, dry Q-tip.
                10.4.3.3     Repeat Sections 10.4.3.1  and  10.4.3.2.
                10.4.3.4     Remove the ocular and repeat the cleaning procedure on the bottom lens
                             of the  ocular.
       10.4.4   Protocol for cleaning objective lenses
                10.4.4.1      Wipe 100X oil  objective with lens paper to remove the bulk of the oil
                             from the objective.
                10.4.4.2     Hold a new Q-tip dampened with MCS at a 45° angle on the objective
                             and twirl.
                10.4.4.3     Repeat Sections 10.4.4.2 with a new, dry Q-tip.
                10.4.4.4     Repeat Sections 10.4.4.2 and  10.4.4.3.
                10.4.4.5     Clean  all objectives whether they are used or not.
       10.4.5   Protocol for cleaning light source lens and filters
                10.4.5.1      Using a Kimwipe dampened with microscope cleaning solution, wipe off
                             the surface of each lens and filter.
                10.4.5.2     Repeat the procedure using a dry Kimwipe.
                10.4.5.3     Repeat Sections 10.4.5.1  and  10.4.5.2.
       10.4.6   Protocol for cleaning microscope stage
                10.4.6.1      Using a Kimwipe dampened with microscope cleaning solution, wipe off
                             the stage and stage clip. Be sure to clean off any  residual immersion oil or
                             fingernail polish. Remove the stage clip if necessary to ensure that it is
                             thoroughly cleaned.
       10.4.7   Use 409 and a paper towel to clean the bench top surrounding the microscope.
       10.4.8   Frequency
                10.4.8.1      Perform Sections 10.4.2, 10.4.3, 10.4.4, 10.4.5 and 10.4.7 after each
December 2005                                 26

-------
                                                         Method 1623 - Cryptosporidium and Giardia
                              microscope session.
                10.4.8.2      Perform complete cleaning each week.
105   Protozoa libraries: Each laboratory is encouraged to develop libraries of photographs and
       drawings for identification of protozoa.
       10.5.1  Take color photographs of Cryptosporidium oocysts and Giardia cysts by FA, 4',6-
               diamidino-2-phcnylindole (DAP1), and DIC that the analysts (Section 22.2) determine
               are accurate (Section  15.2).
       10.5.2  Similarly, take color photographs of interfering organisms and materials by FA, DAPI,
               and DIC that the analysts believe are not Cryptosporidium oocysts or Giardia cysts.
               Quantify the size, shape, microscope settings, and other characteristics that can be used
               to differentiate oocysts and cysts from interfering debris and that will result in accurate
               identification of positive or negative  organisms.
106   Verification of analyst performance: Until standard reference materials, such as National Institute
       of Standards and Technology standard reference materials, are available that contain a reliable
       number of DAPI positive or negative oocysts and cysts, this method shall rely upon the ability of
       the analyst for identification and enumeration  of oocysts and cysts. The goal of analyst
       verification is to encourage comparison and discussion among analysts to continually refine the
       consistency of characterizations between analysts.
       10.6.1  At least monthly when microscopic examinations are being performed, the laboratory
               shall prepare a slide containing 40 to 200 oocysts and 40 to 200 cysts. More than 50% of
               the oocysts and cysts must be DAPI positive and undamaged under DIC.
       10.6.2  Each analyst shall determine the total number of oocysts and cysts detected by FITC on
               the entire slide meeting the criteria in 10.6.1. For the same 10 oocysts and 10 cysts, each
               analyst shall determine the DAPI category (DAPI negative, DAPI positive internal
               intense blue and DAPI positive number of nuclei) and the DIC category (empty,
               containing amorphous structures, or containing identifiable internal structures) of each.
               The DAPI/DIC comparisons may be performed on the slide prepared in 10.6.1,  OPR
               slide, MS slide, or a positive staining control slide.
       10.6.3  Requirements for laboratories with multiple analysts
               10.6.3.1       The total number of oocysts and cysts determined by each analyst
                              (Section 10.6.2.) must be within ±10% of each other. If the number is not
                             within this range, the analysts must identify the source of any variability
                             between analysts' examination criteria, prepare a new slide, and repeat
                             the performance verification (Sections 10.6.1 to 10.6.2). It is
                             recommended that the DAPI and DIC categorization of the same 10
                             oocysts and 10 cysts occur with all analysts at the same time, i.e. each
                             analyst determines the categorizations independently, then the differences
                             in  the DAPI and DIC categorizations among analysts are discussed  and
                             resolved, and these resolutions documented. Alternatively, organism
                             coordinates may be recorded for each analyst to locate and categorize the
                             organisms at different times. Differences among analysts must be
                             discussed and resolved.
               10.6.3.2      Document the date, name(s) of analyst(s), number of total oocysts and
                             cysts, and DAPI and DIC categories determined by the analyst(s),
                             whether the test was passed/failed and the results of attempts before the
                             test was passed.
               10.6.3.3      Only after an analyst has passed the criteria in Section 10.6.3, may
                             oocysts and cysts in QC samples and field samples be identified and
                             enumerated.
                                               27                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
       10.6.4  Laboratories with only one analyst should maintain a protozoa library (Section 10.5) and
               compare the results of the examinations performed in Sections 10.6.1 and 10.6.2 to
               photographs of oocysts and cysts and interfering organisms to verify that examination
               results are consistent with these references. These laboratories also should perform
               repetitive counts of a single verification slide for FITC. These laboratories should also
               coordinate with other laboratories to share slides and compare counts.

11.0  Oocyst and Cyst Suspension  Enumeration and Sample Spiking
•j -j -j   This method requires routine analysis of spiked QC samples to demonstrate acceptable initial and
       ongoing laboratory and method performance (initial precision and recovery samples [Section 9.4],
       matrix spike and matrix spike duplicate samples [Section 9.5], and ongoing precision and
       recovery samples [Section 9.7]). The organisms used for these samples must be enumerated to
       calculate recoveries (and precision) and monitor method performance. EPA recommends that flow
       cytometry be used for this enumeration, rather than manual techniques. Flow cytometer-sorted
       spikes generally are characterized by a relative standard deviation of <2.5%, versus greater
       variability for manual enumeration techniques (Reference 20.11). Guidance on preparing spiking
       suspensions using a flow cytometer is provided in Section 11.2. Manual enumeration procedures
       are provided in  Section 11.3. The  procedure for spiking bulk samples in the laboratory is provided
       in Section 11.4.
11 2   Flow cytometry enumeration guidelines. Although it is unlikely that many  laboratories
       performing Method 1623 will have direct access to a  flow cytometer for preparing spiking
       suspensions, flow-sorted suspensions are available from commercial vendors and other sources
       (Section 7.10.1). The information provided in Sections 11.2.1 through 11.2.4 is simply meant as a
       guideline for preparing spiking suspensions using a flow cytometer. Laboratories performing flow
       cytometry must develop and implement detailed standardized protocols for calibration and
       operation of the flow cytometer.
       11.2.1  Spiking suspensions should be prepared using unstained organisms that have not been
               formalin-fixed.
       11.2.2  Spiking suspensions should be prepared using Cryptosporidium parvum oocysts <3
               months old, and Giardia intestinalis cysts <2 weeks old.
       11.2.3  Initial calibration. Immediately before sorting spiking suspensions, an initial calibration
               of the flow cytometer should be performed by conducting 10 sequential sorts directly
               onto membranes or well  slides. The oocyst and cyst levels used for the initial calibration
               should be the same as the levels used for the spiking suspensions. Each initial calibration
               sample should be stained and manually counted microscopically and the manual counts
               used to verify the accuracy of the system. The relative standard deviation (RSD) of the
               10 counts should be  < 2.5%. If the RSD is > 2.5%, the laboratory should perform the
               initial calibration again, until the RSD of the 10 counts is < 2.5%. In addition to counting
               the organisms, the laboratory also should evaluate the quality of the organisms using
               DAPI fluorescence and DIG to confirm that  the organisms are in good condition.
       11.2.4  Ongoing calibration. When sorting the spiking suspensions for use in QC samples, the
               laboratory should perform ongoing calibration samples at a 10% frequency, at a
               minimum. The laboratory should sort the first run and every eleventh sample directly
               onto a membrane or well slide. Each ongoing calibration sample should be stained and
               manually counted microscopically and the manual counts used to verify the accuracy of
               the system. The mean of the ongoing calibration counts also should be used as the
               estimated spike dose, if the relative standard deviation (RSD) of the ongoing calibration
               counts is <  2.5%. If the RSD is > 2.5%, the  laboratory should discard the batch.
       11.2.5  Method blanks. Depending on the operation of the flow cytometer, method blanks
               should be prepared and examined at the same frequency as the ongoing calibration


December 2005                                 28

-------
                                                 Method 1623 - Cryptosporidium and Giardia
        samples (Section 11.2.4).
11.2.6  Holding time criteria. Flow-cytometer-sorted spiking suspensions (Sections 7.10.1 and
        11.2) used for spiked quality control (QC) samples (Section 9) must be used within the
        expiration date noted on the suspension. The holding time specified by the flow
        cytometry laboratory should be determined based on a holding time study.
Manual enumeration procedures. Two sets of manual enumerations are required per organism
before purified Cryptosporidium oocyst and Giardia cyst stock suspensions (Section 7.10.2)
received from suppliers can be used to spike  samples in the laboratory. First, the stock suspension
must be diluted and enumerated (Section 11.3.3) to yield a suspension at the appropriate oocyst or
cyst concentration for spiking (spiking suspension). Then, 10 aliquots of spiking suspension must
be enumerated to calculate a mean spike dose. Spiking suspensions can be enumerated using
hemacytometer chamber counting (Section 11.3.4), well slide counting (Section 11.3.5), or
membrane filter counting (Section 11.3.6).
11.3.1  Precision criteria. The relative standard deviation (RSD) of the calculated mean spike
        dose for manually enumerated spiking suspensions must be < 16% for Cryptosporidium
        and < 19% for Giardia before proceeding (these criteria are based on the pooled RSDs of
        105 manual Cryptosporidium enumerations and  104 manual Giardia enumerations
        submitted by 20 different laboratories under the EPA Protozoa Performance Evaluation
        Program).
11.3.2  Holding time criteria. Manually enumerated spiking suspensions must be used within
        24 hours of enumeration of the spiking suspension if the hemacytometer chamber
        technique is used (Section 11.3.4); or within  24 hours of application of the spiking
        suspension or membrane filter to the slides if the well slide or membrane filter
        enumeration technique is used (Sections 11.3.5 and 11.3.6).
11.3.3  Enumerating and diluting stock suspensions
        11.3.3.1      Purified, concentrated stock suspensions (Sections 7.10.2.1 and 7.10.2.2)
                      must be diluted and enumerated before the diluted suspensions are used
                      to spike samples in the laboratory. Stock suspensions should be diluted
                      with reagent water/Tween® 20 , 0.01% (Section 7.10.2.3), to a
                      concentration of 20 to 50 organisms per large hemacytometer square
                      before proceeding to Section 11.3.3.2.
        11.3.3.2      Apply a clean hemacytometer coverslip (Section 6.4.5) to the
                      hemacytometer and load the hemacytometer chamber with 10 uL of
                      vortexed suspension per chamber. If this operation has been properly
                      executed, the liquid should amply fill the entire chamber without bubbles
                      or overflowing into the surrounding moats. Repeat this step with a clean,
                      dry hemacytometer and coverslip if loading has been incorrectly
                      performed. See Section 11.3.3.13, below, for the hemacytometer cleaning
                      procedure.
        11.3.3.3      Place the hemacytometer on the microscope stage and allow the oocysts
                      or cysts to settle for 2 minutes. Do not  attempt to adjust the coverslip,
                      apply clips, or in any way disturb the chamber after it has been filled.
        11.3.3.4      Use 200X magnification.
        11.3.3.5      Move the chamber so the ruled area is  centered underneath the objective.
        11.3.3.6      Move the objective close to the coverslip while watching it from the side
                      of the microscope, rather than through  the microscope.
        11.3.3.7      Focus up from the coverslip until the hemacytometer ruling appears.
        11.3.3.8      At each of the four corners of the chamber is a I -square-mm area divided
                      into 16 squares in which organisms are to be counted (Figure 1).

                                       29                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                             Beginning with the top row of four squares, count with a hand-tally
                             counter in the directions indicated in Figure 2. Avoid counting organisms
                             twice by counting only those touching the top and left boundary lines.
                             Count each square millimeter in this fashion.
                11.3.3.9     Use the following formula to determine the number of organisms per uL
                             of suspension:

                             number of            ...        dilution         .    3        number of
                          organisms counted                   factor           mm         organisms
                           number of mm2        .              .            .  ,              .
                              counted           1mm         1            1^L            ^L

                11.3.3.10    Record the result on a hemacytometer data sheet.
                11.3.3.11    A total of six different hemacytometer chambers must be loaded, counted,
                             and averaged for each suspension to achieve optimal counting accuracy.
                11.3.3.12    Based on the hemacytometer counts, the stock suspension should be
                             diluted to a final concentration of between 8 to 12 organisms per uL;
	however, ranges as great as 5 to 15 organisms per uL can be used.	
       NOTE:  If the diluted stock suspensions (the spiking suspensions)  will be enumerated
       using hemacytometer chamber counts (Section 11.3.4) or membrane filter counts (Section
       11.3.6), then the stock suspensions should be diluted with 0.01% Tween® 20 . If the
       spiking suspensions will be enumerated using well slide counts (Section 11.3.5), then the
       stock suspensions should be diluted in reagent water.

                             To calculate the volume (in uL) of stock suspension required per uL of
                             reagent water (or reagent  water/Tween® 20 , 0.01%), use the following
                             formula:

                                                               required number of organisms
                   volume of stock suspension (uL) required =
                                                          number of organisms/ uL of stock suspension

                             If the volume is less than  10 uL, an additional  dilution of the stock
                             suspension is recommended before proceeding.
                             To calculate the dilution factor needed to achieve the required number of
                             organisms per 10 uL, use the following formula:

                                                          number of organisms required x 10uL
                              total volume (uL) =
                                                   predicted number of organisms per 10uL (8 to 12)

                             To calculate the volume of reagent water (or reagent water/Tween® 20 ,
                             0.01%) needed, use the following formula:

                   reagent water volume (uL) =   total volume (uL) -  stock  suspension volume required (uL)


                11.3.3.13    After each use, the hemacytometer and coverslip must be cleaned
                             immediately to prevent the organisms and  debris from drying on it.  Since
                             this apparatus is precisely machined, abrasives cannot be used to clean it,

December 2005                                 30

-------
                                                 Method 1623 - Cryptosporidium and Giardia
                      as they will disturb the flooding and volume relationships.
                      11.3.3.13.1    Rinse the hemacytometer and cover glass first with tap
                                     water, then 70% ethanol, and finally with acetone.
                      11.3.3.13.2    Dry and polish the hemacytometer chamber and cover
                                     glass with lens paper. Store it in a secure place.
        11.3.3.14    Several factors arc known to introduce errors into hemacytometer counts,
                      including:
                      •      Inadequate mixing of suspension before flooding the chamber
                      •      Irregular filling of the  chamber, trapped air bubbles, dust, or oil
                             on the chamber or coverslip
                      •      Total number of organisms counted is too low to provide
                             statistical confidence in the result
                      •      Error in recording tally
                      •      Calculation error; failure to consider dilution factor, or area
                             counted
                      •      Inadequate cleaning and removal of organisms from the previous
                             count
                      •      Allowing filled  chamber to sit too long, so that the chamber
                             suspension dries and concentrates.
11.3.4  Enumerating spiking suspensions using a hemacytometer chamber

NOTE: Spiking suspensions enumerated using  a hemacytometer chamber must be used
within 24 hours of enumeration.

        11.3.4.1      Vortex the tube containing the spiking suspension (diluted stock
                      suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert
                      the tube three times.
        11.3.4.2      To an appropriate-size beaker containing a stir bar, add enough spiking
                      suspension to perform all spike testing and the enumeration as described.
                      The liquid volume and beaker relationship should be such that a spinning
                      stir bar does not splash the sides of the beaker, the stir bar has unimpeded
            •          rotation, and there is enough room to draw sample from the beaker with a
                      10-uL micropipette without touching the stir bar. Cover the beaker with a
                      watch glass or petri dish to prevent evaporation between sample
                      withdrawals.
        11.3.4.3      Allow the beaker contents to stir for a minimum of 30 minutes before
                      beginning enumeration.
        11.3.4.4      While the stir bar is still spinning, remove a 10-uL aliquot and carefully
                      load one side of the hemacytometer. Count all organisms on the platform,
                      at 200X magnification using phase-contrast or darkfield microscopy. The
                      count must include the entire area under the hemacytometer, not just the
                      four outer 1-mm2 squares. Repeat this procedure nine times. This  step
                      allows confirmation of the number of organisms per 10 uL (Section
                      11.3.3.12). Based on the 10 counts, calculate the mean, standard
                      deviation, and RSD of the counts. Record the counts and the calculations
                      on a spiking suspension  enumeration form. The relative standard
                      deviation (RSD) of the calculated mean spike dose must be < 16% for
                      Cryptosporidium and < 19% for Giardia before proceeding. If the RSD is
                      unacceptable, or the mean number is outside the expected range, add

                                       31                                December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                              additional oocysts from stock suspension or dilute the contents of the
                              beaker appropriately with reagent water. Repeat the process to confirm
                              counts. Refer to Section 11.3.3.14 for factors that may introduce errors.
       11.3.5  Enumerating spiking suspensions using well slides

       NOTE:  Spiking suspensions enumerated using well slides must be used within 24 hours
       of application of the spiking suspension to the slides.

                11.3.5.1       Prepare well slides for sample  screening and label the slides.
                11.3.5.2       Vortex the tube containing the spiking suspension (diluted stock
                              suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert
                              the tube three times.
                11.3.5.3       Remove a 10-uL aliquot from the spiking suspension and apply it to the
                              center of a well.
                11.3.5.4       Before removing subsequent aliquots, cap the tube and gently invert it
                              three times to ensure that the oocysts or cysts are in suspension.
                11.3.5.5       Ten wells must be prepared and counted, and the counts averaged, to
                              sufficiently enumerate the spike dose. Air-dry the well slides. Because
                              temperature and humidity varies from laboratory to laboratory, no
                              minimum time is specified. However, the laboratory must take care to
                              ensure that the sample has dried completely before staining to prevent
                              losses during the rinse steps. A slide warmer set at 35°C to 42°C also can
                              be used.
                11.3.5.6       Positive and negative controls  must be prepared.
                              11.3.5.6.1     For the positive control, pipette 10 uL of positive antigen
                                            or 200 to 400 intact oocysts or cysts to the center of a
                                            well and distribute evenly over the well area.
                              11.3.5.6.2     For the negative control, pipette 50 uL of PBS onto the
                                            center of a well and spread it over the  well area with a
                                            pipette tip.
                              11.3.5.6.3     Air-dry the control slides.
                11.3.5.7       Follow the manufacturer's instructions  (Section 7.6) in applying the stain
                              to the slide.
                11.3.5.8       Place the  slides in a humid chamber in the dark and incubate according to
                              manufacturer's directions. The humid chamber consists of a tightly sealed
                              plastic container containing damp paper towels on top  of which the slides
                              are placed.
                11.3.5.9       Apply one drop of wash buffer (prepared according to  the manufacturer's
                              instructions [Section 7.6]) to each well. Tilt each slide on a clean paper
                              towel, long edge down. Gently aspirate the excess detection reagent from
                              below the well using a clean Pasteur pipette or absorb with a paper towel
      	or other absorbent material. Avoid disturbing the sample.	
       NOTE:  If using the MeriFluor® Cryptosporidium/'Giardia stain (Section 7.6.1), do not
       allow slides to dry completely.


                11.3.5.10     Add mounting medium (Section 7.8) to each well.
December 2005                                 32

-------
                                                  Method 1623 - Cryptosporidium and Giardia
        11.3.5.11     Apply a cover slip. Use a tissue to remove excess mounting fluid from the
                      edges of the coverslip. Seal the edges of the coverslip onto the slide using
                      clear nail polish.
        11.3.5.12     Record the date and time that staining was completed. If slides will not be
                      read immediately, store in a humid chamber in the dark between 1°C and
                      !0°C until ready for examination.
        11.3.5.13     After examination of the 10 wells, calculate the mean, standard deviation,
                      and RSD of the 10 replicates. Record the counts and the calculations on a
                      spiking suspension enumeration form. The relative standard deviation
                      (RSD) of the calculated mean spike dose must be < 16% for
                      Cryptosporidium and < 19% for Giardia before proceeding. If the RSD is
                      unacceptable, or the mean number is outside the expected range, add
                      additional oocysts from stock suspension or dilute the contents of the
                      beaker appropriately with reagent water. Repeat the process to confirm
                      counts.
11.3.6  Enumeration of spiking suspensions using membrane filters

NOTE: Spiking suspensions enumerated using membrane filters must be used within 24
hours of application of the filters to the slides.

        11.3.6.1      Precoat the glass funnels with Sigmacote® by placing the funnel in a
                      large petri dish and applying 5-mL of Sigmacoat® to the funnel opening
                      using a pipette and allowing it to run down the inside of the funnel.
                      Repeat for all funnels to be used. The pooled  Sigmacoat® may be
                      returned to the bottle for re-use. Place the funnels at 35°C or 41°C for
                      approximately 5 minutes to dry.
        11.3.6.2      Place foil around the bottoms of the 100 x 15 mm petri dishes.
        11.3.6.3      Filter-sterilize (Section 6.19) approximately 10 mL of PBS (Section
                      7.4.2.1). Dilute detection reagent (Section 7.6) as per manufacturer's
                      instructions using sterile PBS. Multiply the anticipated number of filters
                      to be stained by 100 mL  to calculate total volume of stain required.
                      Divide the total volume required by 5 to obtain the microliters of
                      antibody necessary. Subtract the volume of antibody from the total stain
                      volume to obtain the required microliters of sterile PBS to add to the
                      antibody.
        11.3.6.4      Label the tops of foil-covered, 60 * 15 mm petri dishes for 10 spiking
                      suspensions plus positive and negative staining controls and multiple
                      filter blanks controls (one negative control,  plus a blank after every five
                      sample filters to control for carry-over). Create a humid chamber by
                      laying damp paper towels on the bottom of a stain tray (the inverted foil-
                      lined petri dishes will protect filters from light and prevent evaporation
                      during incubation).
        11.3.6.5      Place a decontaminated and cleaned filter holder base (Section 6.4.8.1)
                      into each of the three ports of the vacuum manifold (Section 6.4.8.2).
        11.3.6.6      Pour approximately 10 mL of 0.01% Tween® 20 into a 60 x 15 mm petri
                      dish.
        11.3.6.7      Using forceps, moisten a 1.2-um cellulose-acetate support membrane
                      (Section 6.4.8.3) in the 0.01% Tween® 20  and place it on the fritted
                      glass support of one of the filter bases. Moisten a polycarbonate filter
                      (Section 6.4.8.4)  the same way and position it on top of the cellulose-


                                       33                                December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                              acetate support membrane. Carefully clamp the glass funnel to the loaded
                              filter support. Repeat for the other two filters.
                11.3.6.8      Add 5 mL of 0.01% Tween® 20 to each of the three filtration units and
                              allow to stand.
                11.3.6.9      Vortex the tube containing the spiking suspension (diluted stock
                              suspension; Section 11.3.3) for a minimum of 2 minutes. Gently invert
                              the tube three times.
                11.3.6.10     Using a micropipettor, sequentially remove two, 10-uL aliquots from the
                              spiking suspension and pipet into the 5 mL of 0.01% Tween® 20
                              standing in the unit. Rinse the pipet tip twice after each addition. Apply
                              10 uL of 0.01% Tween® 20 to the third unit to serve as the negative
                              control. Apply vacuum at 2" Hg and allow liquid to drain to miniscus,
                              then close off vacuum. Pipet 10 mL of reagent water into each funnel and
                              drain to miniscus, closing off the vacuum. Repeat the rinse and drain all
                              fluid, close off the vacuum.
                11.3.6.11     Pipet 100 mL of diluted antibody to the center of the bottom of a 60 * 15
                              mm petri dish for each sample.
                11.3.6.12     Unclamp the top funnel and transfer each cellulose acetate support
                              membrane/ polycarbonate filter combination onto the drop of stain using
                              forceps (apply each membrane/filter combination to a different petri dish
                              containing stain). Roll  the filter into the drop to exclude air. Place the
                              small petri dish containing the filter onto the damp towel and cover with
                              the corresponding labeled foil-covered top. Incubate for approximately 45
                              minutes at room temperature.
                11.3.6.13     Reclamp the top funnels, apply vacuum and rinse each three times, each
                              time with 20 mL of reagent water.
                11.3.6.14     Repeat Sections 11.3.6.4 through 11.3.6.10 for the next three  samples (if
                              that the diluted spiking suspension has sat less than  15 minutes, reduce
                              the suspension vortex time to 60 seconds). Ten, 10-uL spiking suspension
                              aliquots must be prepared and counted, and the counts averaged, to
                              sufficiently enumerate  the spike dose. Include a filter blank sample at a
                              frequency of every five samples; rotate the position of filter blank to
                              eventually include all three filter placements.
                11.3.6.15     Repeat Sections 11.3.6.4 through 11.3.6.10 until the  10-uL  spiking
                              suspensions have been filtered. The last batch should include a 10-uL
                              0.01 Tween® 20 blank control and 20 uL of positive control antigen as a
                              positive staining control.
                11.3.6.16     Label slides. After incubation is complete, for each sample, transfer the
                              cellulose acetate filter support and polycarbonate filter from drop of stain
                              and place on fritted glass support. Cycle vacuum on and off briefly to
                              remove excess fluid. Peel the top polycarbonate filter off the supporting
                              filter and place on labeled slide. Discard cellulose acetate filter support.
                              Mount and apply coverslips to the filters immediately to avoid drying.
                11.3.6.17     To each slide, add 20 uL of mounting medium (Section 7.8).
                11.3.6.18     Apply a coverslip. Seal the edges of the coverslip onto the slide using
                              clear nail polish. (Sealing may be delayed until cover slips are applied to
                              all slides.)
December 2005                                 34

-------
                                                     Method 1623 - Cryptosporidium and Giardia
             11.3.6.19    Record the date and time that staining was completed. If slides will not be
                          read immediately, store sealed slides in a closed container in the dark
                          between 1°C and 10°C until ready for examination.
             11.3.6.20    After examination of the 10 slides, calculate the mean, standard
                          deviation, and RSD of the 10 replicates. Record the counts and the
                          calculations on a spiking suspension enumeration form. The relative
                          standard deviation (RSD) of the calculated mean spike dose must be
                          < 16% for Cryptosporidium and < 19% for Giardia before proceeding. If
                          the RSD is unacceptable, or the mean number is outside the expected
                          range, add additional oocysts from stock suspension or dilute the contents
                          of the beaker appropriately with reagent water. Repeat the process to
                          confirm counts.
             11.3.6.21     If oocysts or cysts are detected on the filter blanks, modify the rinse
                          procedure to ensure that no carryover occurs and repeat enumeration.
4   Procedure for spiking samples in the laboratory with enumerated spiking suspensions.
    11.4.1   Arrange a disposable cubitainer or bottom-dispensing container to feed the filter or insert
             the influent end of the tube connected to the filter through the top of a carboy to allow
             siphoning of the sample.
    11.4.2   For initial precision and  recovery (Section 9.4) and ongoing precision and recovery
             (Section 9.7) samples, fill the container with 10 L of reagent water or a volume of
             reagent water equal to the volume of the field samples analyzed in the analytical batch.
             For matrix spike samples (Section 9.5), fill the container with the field sample to be
             spiked. Continuously mix the sample (using a stir bar and stir plate for smaller-volume
             samples and alternate means for larger-volume samples).
    11.4.3   Follow the procedures in Section 11.4.3.1 or manufacturer's instructions for flow
             cytometer-enumerated suspensions and the procedures in Section 11.4.3.2 for manually
             enumerated suspensions.
             11.4.3.1      For flow cytometer-enumerated suspensions (where the entire volume of
                          a spiking suspension tube will be used):
                          11.4.3.1.1     Add 400 uL of Antifoam A to 100 mL of reagent water,
                                         and mix well to emulsify.
                          11.4.3.1.2     Add 500 uL of the diluted antifoam to the tube
                                         containing the spiking suspension and vortex  for 30
                                         seconds.
                          11.4.3.1.3     Pour the suspension into the sample container.
                          11.4.3.1.4     Add 20 mL of reagent water to the empty tube, cap,
                                         vortex 10 seconds to rinse, and add the rinsate to the
                                         carboy.
                          11.4.3.1.5     Repeat this rinse using another 20 mL of reagent water.
                          11.4.3.1.6     Record the estimated number of organisms spiked, the
                                         date and time the  sample was spiked, and the sample
                                         volume  spiked on a bench sheet. Proceed to Section
                                         11.4.4.
             11.4.3.2      For manually enumerated spiking  suspensions:
                          11.4.3.2.1      Vortex the spiking suspension(s) (Section 11.2 or Section
                                         11.3) for a minimum of 30  seconds.
                          11.4.3.2.2     Rinse a pipette tip with 0.01% Tween® 20 once, then
                                         repeatedly pipette the well-mixed spiking suspension a

                                           35                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                                           minimum of five times before withdrawing an aliquot to
                                           spike the sample.
                             11.4.3.2.3     Add the spiking suspension(s) to the carboy, delivering
                                           the aliquot below the surface of the sample.
                             11.4.3.2.4     Record the estimated number of organisms spiked, the
                                           date and time the sample was spiked, and the sample
                                           volume spiked on a bench sheet. Proceed to Section
                                           11.4.4
       11.4.4  Allow the spiked sample to mix for approximately I minute in the container.
       11.4.5  Turn on the pump and allow the flow rate to stabilize. Set flow at the rate designated for
               the filter being used. As the carboy is depleted, check the flow rate and adjust if
               necessary.
       11.4.6  When the water level approaches the discharge port of the carboy, tilt the container so
               that it is completely emptied. At that time, turn off the pump and add 1-L PBST or
               reagent water to the 10-L carboy to rinse (5 L PBST or reagent water rinse to 50-L
               carboy). Swirl the contents to rinse down the sides. Additional rinses may be performed.
       11.4.7  Turn on the pump. Allow all of the water to flow through the filter and turn off the
               pump.
       11.4.8  Proceed to filter disassembly.

12.0  Sample Filtration and Elution

-------
                                                  Method 1623 - Cryptosporidium and Giardia
                       transfer the sample water to the laboratory carboy used in Section
                       12.2.1.1. If the sample will be filtered from a field carboy, a spigot
                       (Section 6.2.1) can be used with the carboy to facilitate sample filtration.
 NOTE:  If the bulk field sample is transferred to a laboratory carboy, the laboratory
 carboy must be cleaned and disinfected before it is used with another field sample.
         12.2.4.2
         12.2.4.3
         12.2.4.4
12.2.5
12.2.6
        12.2.4.5


        12.2.4.6

        Disassembly
        12.2.5.1
         12.2.5.2


         12.2.5.3
         Elution
Place the drain end of the sampling system tubing into an empty
graduated container with a capacity of 10 to 15 L, calibrated at 9.0, 9.5,
10.0, 10.5, and 11.0 L (Section 6.18). This  container will be used to
determine the sample volume filtered. Alternately, connect a flow meter
(Section 6.3.4) downstream of the filter, and record  the initial meter
reading.
Allow the carboy discharge tube and capsule to fill with sample water by
gravity. Vent residual air using the bleed valve/vent port, gently shaking
or tapping the capsule, if necessary. Turn on the pump to start water
flowing through the filter. Verify that the flow rate is 2 L/min.
After all of the sample has passed through the filter, turn off the pump.
Allow the pressure to decrease until flow stops. (If the sample was
filtered in the field, and excess sample remains in the filter capsule upon
receipt in the laboratory, pull the remaining sample volume through the
filter before eluting the filter [Section 12.2.6].)
Turn off stir plate; add 1 L PBST or reagent water rinse (to 10-L carboy)
or 5 L PBST or reagent water rinse (to 50-L carboy). Swirl or shake the
carboy to rinse down the side walls.
Reconnect to pump, turn on pump and allow pump to pull all water
through filter; turn off pump.

Disconnect the inlet end of the capsule filter assembly while maintaining
the level of the inlet fitting above the level  of the outlet fitting to prevent
backwashing and the loss of oocysts and cysts  from  the filter. Restart the
pump and allow as much water to drain as possible.  Turn off the pump.
Based on the water level in the graduated container and Vi-L hash marks
or meter reading, record the volume filtered on the bench sheet to the
nearest quarter liter. Discard the contents of the graduated container.
Loosen the outlet fitting, then cap the inlet  and outlet fittings.
NOTE:  The laboratory must complete the elution, concentration, and purification
(Sections 12.2.6 through 13.3.3.11) in one workday. It is critical that these steps be
completed in one work day to minimize the time that any target organisms present in the
sample sit in eluate or concentrated matrix. This process  ends with the application of the
purified sample on the slide for drying.
         12.2.6.1
                      Setup
                      12.2.6.1.1
                                     Assemble the laboratory shaker with the clamps aligned
                                     vertically so that the filters will be aligned horizontally.
                                     Extend the clamp arms to their maximum distance from
                                     the horizontal shaker rods to maximize the shaking
                                     action.
                                        37
                                                                           December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                             12.2.6.1.2     Prepare sufficient quantity of elution buffer to elute all
                                            samples that are associated with the OPR/MB which used
                                            that batch of elution buffer. Elution may require up to
                                            275 mL of buffer per sample.
                             12.2.6.1.3     Designate at least one 250-mL conical centrifuge tube for
                                            each sample and label with the sample number.
                12.2.6.2     Elution
                             12.2.6.2.1     Record the elution date and time on the  bench sheet.
                                            Using a ring stand or other means, clamp each capsule in
                                            a vertical position with the inlet  end up.
                             12.2.6.2.2     Remove the inlet cap, pour elution buffer through the
                                            inlet fitting, and allow the liquid level to stabilize.
                                            Sufficient elution  buffer must be added to cover the
                                            pleated white membrane with buffer solution or elution
                                            buffer may be measured to ensure the  use of one 250-mL
                                            centrifuge tube. Replace the inlet cap.
                             12.2.6.2.3     Securely clamp the capsule in one of the clamps on the
                                            laboratory shaker  with the bleed valve positioned at the
                                            top on  a vertical axis (in the 12 o'clock position). Turn on
                                            the shaker and set the speed to maximum (approximately
                                            900 rpm or per manufacturer's instructions). Agitate the
                                            capsule for approximately 5 minutes. Time the agitation
                                            using a lab timer, rather than the timer on the shaker to
                                            ensure accurate time measurement.
                             12.2.6.2.4     Remove the filter  from  the shaker, remove the inlet cap,
                                            and pour the contents of the capsule into the 250-mL
                                            conical centrifuge tube.
                             12.2.6.2.5     Clamp the capsule vertically with the inlet end up and
                                            add sufficient volume of elution buffer through the inlet
                                            fitting to cover the pleated membrane. Replace the inlet
                                            cap.
                             12.2.6.2.6     Return the capsule to the shaker with the bleed valve
                                            positioned at the 4 o'clock position. Turn on the shaker
                                            and agitate the capsule for approximately 5 minutes.
                             12.2.6.2.7     Remove the filter  from  the shaker, but leave the elution
                                            buffer in the capsule. Re-clamp the capsule to the shaker
                                            at the 8 o'clock position. Turn on the shaker and agitate
                                            the capsule for a final 5 minutes.
                             12.2.6.2.8     Remove the filter  from  the shaker and pour the contents
                                            into the 250-mL centrifuge tube. Rinse down the inside
                                            of the capsule filter walls with reagent water or elution
                                            buffer using a squirt bottle inserted in the inlet end of the
                                            capsule. Invert the capsule filter over the centrifuge tube
                                            and ensure that as much of the eluate as possible has been
                                            transferred.
       12.2.7  Proceed to Section 13.0 for concentration and separation (purification).
123   Sample filtration using the Filta-Max® foam filter. This procedure was validated using 50-L
       sample volumes. Alternate sample volumes may be used, provided the laboratory demonstrates
December 2005                                 38

-------
                                                  Method 1623 - Cryptosporidium and Giardia
acceptable performance on initial and ongoing spiked reagent water and source water samples
(Section 9.1.2).
NOTE:  The filtration procedures specified in Sections 12.3.1.2 - 12.3.1.6.3 are specific
to laboratory filtration of a bulk sample. These procedures may require modification if
samples will be filtered in the field.

12.3.1   Filtration
         12.3.1.1      Flow rate adjustment
                       12.3.1.1.1     Connect the sampling system, minus the filter housing, to
                                     a carboy filled with reagent water.
                       12.3.1.1.2    Place the peristaltic pump upstream of the filter housing.
                       12.3.1.1.3    Turn on the pump and adjust the flow rate to 1 to 4 L per
                                     minute.
NOTE:  A head pressure of 0.5 bar (7.5 psi) is required to create flow through the filter,
and the recommended pressure of 5 bar (75 psi) should produce the flow rate of 3 to 4 L
per minute.  The maximum operating pressure of 8 bar (120 psi) should not be exceeded.

                      12.3.1.1.4    Allow 2 to  10 L of reagent water to flush the system.
                                     Adjust the pump speed as necessary during this period.
                                     Turn off the pump when the flow rate has been adjusted.
         12.3.1.2      Place filter module into the filter housing bolt head down and secure lid,
                      hand tighten housings, apply gentle pressure to create the seal between
                      the module and the 'O' rings in the base and the lid of the housing.
                      Excessive tightening is not necessary, and may shorten the life of the 'O'
                      rings. Tools may be used to tighten housing to the alignment marks (refer
                      to manufacturer's instructions). 'O' rings should be lightly greased before
                      use (refer to manufacturer's instructions).
         12.3.1.3      Install the filter housing in the line, securing the inlet and outlet ends with
                      the appropriate clamps/fittings. Verify that the filter housing is installed
                      so that the end closest to the screw top cap is the inlet and the opposite
                      end is the outlet.
         12.3.1.4      Record the sample number, sample turbidity (if not provided with the
                      field sample), and the name of the analyst filtering the sample on a bench
                      sheet.
         12.3.1.5      Filtration
                      12.3.1.5.1     Connect the sampling system to the field carboy of
                                     sample water, or transfer the  sample water to the
                                     laboratory carboy used in Section 12.3.1.1.1. If the
                                     sample will be filtered from a field carboy, a spigot can
                                     be used with the carboy to facilitate sample filtration.
NOTE:  If the bulk field sample is transferred to a laboratory carboy, the laboratory
carboy must be cleaned and disinfected before it is used with another field sample.

                      12.3.1.5.2     Place the drain end of the sampling system tubing into an
                                     empty graduated container with a capacity greater than or
                                     equal to the volume to be filtered. This container will be
                                     used to determine the sample volume filtered.
                                     Alternately, connect a flow meter downstream  of the
                                     filter, and record the initial meter reading.

                                        39                                December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                             12.3.1.5.3     Allow the carboy discharge tube and filter housing to fill
                                            with sample water. Turn on the pump to start water
                                            flowing through the filter. Verify that the  flow rate is
                                            between 1 and 4 L per min.
                             12.3.1.5.4     After all of the sample has passed through the filter, turn
                                            off the pump. Allow the pressure to decrease until flow
                                            stops.
                12.3.1.6     Disassembly
                             12.3.1.6.1     Disconnect the inlet end of the filter housing assembly
                                            while maintaining the level of the inlet fitting above the
                                            level of the outlet fitting to prevent backwashing and the
                                            loss of oocysts and cysts from the filter. Restart the pump
                                            and allow as much water to drain as possible. Turn off
                                            the pump.
                             12.3.1.6.2     Based on the water level in the graduated  container or the
                                            meter reading, record the volume filtered  on a bench
                                            sheet to the nearest quarter liter.
                             12.3.1.6.3     Loosen the  outlet fitting, the filter housing should be
                                            sealed with rubber plugs.
       NOTE:  Filters should be prevented from drying out, as this can impair  their ability to
       expand when decompressed.

       12.3.2  Elution
                12.3.2.1      The filter is eluted to wash the oocysts from the filter. This can be
                             accomplished using the Filta-Max® wash station, which moves a plunger
                             up and down a tube  containing the filter and eluting solution (Section
                             12.3.2.2), or a stomacher, which uses paddles to agitate the stomacher bag
                             containing the foam filter in the eluting solution (Section  12.3.2.3). If the
                             Filta-Max® automatic wash station is used please see the  manufacturer's
                             operator's guide for instructions on its use. If Filta-Max® Quick Connect
                             kit is used please follow manufacturer's instructions.
                12.3.2.2     Filta-Max® wash station elution procedure
                             12.3.2.2.1     First wash

                                            (a)  Detach the removable plunger head using the tool
                                                provided, and remove the splash guard.
                                            (b)  Place the filter membrane flat in the concentrator
                                                base with the rough side up.  Locate the concentrator
                                                base in the jaws of the wash  station and screw on the
                                                concentrator tube (the longer of the two tubes),
                                                creating a tight seal at the membrane. Take the
                                                assembled concentrator out of the jaws and place on
                                                the bench.
                                            (c)  Replace the splash guard and temporarily secure it at
                                                least 15 cm above the  end of the rack. Secure the
                                                plunger head with the tool provided ensuring that the
                                                lever is fully locked down.
                                            (d)  Remove the filter module from the filter housing or
                                                transportation container. Pour excess  liquid into  the
                                                assembled concentrator, then rinse the housing or

December 2005                                 40

-------
                                                  Method 1623 - Cryptosporidium and Giardia
                                        container with PBST and add the rinse to the
                                        concentrator tube. Screw the filter module onto the
                                        base of the plunger. Locate the elution tube base in
                                        the jaws of the wash station and  screw the elution
                                        tube (the shorter of the two tubes) firmly in place.
                                     (e) Pull the plunger down until the filter module sits at
                                        the bottom of the elution tube; the locking pin (at the
                                        top left of the wash station) should "click" to lock  the
                                        plunger in position.
                                     (f) Remove the filter module bolt by turning the adapted
                                        alien key (provided) in a clockwise direction (as seen
                                        from above). Attach the steel tube to the elution tube
                                        base.
                                     (g) Add 600 mL of PBST to the assembled concentrator.
                                        If more than 50 mL of liquid has been recovered from
                                        the shipped filter module, reduce the volume of
                                        PBST accordingly. Screw the concentrator tube onto
                                        the base beneath the elution tube. Release the locking
                                        pin.
NOTE:  Gentle pressure on the lever, coupled with a pulling action on the locking pin
should enable the pin to be easily released.


                                     (h) Wash the foam disks by moving the plunger up and
                                        down 20 times. Gentle movements of the plunger are
                                        recommended to avoid generating excess foam.

NOTE:  The plunger has an upper movement limit during the wash process to prevent it
popping out of the top of the chamber.

                                     (i) Detach the concentrator and hold it such that the
                                        stainless steel tube is just above the level of the
                                        liquid. Purge the remaining liquid from the elution
                                        tube by  moving the plunger up and down 5 times,
                                        then lock the plunger in place. To prevent drips,
                                        place the plug provided in the end of the steel tube.
                                     (j) Prior to  the second wash the eluate from the first
                                        wash can be concentrated using the Filta-Max®
                                        apparatus according to Section  12.3.3.2.1 or the
                                        eluate can be decanted into a 2-L pooling beaker and
                                        set aside.
                      12.3.2.2.2     Second wash

                                     (a) Add an additional 600 mL of PBST to the
                                        concentrator module, remove the plug from the end
                                        of the steel tube and screw the concentrator tube back
                                        onto the elution module base. Release the locking
                                        pin.
                                     (b) Wash the foam disks by moving the plunger up and
                                        down 10 times. Gentle movements of the plunger are
                                        recommended to avoid generating excess foam.
                                       41                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                                            (c) The eluate can be concentrated using the Filta-Max®
                                               apparatus according to Section 12.3.3.2.2 or the
                                               eluate can be decanted into the 2-L pooling beaker
                                               containing the eluate from the first wash and
                                               concentrated using centrifugation, as described in
                                               Section 12.3.3.3.
                12.3.2.3     Stomacher elution procedure
                             12.3.2.3.1     First wash

                                            (a) Place the filter module in the stomacher bag then use
                                               the alien key to remove the bolt from the filter
                                               module, allowing the rings to expand. Remove the
                                               end caps from the stomacher bag and rinse with
                                               PBST into the stomacher bag.
                                            (b) Add 600 mL of PBST to stomacher bag containing
                                               the filter pads. Place bag in stomacher and wash for 5
                                               minutes on a normal setting.
                                            (c) Remove the  bag from the stomacher and decant the
                                               eluate into a 2-L pooling beaker.
                             12.3.2.3.2     Second wash

                                            (a) Add a second 600-mL aliquot of PBST to the
                                               stomacher bag. Place bag in stomacher and wash for
                                               5 minutes on a normal setting. Remove the bag from
                                               the stomacher and decant the eluate from the
                                               stomacher bag into the 2-L pooling beaker. Wring the
                                               stomacher bag by hand to remove eluate from the
                                               foam filter and add to the pooling beaker. Remove
                                               the foam filter from the bag and using a squirt bottle,
                                               rinse the stomacher bag with reagent water and add
                                               the rinse to the pooling beaker.
                                            (b) Proceed to concentration (Section 12.3.3).
        12.3.3  Concentration
                12.3.3.1      The eluate can be concentrated using the Filta-Max® concentrator
                             apparatus, which pulls most of the eluate through a membrane filter
                             leaving the oocysts concentrated in a small volume of the remaining
                             eluting solution (Section 12.3..2), or by directly centrifuging all of the
                             eluting solution used to wash the filter (Section 12.3.2.3).
                12.3.3.2     The Filta-Max® concentrator procedure
                             12.3.3.2.1     Concentration of first wash
                                            (a) If the stomacher was used to elute the sample
                                               (Section 12.3.2.3), transfer 600 mL of eluate from the
                                               pooling beaker to the concentrator tube. Otherwise
                                               proceed to Step (b).
                                            (b) Stand the concentrator tube on a magnetic stirring
                                               plate and attach the lid (with magnetic stirrer bar).
                                               Connect the  waste bottle trap and  hand or electric
                                               vacuum pump to the valve on the concentrator base.
                                               Begin stirring and open the tap. Increase the vacuum
                                               using the hand pump.
December 2005                                42

-------
                                                 Method 1623 - Cryptosporidium and Giardia
NOTE:  The force of the vacuum should not exceed 30 cmHg.
                                    (c) Allow the liquid to drain until it is approximately
                                        level with the middle of the stirrer bar then close the
                                        valve. Remove the magnetic stirrer, and rinse it with
                                        PBST or distilled water to recover all oocysts. Decant
                                        the concentrate into a 50-mL tube, then rinse the
                                        sides of the concentration tube and add the rinsate to
                                        the 50-mL tube.
                      12.3.3.2.2    Concentration of second wash

                                    (a) If the stomacher was used to elute the sample
                                        (Section 12.3.2.3), transfer the remaining 600 mL of
                                        eluate from the pooling beaker to the concentrator
                                        tube. Otherwise proceed to Step (b).
                                    (b) Add the concentrate, in the 50-mL tube, retained
                                        from the first concentration (Section 12.3.3.2.1 (c)) to
                                        the 600 mL of eluate from the second wash, then
                                        repeat concentration steps from Sections 12.3.3.2.1
                                        (b) and 12.3.3.2.1 (c). The final sample can be poured
                                        into the same 50-mL tube used to retain the first
                                        concentrate. Rinse the sides of the concentrator tube
                                        with PBST and add  the rinse to the 50-mL tube.
                                    (c) Remove the magnetic stirrer. Insert the empty
                                        concentrator module into the jaws of the wash station
                                        and twist off the concentrator tube.
                                    (d) Transfer the membrane from the concentrator base to
                                        the bag provided using membrane forceps.
                      12.3.3.2.3    Membrane elution. The  membrane can be washed
                                    manually or using a stomacher:
                                        Manual wash. Add 5 mL of PBST to the bag
                                        containing the membrane. Rub the surface of the
                                        membrane through the bag until the membrane
                                        appears clean. Using a pipette, transfer the eluate to a
                                        50-mL tube. Repeat the membrane wash with another
                                        5 mL of PBST and transfer the eluate to the 50-mL
                                        tube. (Optional: Perform a third wash using another 5
                                        mL of PBST, by hand-kneading an additional minute
                                        or placing the bag on a flat-headed vortexer and
                                        vortexing for one minute. Transfer the eluate to the
                                        50-mL tube.)
NOTE: Mark the bag with an "X" to note which side of the membrane has the oocysts to
encourage the hand-kneading to focus on the appropriate side of the membrane.


                                        Stomacher wash. Add  5 mL of PBST to the bag
                                        containing the membrane. Place the bag containing
                                        the membrane into a small stomacher and stomach
                                        for 3 minutes. Using a pipette transfer the eluate to a
                                        50-mL tube. Repeat  the wash two times using the
                                        stomacher and 5-mL aliquots of PBST. (Optional:

                                       43                                December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                                               Perform a fourth wash using another 5 mL of PBST,
                                               by hand-kneading an additional minute or placing the
                                               bag on a flat-headed vortexer and vortexing for one
                                               minute. Transfer the eluate to the 50-mL tube.)
                             12.3.3.2.4    If the membrane filter clogs before concentration is
                                           complete, there are two possible options for completion
                                           of concentration. One option is replacing the membrane
                                           as often as necessary. Filter membranes may be placed
                                           smooth side up during the second concentration step.
                                           Another option is concentrating the remaining eluate
                                           using centrifugation. Both options are provided below.
                                           •   Using multiple membranes. Disassemble the
                                               concentrator tube and pour any remaining eluate back
                                               into the pooling beaker. Remove the membrane using
                                               membrane forceps,  placing it in the bag provided.
                                               Place a new membrane in the concentrator tube
                                               smooth side up, reassemble, return the eluate to the
                                               concentrator tube, rinse the pooling beaker and add
                                               rinse to the eluate, and continue the concentration.
                                               Replace the membrane as often as necessary.
                                               Centrifuging remaining volume.  Decant the
                                               remaining eluate into a 2-L pooling beaker.  Rinse the
                                               sides of the concentrator tube and add to the pooling
                                               beaker. Remove the filter membrane and place it in
                                               the bag provided. Wash the membrane as described
                                               in Section 12.3.3.2.3, then concentrate the sample as
                                               described in Section 12.3.3.3.1.
                12.3.3.3      If the Filta-Max® concentrator is not used for sample concentration, or if
                             the membrane filter clogs before sample concentration is complete, then
                             the procedures described in Section  12.3.3.3.1 should be used to
                             concentrate the sample. If less than 50 mL of concentrate has been
                             generated, the sample can be further concentrated, as described in Section
                             12.3.3.3.2, to reduce the volume of sample to be processed through IMS.
       NOTE:  The volume must not be reduced to less than 5 mL above the packed pellet. The
       maximum amount of pellet that should be processed through IMS is 0.5 mL.  If the packed
       pellet is greater than 0.5 mL then the pellet may be  subsampled  as described in  Section
       13.2.4.

                             12.3.3.3.1     Centrifugation of greater than 50 mL of eluate

                                           (a) Decant the eluate from the 2-L pooling beaker into
                                               250-mL conical centrifuge tubes. Make sure that the
                                               centrifuge tubes are balanced.

                                           (b) Centrifuge the 250-mL centrifuge tubes containing
                                               the eluate at 1500 x G for 15 minutes. Allow the
                                               centrifuge to coast to a stop.
                                           (c) Using a Pasteur pipette, carefully aspirate off the
                                               supernatant to 5 mL above the pellet. If the  sample is
                                               reagent water (e.g.  initial or ongoing precision and
                                               recovery sample) extra care must be taken to avoid
                                               aspirating oocysts and cysts during this step.

December 2005                                44

-------
                                                         Method 1623 - Cryptosporidium and Giardia
                                            (d) Vortex each 250-mL tube vigorously until pellet is
                                                completely resuspendcd. Swirl the centrifuge tube
                                                gently to reduce any foaming after vortexing.
                                                Combine the contents of each 250-mL centrifuge
                                                tube into a 50-mL centrifuge tube. Rinse each of the
                                                250-mL centrifuge tubes with PBST and add the
                                                rinse to the 50-mL tube.
                                            (e) Proceed to Section 12.3.3.3.2.
                              12.3.3.3.2     Centrifugation of less than 50 mL of eluate

                                            (a) Centrifuge the 50-mL centrifuge tube  containing the
                                                combined  concentrate at 1500 x G for 15 minutes.
                                                Allow the  centrifuge to coast to a stop. Record the
                                                initial pellet volume (volume of solids) and the date
                                                and time that concentration was completed on a
                                                bench sheet.
                                            (b) Proceed to Section 13.0 for concentration and
                                                separation (purification).
       12.3.4  Maintenance and cleaning
                12.3.4.1       Maintenance of O-rings
                              12.3.4.1.1     Check all rubber O-rings for wear or deterioration prior
                                            to each use and replace as necessary.
                              12.3.4.1.2     Lubricate the plunger head O-ring inside and out with
                                            silicon before  each use.
                              12.3.4.1.3     Lubricate all other O-rings (concentrator tube set, filter
                                            housing) regularly in order to preserve their condition.
                12.3.4.2       Cleaning
                              12.3.4.2.1     All components of the Filta-Max® system can be cleaned
                                            using warm water and laboratory detergent.  After
                                            washing, rinse all components with oocyst and cyst free
                                            reagent water and dry them. All O-rings should be re-
                                            lubricated. Alternatively a mild (40°C) dishwasher cycle
                                            without bleach or rinse aid can be used.
                              12.3.4.2.2     To wash the detachable plunger head slide the locking
                                            pin out and wash the plunger head and locking pin  in
                                            warm water and laboratory detergent. Rinse the plunger
                                            head and locking pin with oocyst and cyst free reagent
                                            water and dry. Lightly lubricate the locking  pin and re-
                                            assemble the plunger head.
124   Sample collection (filtration and concentration) using portable continuous-flow centrifugation.
       Please follow manufacturer's instructions. This procedure was validated for the detection of
       Cryptosporidium using 50-L sample volumes. Alternate sample volumes may be used, provided
       the laboratory demonstrates acceptable performance on initial and ongoing spiked  reagent water
       and source water samples (Section 9.1.2). Laboratories are permitted to demonstrate  acceptable
       performance for Giardia in their individual laboratory.
                                               45                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
13.0  Sample Concentration and Separation (Purification)
•|3 -j   During concentration and separation, the filter eluate is concentrated through centrifugation, and
       the oocysts and cysts in the sample are separated from other participates through immunomagnetic
       separation (IMS). Alternate procedures and products may be used if the laboratory first
       demonstrates equivalent or superior performance as per Section 9.1.2.
132   Adjustment of pellet volume
       13.2.1  Centrifuge the 250-mL centrifuge tube containing the capsule filter eluate at 1500 x G
               for 15 minutes. Allow the centrifuge to coast  to a stop—do not use the brake. Record the
               pellet volume (volume of solids) on the bench sheet.
       NOTE: Recoveries may be improved if centrifugation force is increased to 2000 x  G.
       However, do not use this higher force if the sample contains sand or other gritty material
       that may degrade the condition of any oocysts and/or cysts in the sample.

       13.2.2  Using a Pasteur pipette, carefully aspirate the supernatant to 5  mL above the pellet. Extra
               care must be taken to avoid aspirating oocysts and cysts during this step, particularly if
               the sample is reagent water (e.g. initial or ongoing precision and recovery sample).
       13.2.3  If the packed  pellet volume is < 0.5 mL, vortex the tube vigorously until pellet is
               completely resuspended. Swirl the centrifuge tube gently to reduce any foaming after
               vortexing. Record the resuspended  pellet volume on the bench sheet. Proceed to Section
               13.3.
       NOTE: Extra care must be taken with samples containing sand or other gritty material
       when vortexing to ensure that the condition of any oocysts and/or cysts  in the sample is
       not compromised.

       13.2.4  If the packed pellet volume is > 0.5 mL, the concentrate must be separated into
               multiple subsamples (a subsample is equivalent to no greater than 0.5 mL of packed
               pellet material, the recommended maximum amount of particulate material to process
               through the subsequent purification and examination steps in the method). Use the
               following formula to  determine the total volume required in the centrifuge tube before
               separating the concentrate into two or more subsamples:

                                                           pellet volume
                               total volume (mL) required =                     x 5 mL
                                                             0.5 mL

               (For example, if the packed pellet volume is 1.2 mL, the total volume required is 12 mL.)
               Add reagent water to the centrifuge tube to bring the total volume to the level calculated
	above.	
       NOTE: Extra care must be taken with samples containing sand or other gritty material
       when vortexing to ensure that the condition of any oocysts in the sample is not
       compromised.

               13.2.4.1      Analysis of entire sample. If analysis of the entire sample is required,
                             determine the number of subsamples to be processed independently
                             through the remainder of the method:
                             13.2.4.1.1     Calculate number of subsamples: Divide the total
                                           volume in the centrifuge tube by 5 mL and round up to
                                           the nearest integer (for example, if the resuspended
                                           volume in Section 13.2.4 is 12 mL, then the number of
December 2005                                46

-------
                                                        Method 1623 - Cryptosporidium and Giardia
                                            subsamples would be 12 mL / 5 mL = 2.4, rounded = 3 subsamples).
                             13.2.4.1.2     Determine volume of resuspended concentrate per
                                            subsample. Divide the total volume in the centrifuge
                                            tube by the calculated number of subsamples (for
                                            example, if the resuspended volume in Section 13.2.4 is
                                            12 mL, then the volume to use for each subsample = 12
                                            mL / 3 subsamples = 4 mL).
                             13.2.4.1.3     Process subsamples through IMS. Vortex the tube
                                            vigorously for 10 to 15 seconds to completely resuspend
                                            the pellet. Record the resuspended pellet volume on the
                                            bench sheet. Proceed immediately to Section 13.3, and
                                            transfer aliquots  of the resuspended concentrate
                                            equivalent to  the volume in the previous step to multiple,
                                            flat-sided sample tubes in Section 13.3.2.1. Process the
                                            sample as multiple, independent subsamples from Section
                                            13.3 onward,  including the preparation and examination
                                            of separate slides for each aliquot. Record the volume of
                                            resuspended concentrate transferred to IMS on the bench
                                            sheet (this will be equal to the volume recorded in
                                            Section 13.2.4). Also record the number of subsamples
                                            processed independently through the method on the
                                            bench sheet.
               13.2.4.2      Analysis of partial sample. If not all of the concentrate will be
                             examined, vortex the tube vigorously for 10 to 15 seconds to completely
                             resuspend the pellet. Record the resuspended pellet volume on the bench
                             sheet. Proceed immediately to Section 13.3, and transfer one or more 5-
                             mL aliquots of the resuspended concentrate to one or more flat-sided
                             sample tubes in  Section 13.3.2.1. Record the volume of resuspended
                             concentrate transferred to IMS on the bench  sheet. To determine the
                             volume analyzed, calculate the percent of the concentrate examined using
                             the following formula:

                                    total volume of resuspended concentrate transferred to IMS
                percent examined =                                                       x 100%
                                    total volume of resuspended concentrate in Section 13.2.4
                             Then multiply the volume filtered (Section 12.2.5.2) by this percentage to
                             determine the volume analyzed.
133   IMS procedure (adapted from Reference 20.13)

       NOTE: The IMS procedure should be performed on a bench top with all materials at
       room temperature, ranging from 15°C to 25° C.

       13.3.1  Preparation and addition of reagents
               13.3.1.1      Prepare a IX dilution of SL-buffer-A from the 10X SL-buffer-A (clear,
                             colorless solution) supplied. Use reagent water (demineralized; Section
                             7.3) as the diluent. For every I mL of IX SL-buffer-A required, mix 100
                             ^L of 10X SL-buffer-A and 0.9 mL diluent water. A volume of 1.5 mL of
                             IX SL-buffer-A will be required per sample or subsample on which the
                             Dynal IMS procedure is performed.
                                              47                                December 2005

-------
Method 1623 - Cryptosporidium and Giardia
               13.3.1.2      For each lOmL sample or subsample (Section 13.2) to be processed
                             through IMS, add 1 mL of the 10X SL-buffer-A (supplied—not the
                             diluted IX SL-buffer-A) to a flat-sided tube (Section 6.5.4).
               13.3.1.3      For each subsample, add  I mL of the 10X SL-buffer-B (supplied—
                             magenta solution) to the flat-sided tube containing the iOX SL-buffer-A.
       13.3.2  Oocyst and cyst capture
               13.3.2.1      Use a graduated, 10-mL pipette that has been pre-rinsed with elution
                             buffer to transfer the water sample concentrate from Section 13.2 to the
                             flat-sided tube(s) containing the SL-buffers. If all of the concentrate is
                             used, rinse the centrifuge tube twice with reagent water and add the
                             rinsate to the flat-sided tube containing the concentrate (or to the tube
                             containing the first subsample, if multiple subsamples will be processed).
                             Each of the two rinses should be half the volume needed to bring the total
                             volume in the flat-sided sample tube to 12 mL (including the buffers
                             added in Sections 13.3.1.2 and 13.3.1.3). (For example, if the tube
                             contained 1 mL of SL-buffer-A and 1 mL of SL-buffer-B, and 5 mL of
                             sample was transferred after resuspension of the pellet, for a total of 7
                             mL, the centrifuge tube would be rinsed twice with 2.5 mL of reagent
                             water to bring the total volume in the flat-sided tube to 12 mL.) Visually
                             inspect the centrifuge tube after  completing the transfer to ensure that no
                             concentrate remains. If multiple  subsamples will be processed, bring the
                             volume in the remaining flat-sided tubes to 12 mL with reagent water.
                             Label the flat-sided tube(s) with the sample number (and subsample
                             letters).
               13.3.2.2      Vortex the Dynabeads®Crypto-Combo vial from the IMS kit for
                             approximately 10 seconds to suspend the beads. Einsure that the beads are
                             fully resuspended by inverting the sample tube and making sure that there
                             is no residual pellet at the bottom.
               13.3.2.3      Add  100 uL of the resuspended  Dynabeads®Crypto-Combo (Section
                             13.3.2.2) to the sample tube(s) containing the water sample concentrate
                             and SL-buffers.
               13.3.2.4      Vortex the Dynabeads®Giardia-Combo vial from the IMS kit for
                             approximately 10 seconds to suspend the beads. Ensure that the beads are
                             fully resuspended by inverting the tube and making sure that there  is no
                             residual pellet at the bottom.
               13.3.2.5      Add  100 \iL of the resuspended  Dynabeads®Giardia-Combo  (Section
                             13.3.2.4) to the sample tube(s) containing the water sample concentrate,
                             Dynabeads®Crypto-Combo, and SL-buffers.
               13.3.2.6      Affix the sample tube(s) to a rotating mixer and rotate at approximately
                             18 rpm for 1 hour at room temperature.
               13.3.2.7      After rotating for 1 hour, remove each sample tube from the mixer and
                             place the tube in the magnetic particle concentrator (MPC®-1 or MPC®-
                             6) with flat side of the tube toward the magnet.
               13.3.2.8      Without removing the sample tube from the MFC®-1, place the magnet
                             side of the MPC®-1 downwards, so the tube is horizontal and the flat side
                             of the tube is facing down.
               13.3.2.9      Gently rock the  sample tube by hand end-to-end through approximately
                             90°, tilting the cap-end and base-end of the tube up and down in turn.
December 2005                                48

-------
                                                 Method 1623 - Cryptosporidium and Giardia
                      Continue the tilting action for 2 minutes with approximately one tilt per
                      second.
        13.3.2.10     Ensure that the tilting action is continued throughout this period to
                      prevent binding of low-mass, magnetic or magnetizable material. If the
                      sample in the MPC®-1 is allowed to stand motionless for more than 10
                      seconds, remove the flat-sided tube from the MFC®-1, shake the tube to
                      resuspend all material, replace the sample tube in the MPC®-1 and repeat
                      Section 13.3.2.9 before continuing to Section 13.3.2.11.
        13.3.2.11     Return the MFC®-1 to the upright position, sample tube vertical, with
                      cap at top. Immediately remove the cap and, keeping the flat side of the
                      tube on top, pour off all of the  supernatant from the tube held in the
                      MPC®-1 into a suitable container. Do not shake the tube and do not
                      remove the tube from MPC®-1 during this step. Allow more supernatant
                      to settle; aspirate additional supernatant with pipette.
        13.3.2.12     Remove the sample tube from the MPC®-1 and resuspend the sample in
                      0.5 mL IX SL-buffer-A (prepared from 10X SL-buffer-A
                      stock—supplied). Mix very gently to resuspend all material in the tube.
                      Do not vortex.
        13.3.2.13     Quantitatively transfer (transfer followed by two rinses)  all the liquid
                      from the sample tube to a labeled, 1.5-mL microcentrifuge tube. Use 0.5
                      mL of IX SL-buffer-A to perform the first rinse and 0.5  mL of IX SL-
                      buffer-A for the second rinse.  Allow the flat-sided sample tube to sit for
                      a minimum of 1 minute after transfer of the second rinse volume, then use
                      a pipette to collect any residual volume that drips down to the bottom of
                      the tube to ensure that as much sample volume is recovered as possible.
                      Ensure that all of the liquid and beads are  transferred.
        13.3.2.14     Place the microcentrifuge tube into the second magnetic  particle
                      concentrator (MPC®-M or MPC®-S), with its magnetic  strip in place.
        13.3.2.15     Without removing the microcentrifuge tube from MPC®-M, gently
                      rock/roll the tube through 180° by hand. Continue for approximately 1
                      minute with approximately one 180° roll/rock per second. At the end of
                      this step, the beads should produce a distinct brown dot at the back of the
                      tube.
        13.3.2.16     Immediately aspirate the supernatant from the tube and cap held in the
                      MPC®-M. If more than one sample is being processed, conduct three 90°
                      rock/roll actions before removing the supernatant from each tube. Take
                      care not to disturb the material attached to the wall of the tube adjacent to
                      the magnet. Do not shake the tube. Do not remove the tube from MPC®-
                      M while conducting these steps.
13.3.3  Dissociation of beads/oocyst/cyst complex

NO TE: Two acid dissociations are required.

        13.3.3.1      Remove the magnetic strip from the MPC®-M.
        13.3.3.2      Add 50 uL of 0.1 N HC1, then vortex at the highest setting for
                      approximately 50 seconds.
NOTE: The laboratory must use 0.1-N standards purchased directly from a vendor,
rather than adjusting the normality in-house.
                                       49                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                13.3.3.3      Place the tube in the MPC®-M without the magnetic strip in place and
                             allow to stand in a vertical position for at least 10 minutes at room
                             temperature.
                13.3.3.4      Vortex vigorously for approximately 30 seconds.
                13.3.3.5      Ensure that all of the sample is at the base of the tube. Place the
                             microcentrifuge tube in the MPC®-M.
                13.3.3.6      Replace magnetic strip in MPC®-M and allow the tube to stand
                             undisturbed for a minimum of 10 seconds.
                13.3.3.7      Prepare a well slide for sample screening and label the slide.
                13.3.3.8      Add 5 uL of 1.0 N NaOH to the sample wells of two well slides (add 10
                             uL to the sample well of one well slide if the volume from the two
                             required dissociations will be added to the same slide).
       NOTE:  The laboratory must use l.O-N standards purchased directly from a vendor
       rather than adjusting the normality in-house.

                13.3.3.9      Without removing the microcentrifuge tube from the MPC®-M, transfer
                             all of the sample from the microcentrifuge tube in the MPC®-M to the
                             sample well with the NaOH. Do not disturb the beads at the back wall of
                             the tube. Ensure that all of the fluid is transferred.
                13.3.3.10    Do not discard the beads or microcentrifuge tube after transferring the
                             volume from the first acid dissociation to the well slide. Perform the steps
                             in Sections 13.3.3.1 through 13.3.3.9 a second time. The volume from the
                             second dissociation can be added to  the slide containing the volume from
                             the first dissociation, or can be applied to a second slide.
       NOTE:  The wells on Dynal Spot-On slides are likely to be too small to accommodate the
       volumes from both dissociations.

                13.3.3.11    Record the date  and time the purified sample was applied to the slide(s).
                13.3.3.12    Air-dry the sample on the well slide(s). Because temperature and
                             humidity vary from laboratory to laboratory, no minimum time is
                             specified. However, the laboratory must take care to ensure  that the
                             sample has dried completely before staining to prevent losses during the
                             rinse steps. A slide warmer set at 35°C to 42°C also can be used.
       13.3.4   Tips for minimizing carry-over of debris onto microscope slides after IMS

                •  Make sure the resuspended pellet is fully homogenized before placing the tube in the
                  MPC®-1 or MPC®-M to avoid trapping "clumps" or a dirty layer between the beads
                  and the side of the tube.
                •  When using the MFC®-1 magnet, make sure that the tube is snugged flat against the
                  magnet. Push the tube flat if necessary.  Sometimes the magnet is not flush with the
                  outside of the holder and, therefore, the attraction between the beads and the magnet
                  is not as strong as it should be. However, it can be difficult to  determine this if you do
                  not have more than one MPC®-1 to make comparisons.
                •  After the supernatant has been poured off at Section 13.3.2.11, leave the tube in the
                  MPC®-1 and allow time for any supernatant remaining in the  tube to settle down to
                  the bottom. Then aspirate the settled supernatant and associated particles from the
                  bottom of the tube.  The same can be done at Section 13.3.2.16 with the
                  microcentrifuge tube.
December 2005                                50

-------
                                                         Method 1623 - Cryptosporidium and Giardia
                •  An additional rinse can also be performed at Section 13.3.2.11. After the supernatant
                   has been poured off and any settled material is aspirated off the bottom, leave the tube
                   in the MPC®-1 and add an additional 10 mL of reagent water or PBS to the tube and
                   repeat Sections 13.3.2.9 and 13.3.2.11. Although labs have reported successfully
                   using this technique to reduce carryover, because the attraction between the MPC®-1
                   and the beads is not as great as the attraction between the MPC®-M and the beads,
                   the chances would be greater for loss of cysts and oocysts doing the rinse at this step
                   instead of at Section 13.3.2.16.

                •  After the supernatant has been aspirated from the tube at Section 13.3.2.16, add 0.1
                   mL of PBS, remove the tube from the MPC®-M, and resuspend. Repeat Sections
                   13.3.2.15 and 13.3.2.16.
                •  Use a slide with the largest diameter well available to spread out the sample as much
                   as possible.

14.0  Sample Staining
        NOTE:  The sample must be stained within  72 hours of application of the purified sample
        to the slide.

14
-------
Method 1623 - Cryptosporidium and Giardia
       NOTE: If us ing the MeriFluor® Cryptosporidium/Giardia (Section 7.6.1), do not allow
       slides to dry completely.

•J4 g   Add mounting medium (Section 7.8) to each well.
149   Apply a cover slip. Use a tissue to remove excess mounting fluid from the edges of the coverslip.
       Seal the edges  of the coverslip onto the slide using clear nail polish.
14.10  Record the date and time that staining was completed on the bench sheet. If slides will not be read
       immediately, store in a humid chamber in the dark  between 1°C and  10°C until ready for
       examination.

15.0  Examination
       NOTE: Although immunofluorescence assay (FA) and 4',6-diamidino-2-phenylindole
       (DAPI) and differential interference contrast (DIC) microscopy examination should be
       performed immediately after staining is complete, laboratories have  up to 168 hours (7
       days) from completion of sample staining to complete the examination and verification of
       samples. However, if fading/diffusion of FITC or DAPI fluorescence is noticed, the
       laboratory must reduce this holding time. In addition the laboratory  may adjust the
       concentration of the DAPI staining solution (Sections 7.7.2) so that fading/diffusion does
       not occur.

15 -J   Scanning technique: Scan each well in a systematic fashion. An up-and-down or a side-to-side
       scanning pattern may be used (Figure 4).
152   Examination using immunofluorescence assay (FA), 4',6-diamidino-2-phenylindole (DAPI)
       staining characteristics, and differential interference contrast (DIC) microscopy. The minimum
       magnification requirements for each type of examination are noted below.
       NOTE: All characterization (DAPI and DIC) and size measurements must be determined
       using 1OOOX magnification and reported to the nearest 0.5 fjm.

       Record examination results for Cryptosporidium oocysts on a Cryptosporidium examination form;
       record examination results for Giardia cysts on a Giardia examination results form. All
       organisms that meet the criteria specified in Sections 15.2.2 and 15.2.3, less atypical organisms
       specifically identified as non-target organisms by DIC or DAPI (e.g.  possessing spikes, stalks,
       appendages, pores, one or two large nuclei filling the cell, red fluorescing chloroplasts, crystals,
       spores, etc),  must be reported.
       15.2.1  Positive and negative staining control. Positive and negative staining controls must be
               acceptable before proceeding with examination of field sample slides.
               15.2.1.1     Each analyst must characterize a minimum of three Cryptosporidium
                            oocysts and three Giardia  cysts on the positive staining control slide
                            before examining field sample slides. This characterization must be
                            performed by each analyst during each microscope examination session.
                            FITC examination must be conducted at a minimum of 200X total
                            magnification, DAPI examination must be conducted at a minimum of
                            400X, and DIC examination and size  measurements must be conducted at
                            a minimum of 1000X. Size, shape, and DIC  and DAPI characteristics of
                            three Cryptosporidium oocysts and three Giardia cysts must be recorded
                            by the analyst on a microscope log. The analyst also must indicate on
                            each sample examination form whether the positive staining control was
                            acceptable.
December 2005                                52

-------
                                                  Method 1623 - Cryptosporidium and Giardia
        15.2.1.2      Examine the negative staining control to confirm that it does not contain
                      any oocysts or cysts (Section 14.1). Indicate on each sample examination
                      form whether the negative staining control was acceptable.
        15.2.1.3      If the positive staining control contains oocysts and cysts within the
                      expected range and at the appropriate fluorescence for both FA and
                      DAPI, and the negative staining control does not contain  any oocysts or
                      cysts (Section  14.1), proceed to Sections 15.2.2 and 15.2.3.
15.2.2  Sample examination—Cryptosporidium
        15.2.2.1      FITC examination (the analyst must use a minimum of 200X total
                      magnification). Use epifluorescence to scan the entire well for apple-
                      green fluorescence of oocyst and cyst shapes. When brilliant apple-green
                      fluorescing ovoid or spherical objects 4 to 6 urn in diameter are observed
                      with brightly highlighted edges, increase magnification to 400X and
                      switch the microscope to the UV filter block for DAPI (Section 15.2.2.2),
                      then to DIC (Section 15.2.2.3) at 1000X.
        15.2.2.2      DAPI fluorescence examination (the analyst must use a minimum of
                      400X total magnification).  Using the UV filter block for DAPI, the
                      object will exhibit one of the following characteristics:
                      (a)  Light blue internal staining (no distinct nuclei) with a green rim
                      (b)  Intense blue internal staining
                      (c)  Up to four distinct, sky-blue nuclei

                      Look  for atypical DAPI fluorescence, e.g., more than four stained nuclei,
                      size of stained nuclei, and wall structure and color.  Record oocysts in
                      category (a) as DAPI-negative; record oocysts in categories (b) and (c) as
                      DAPI-positive.
        15.2.2.3      DIC examination (the analyst must use a minimum of 1000X total
                      magnification [oil immersion lens]). Using DIC, look for  external or
                      internal morphological characteristics atypical of Cryptosporidium
                      oocysts (e.g., spikes, stalks, appendages, pores, one or two large nuclei
                      filling the cell, red fluorescing chloroplasts, crystals, spores, etc.)
                      (adapted from Reference 20.10). If atypical structures are not  observed,
                      then categorize each apple-green fluorescing object as:
                      (a)  An empty Cryptosporidium oocyst
                      (b)  A Cryptosporidium oocyst with amorphous structure
                      (c)  A Cryptosporidium oocyst with internal structure (one to  four
                          sporozoites/oocyst)
                      Using 1000X total magnification, record the shape, measurements (to the
                      nearest 0.5 um), and number of sporozoites (if applicable) for each  apple-
                      green fluorescing object meeting the size and shape characteristics.
                      Although not a defining characteristic, surface oocyst folds may be
                      observed in some specimens.
        15.2.2.4      A positive result is a Cryptosporidium oocyst which exhibits typical IFA
                      fluorescence, typical size and shape and exhibits nothing  atypical on IFA,
                      DAPI fluorescence, or DIC microscopy.  A positive result must be
                      characterized and assigned to one of the DAPI and DIC categories in
                      Sections 15.2.2.2 and  15.2.2.3.
15.2.3  Sample examination—-Giardia
        15.2.3.1      FITC examination (the analyst must use a minimum of 200X total
                      magnification). When brilliant apple-green fluorescing round to ovoid

                                        53                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
                             objects (8-18 (im long by 5 - 15 um wide) are observed with brightly
                             highlighted edges, increase magnification to 400X and switch the
                             microscope to the UV filter block for DAPI (Section 15.2.3.2) then to
                             DIG (Section 15.2.3.3) at 1000X.
                15.2.3.2      DAPI fluorescence examination (the analyst must use a minimum of
                             400X total magnification). Using the UV filter block for DAPI, the object
                             will exhibit one or more of the following characteristics:
                             (a)  Light blue internal staining (no distinct nuclei) and a green rim
                             (b)  Intense blue internal staining
                             (c)  Two to four sky-blue nuclei

                             Look for atypical DAPI fluorescence, e.g., more than four stained nuclei,
                             size of stained nuclei, and wall structure and color. Record cysts in
                             category (a) as DAPI negative; record cysts in categories (b) and (c) as
                             DAPI positive.
                15.2.3.3      DIG examination (the analyst must use a minimum of 1000X total
                             magnification [oil immersion lens]). Using DIC microscopy, look for
                             external or internal morphological characteristics atypical  of Giardia
                             cysts (e.g., spikes, stalks, appendages, pores, one or two large nuclei
                             filling the cell, red fluorescing chloroplasts, crystals, spores, etc.)
                             (adapted from Reference 20.10). If atypical structures are not observed,
                             then categorize each object meeting the criteria specified in Sections
                             15.2.3.1  through 15.2.3.3 as one of the following, based on DIC
                             examination:
                             (a)  An empty Giardia cyst
                             (b)  A Giardia cyst with amorphous structure
                             (c)  A Giardia cyst with one type of internal structure (nuclei, median
                                 body, or axonemes), or
                             (d)  A Giardia cyst with more than one type of internal structure
                             Using 1000X total magnification, record the shape, measurements (to the
                             nearest 0.5 urn), and number of nuclei and presence of median body or
                             axonemes (if applicable) for each apple-green fluorescing  object meeting
                             the  size  and shape characteristics.

                15.2.3.4      A positive result is a Giardia cyst which exhibits typical IFA
                             fluorescence, typical size and shape and exhibits nothing atypical on IFA,
                             DAPI fluorescence, or DIC microscopy. A positive result  must be
                             characterized and assigned to one of the DAPI and DIC categories in
                             Section  15.2.3.2 and 15.2.3.3.
       15.2.4   Record the date and time that sample examination was completed on the examination
                form.
       15.2.5   Report Cryptosporidium and Giardia concentrations as oocysts/L and cysts/L,
                respectively.
       15.2.6   Record analyst name


16.0  Analysis  of Complex Samples
•|g -j   Some samples may contain high levels  (>1000/L) of oocysts and cysts and/or interfering
       organisms, substances, or materials. Some samples may clog the filter (Section 12.0); others will
       not allow separation of the oocysts and cysts from the retentate or eluate; and others may contain
       materials that preclude or confuse microscopic examination.


December 2005                                 54

-------
                                                        Method 1623 - Cryptosporidium and Giardia
 16.2   If the sample holding time has not been exceeded and a full-volume sample cannot be filtered,
        dilute an aliquot of sample with reagent water and filter this smaller aliquot (Section 12.0). This
        dilution must be recorded and reported with the results.
 1g3   If the holding times for the sample and for microscopic examination of the cleaned up
        retentate/eluate have been exceeded, the site should be re-sampled. If this is not possible, the
        results should be qualified accordingly.
 164   Some samples may adhere to the centrifuge tube walls.  The use of siliconized or low-adhesion
        centrifuge tubes (Fisherbrand siliconized/low retention microcentrifuge tubes, 02-681-320 or
        equivalent) may reduce adhesion. Alternately, rinse centrifuge tubes with PBST elution buffer or
        Sigmacote® prior to use.

 17.0  Method Performance
 171    Method acceptance criteria are shown in Tables 3 and 4 in Section 21.0. The initial and ongoing
        precision and recovery criteria are based on the results of spiked reagent water samples analyzed
        during the Information Collection Rule Supplemental Surveys (Reference 20.11). The matrix
        spike and matrix spike duplicate criteria are based on spiked source water data generated during
        the interlaboratory validation study of Method 1623 involving 11 laboratories and 11 raw surface
        water matrices across the U.S. (Reference 20.14).
        NOTE:  Some sample matrices may prevent the MS acceptance criteria in Tables 3 and 4
        to be met. An assessment of the distribution of MS recoveries  across 430 MS samples
        from 87 sites  during the ICR Supplemental Surveys is provided in  Table 5.


 18.0  Pollution Prevention
 181    The solutions and reagents used in this method pose little threat to the environment when recycled
        and managed properly.
 18.2    Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
        the volume of expired materials that need to be discarded.

 19.0  Waste Management
 191    It is the laboratory's responsibility to comply with all federal,  state, and local regulations govern-
        ing waste management, particularly the biohazard and hazardous waste identification rules and
        land disposal  restrictions, and to protect the air, water, and land by minimizing and controlling all
        releases from fume hoods and bench operations. Compliance with all sewage discharge permits
        and regulations is also required. An overview of these requirements can be found in the
        Environmental Management Guide for Small Laboratories (EPA 233-B-98-001).
 19.2    Samples, reference materials, and equipment known or suspected to have viable oocysts or cysts
        attached or contained must be sterilized prior to disposal.
 19.3    For furtrier information on waste management, consult  The Waste Management Manual for
       Laboratory Personnel and Less is Better: Laboratory Chemical Management for Waste
       Reduction, both available from the American Chemical Society's Department of Government
       Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.

20.0  References
20.1   Morgan-Ryan, UM, A. Fall, L.A. Ward, N. Hijjawi, I. Sulaiman, R. Payer, R.C.Thompson, M.
       Olson, A. Lai, L. Xiao. 2002. Cryptosporidium hominis n. sp.  (Apicomplexa: Cryptosporidiidae
       from Homo sapiens). Journal Eukaryot Microbiol 49(6):433450.
20.2   Adam, R.D. 2001. Biology of Giardia lamblia. Clinical Microbiology Review 14(3):447-475.
                                              55                               December 2005

-------
Method 1623 - Cryptosporidium and Giardia
20.3   Rodgers, Mark R., Flanigan, Debbie J., and Jakubowski, Walter, 1995. Applied and
       Environmental Microbiology 6J. (10), 3759-3763.
20.4   Fleming, Diane O., et al.(eds.), Laboratory Safety: Principles and Practices, 2nd edition. 1995.
       ASM Press, Washington, DC
20.5   "Working with Carcinogens," DHEW, PHS, CDC, NIOSH, Publication 77-206,  (1977).
20.6   "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910 (1976).
20.7   "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety (1979).
20.8   APHA, AWWA, and WEF. 2005. Standard Methods for the Examination of Water and
       Wastewater; 21th Edition. American Public Health Association, American Water Works
       Association, Washington, D.C.
20 9   USEPA 2005. Manual for the Certification of Laboratories Analyzing Drinking Water; Criteria
       and Procedures; Quality Assurance. Fifth Edition. EPA 815-R-05-004. Office of Ground Water
       and Drinking Water, U.S. Environmental Protection Agency, 26 West Martin Luther King Drive,
       Cincinnati, OH 45268.
20.10  ICR Microbial Laboratory Manual, EPA/600/R-95/178, National Exposure Research Laboratory,
       Office  of Research and Development, U.S. Environmental Protection Agency, 26 Martin Luther
       King Drive, Cincinnati, OH 45268 (1996).
20.11  Connell, K., C.C. Rodgers, H.L.  Shank-Givens, J Scheller, M.L Pope, and K. Miller, 2000.
       Building a Better Protozoa Data Set. Journal AWWA, 92:10:30.
20.12  "Envirochek™ Sampling Capsule," PN 32915, Gelman Sciences, 600 South Wagner Road, Ann
       Arbor,  MI 48103-9019  (1996).
20.13  "Dynabeads® GC-Combo," Dynal Microbiology R&D, P.O. Box 8146 Dep., 0212 Oslo, Norway
       (September 1998, Revision no. 01).
20 14  USEPA. Results of the Interlaboratory Method Validation Study for Determination of
       Cryptosporidium and Giardia Using USEPA Method 1623, EPA-821-R-01-028.  Office of Water,
       Office  of Science and Technology, Engineering and Analysis Division, Washington, DC (2001).
20 15  USEPA. Implementation and Results of the Information Collection Rule Supplemental Surveys.
       EPA-815-R-01-003. Office of Water, Office of Ground Water and Drinking Water, Standards and
       Risk Management Division, Washington, DC (2001).
20 16  Connell, K., J.  Scheller, K. Miller, and C.C. Rodgers, 2000. Performance of Methods 1622 and
       1623 in the ICR Supplemental Surveys. Proceedings, American Water Works Association Water
       Quality Technology Conference, November 5 - 9, 2000, Salt Lake City, UT.
December 2005                               56

-------
         Method 1623 - Cryptosporidium and Giardia
21.0 Tables and Figures
Table 1. Method Holding Times (See Section 8.2 for details)
Sample Processing Step
Collection
Maximum Allowable Time between Breaks
(Samples should be processed as soon as possible)

Filtration
>• Up to 96 hours are permitted between sample collection (if shipped to the laboratory as a bulk
sample) or filtration (if filtered in the field) and initiation of elution
Elution
Concentration
Purification
Application of purified sample to slide
Drying of sample
These steps must be completed in 1 working day

> Up to 72 hours are permitted from application of the purified sample to the slide to staining
Staining
>• Up to 7 days are permitted between sample staining and examination
Examination
57
December 2005

-------
Method 1623 - Cryptosporidium and Giardia
Table 2.    Tier 1 and Tier 2 Validation/Equivalency Demonstration Requirements
Test
IPR
(Section 9.4)
Method blank
(Section 9.6)
MS
(Section 9.5.1)
MS/MSD
(Section 9.5)
Description
4 replicates of spiked
reagent water
Unspiked reagent
water
Spiked matrix water
2 replicates of spiked
matrix water
Tier 1 modification*1'
Required. Must be accompanied by a
method blank.
Required
Required on each water to which the
modification will be applied and on every
20th sample of that water thereafter. Must be
accompanied by an unspiked field sample
collected at the same time as the MS sample
Recommended, but not required. Must be
accompanied by an unspiked field sample
collected at the same time as the MS sample
Tier 2 modification'21
Required per laboratory
Required per laboratory
Not required
Required per laboratory.
Each laboratory must
analyze a different water.
(1)   If a modification will be used only in one laboratory, these tests must be performed and the results must meet all
     of the QC acceptance criteria in the method (these tests also are required the first time a laboratory uses the
     validated version of the method)
(2)   If nationwide approval of a modification is sought for one type of water matrix (such as surface water), a
     minimum of 3 laboratories must perform the tests and the results from each lab individually must meet all QC
     acceptance criteria in the method. If more than 3 laboratories are used in a study, a minimum of 75% of the
     laboratories must meet all QC acceptance criteria.

        NOTE: The  initial precision and recovery and ongoing precision and recovery (OPR)

        acceptance criteria listed in Tables 3 and 4 are based on results from 293
        Cryptosporidium OPR samples and 186 Giardia OPR samples analyzed by six
        laboratories during the Information Collection Rule Supplemental Surveys (Reference
        20.15). The matrix spike acceptance criteria are based on data generated through
        interlaboratory validation of Method 1623 (Reference 20.14).
December 2005
58

-------
                                                            Method 1623 - Cryptosporidium and Giardia
Table 3. Quality Control Acceptance Criteria for Cryptosporidium
Performance test
Initial precision and recovery
Mean recovery (percent)
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (percent)
Matrix spike/matrix spike duplicate (for method modifications)
Mean recovery1 2 (as percent)
Precision (as maximum relative percent difference)
Section
9.4
9.43
9.4.3
9.7
9.5
9.5.2.2
9.5.2.3
Acceptance criteria

24-100
55
11-100

13-111
61
(1)  The acceptance criteria for mean MS/MSD recovery serves as the acceptance criteria for MS recovery during
    routine use of the method (Section 9.5.1).
(2)  Some sample matrices may prevent the acceptance criteria from being met. An assessment of the distribution of
    MS recoveries from multiple MS samples from 87 sites during the ICR Supplemental Surveys is provided in Table
    5.
Table 4. Quality Control Acceptance Criteria for Giardia
Performance test
Initial precision and recovery
Mean recovery (percent)
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (percent)
Matrix spike/matrix spike duplicate (for method modifications)
Mean recovery1 2 (as percent)
Precision (as maximum relative percent difference)
Section
9.4
9.4.3
9.4.3
9.7
9.5
9.5.2.2
9.5.2.3
Acceptance criteria

24-100
49
14-100

15-118
30
(1)  The acceptance criteria for mean MS/MSD recovery serves as the acceptance criteria for MS recovery during
    routine use of the method (Section 9.5.1).
(2)  Some sample matrices may prevent the acceptance criteria from being met. An assessment of the distribution of
    MS recoveries across multiple MS samples from 87 sites during the ICR Supplemental Surveys is provided in
    Table 5.
                                                 59
December 2005

-------
Method 1623 - Cryptosporidium and Giardia
Table 5. Distribution of Matrix Spike Recoveries from Multiple Samples Collected from 87 Source Waters
       During the ICR Supplemental Surveys (Adapted from Reference 20.16)
MS Recovery Range
<10%
>10%-20%
>20% - 30%
>30% - 40%
>40% - 50%
>50% - 60%
>60% - 70%
>70% - 80%
>80% - 90%
>90%
Percent of 430 Cryptosporidium MS
Samples in Recovery Range
6.7%
6.3%
14.9%
14.2%
18.4%
17.4%
11.2%
8.4%
2.3%
0.2%
Percent of 270 Giardia MS
Samples in Recovery Range
5.2%
4.8%
7.0%
8.5%
17.4%
16.3%
16.7%
14.1%
6.3%
3.7%
December 2005
60

-------
                                      Method 1623 - Cryptosporidium and Giardia
                              1 mm
                                                     9
                       A
        1/5 mm
                       D
B
C
Figure 1.  Hemacytometer Platform Ruling. Squares 1, 2, 3, and 4
          are used to count stock suspensions of Cryptosporidium
          oocysts and Giardia cysts (after Miale, 1967)
                               61
                December 2005

-------
Method 1623 - Cryptosporidium and Giardia
         Figure 2. Manner of Counting Oocysts and Cysts in 1
                  Square mm. Dark organisms are counted and
                  light organisms are omitted (after Miale,
                  1967).
December 2005
62

-------
                                             Method 1623 - Cryptosporidium and Giardia
                                       Flow rate meter
                                          iih value  \
Sample
                                                                Effluent tuNng
                                                         (Wive)
Influent
tubing
tubing
                                   Centrifugal
                                    pump
                                  DIRECTION OF fLOIN
                                     Outlet tubing
                                                 Ftow rate meter-
                                                   with valve

                                                               Eluent tubing
           Pressure   Wet tubing
              Envirochefclu
               capsule
                                              Flow totalizer
                                                       *—;
                                                                 \
                                                                 (Vtalw)
                                   DIRECTION OF FLOW            ,	
Figure 3a. Filtration Systems for Envirochek™ or
            Envirochek™HV Capsule (unpressurized source - top,
            pressurized source - bottom)
                                     63
                                                               December 2005

-------
Method 1623 - Cryptosporidium and Giardia
     Sample
             Influent tubing
              Inlet tubing
Outlet tubing
                                   ERECTION OP FLOIA)
                                         Outlet tubing
                                                      Flow rate meter*-
                                                        with valve
                                                          Effluent tubing
    Influent
    tubing
Pressure   Inlet tubing
regulator             Fita-Max™
                  filter housing
 Row totalizer
                                                                        FtowcwiW
                                                                        •jal'je may be
                                                                        used in place sf
                                                                        flow rale meter
  Figure 3b. Filtration Systems for FiIta-Max® filters (unpressurized
               source - top, pressurized source - bottom)
December 2005
                            64

-------
                                            Method 1623 - Cryptosporidium and Giardia
Figure 4.  Methods for Scanning a Well Slide
                                    65
December 2005

-------
Method 1623 - Cryptosporidium and Giardia
22.0  Glossary of Definitions and Purposes

       These definitions and purposes are specific to this method but have been conformed to common
       usage as much as possible.

22 1   Units of weight and measure and their abbreviations
       22.1.1  Symbols
               °C    degrees Celsius
               uL    microliter
               <      less than
               >      greater than
               %     percent
       22.1.2  Alphabetical characters
               cm    centimeter
               g      gram
               G     acceleration due to gravity
               hr     hour
               ID    inside diameter
               in.    inch
               L      liter
               m     meter
               MCS  microscope cleaning solution
               mg    milligram
               mL    milliliter
               mm    millimeter
               mM    millimolar
               N     normal; gram molecular weight of solute divided by hydrogen equivalent of
                     solute, per liter of solution
               RSD  relative standard deviation
               sr      standard deviation of recovery
               X     mean percent recovery

22 2   Definitions,  acronyms, and  abbreviations (in alphabetical order)

       Analyst—The analyst should have at least 2 years of college in microbiology or equivalent or
       closely related field. The analyst also should have a minimum of 6 months of continuous bench
       experience with Cryptosporidium and IFA microscopy. The analyst should have a minimum of 3
       months experience using EPA Method 1622 and/or EPA Method 1623 and should have
       successfully  analyzed a minimum of 50 samples using EPA Method 1622 and/or EPA Method
       1623.

       Analyte—A  protozoan parasite tested for by this method. The analytes in this method are
       Cryptosporidium and Giardia.

       Axoneme—An internal flagellar structure that occurs in some protozoa, such as Giardia,
       Spironucleous, and Trichonmonas.

       Cyst—A phase or a form of an organism produced either in response to environmental conditions
       or as a normal part of the life cycle of the organism. It is characterized by a thick and
       environmentally resistant cell wall.
December 2005                                66

-------
                                                  Method 1623 - Cryptosporidium and Giardia
Flow cytometer—A particle-sorting instrument capable of counting protozoa.

Immunomagnetic separation (IMS)—A purification procedure that uses microscopic, magnetically
responsive particles coated with an antibodies targeted to react with a specific pathogen in a fluid
stream. Pathogens are selectively removed from other debris using a magnetic field.

Initial precision and recovery (IPR)—Four aliquots of spiking suspension analyzed to establish
the ability to generate acceptable precision and accuracy. An IPR is performed prior to the first
time this method is used and any time the method or instrumentation is modified.

Laboratory blank—See Method blank

Laboratory control sample (LCS)—See Ongoing precision and recovery (OPR) standard

Matrix spike (MS)—A sample prepared by adding a known quantity of organisms to a specified
amount of sample matrix for which an  independent estimate of target analyte concentration is
available. A matrix spike is used to determine the effect of the matrix on a method's recovery
efficiency.

May—This action, activity, or procedural step is neither required nor prohibited.

May not—This action, activity, or procedural step is prohibited.

Median bodies—Prominent,  dark-staining, paired organelles consisting of microtubules and found
in the posterior half of Giardia. In G. intestinalis (from humans), these structures often have a
claw-hammer shape, while in G. muris (from mice), the median bodies are round.

Method blank—An aliquot of reagent water that is treated exactly as a sample, including exposure
to all glassware, equipment, solvents, and procedures that are used with samples. The method
blank is used to determine if analytes or interferences are present in the laboratory environment,
the reagents, or the apparatus.

Must—This action, activity, or procedural step is  required.

Negative control—See Method blank

Nucleus—A membrane-bound organelle containing genetic material. Nuclei are a prominent
internal structure seen both in Cryptosporidium oocysts and Giardia cysts. In Cryptosporidium
oocysts, there is one nucleus per sporozoite.  One to four nuclei can be seen in Giardia cysts.

Oocyst—The encysted zygote of some sporozoa; e.g.,  Cryptosporidium. The oocyst is a phase or
form of the organism produced as a normal part of the life cycle of the organism. It is
characterized by a thick and environmentally resistant outer wall.

Ongoing precision and recovery (OPR) standard—A method blank spiked with known quantities
of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results
produced by the laboratory remain within  the limits specified in this method for precision and
recovery.

Oocyst and cyst spiking suspension—See  Spiking suspension

Oocyst and cyst stock suspension—See Stock suspension
                                        67                                 December 2005

-------
Method 1623 - Cryptosporidium and Giardia
       Positive control—See Ongoing precision and recovery standard

       Principal analyst—The principal analyst (may not be applicable to all monitoring programs)
       should have a BS/BA in microbiology or closely related field and a minimum of I  year of
       continuous bench experience with Cryptosporidium and IFA microscopy. The principal analyst
       also should have a minimum of 6 months experience using EPA Method 1622 and/or EPA
       Method 1623 and should have analyzed a minimum of 100 samples using EPA Method 1622
       and/or EPA Method  1623.

       PTFE—Polytetrafluoroethylene

       Quantitative transfer—The process of transferring a solution from one container to another using
       a pipette in which as much solution as possible is transferred, followed by rinsing of the walls of
       the source container with a small volume of rinsing solution (e.g., reagent water, buffer, etc.),
       followed by transfer of the rinsing solution, followed by a second rinse and transfer.

       Reagent water—Water demonstrated to be free from the analytes of interest and potentially
       interfering substances at the method detection limit for the analyte.

       Reagent water blank—see Method blank

       Relative standard deviation (RSD)—The standard deviation divided by the mean times 100.

       RSD—See Relative standard deviation

       Should—This action, activity, or procedural step is suggested but not required.

       Spiking suspension—Diluted stock suspension containing the organism(s) of interest at a
       concentration appropriate for spiking samples.

       Sporozoite—A motile, infective stage of certain protozoans; e.g., Cryptosporidium. There are four
       sporozoites  in each Cryptosporidium oocyst, and they are generally banana-shaped.

       Stock suspension—A concentrated suspension containing the organism(s) of interest that is
       obtained from a source that will attest to the host source, purity, authenticity, and viability of the
       organism(s).

       Technician—The technician filters samples, performs centrifugation, elution, concentration, and
       purification using MS, and places purified samples on slides for microscopic examination, but
       does not perform microscopic protozoan detection and identification. No minimum education or
       experience requirements with Cryptosporidium and IFA microscopy apply to the technician. The
       technician should have at least 3 months of experience in filter extraction and processing of
       protozoa samples by  EPA Method 1622/1623 and should have successfully processed a minimum
       of 50 samples using EPA Method 1622/1623.
December 2005                                 68

-------
                  Appendix D



Abbreviated Checklist for Cryptosporidium Sample Results

-------
This page intentionally left blank

-------
Abbreviated Checklist for Cryptosporidium Sample Results
Sample analyzed by EPA Method 1622 or 1623
Laboratory has current approval status through Lab QA Evaluation Program
•
•
Quality Control (QC) Requirements Before Sample Processing
Frequency
Method blank results
OPR sample results
Spike for OPR
Analyst Performance
The ongoing precision and recovery (OPR) and method blank samples associated
with the sample were performed within the same week and associated with no
more than 19 other samples
No Cryptosporidium oocysts or potentially interfering materials were found in the
method blank
Recovery for the OPR sample was at least 11%)
The associated OPR was spiked with no more than 500 oocysts as counted by
flow-cytometry
Analysts' examination criteria was verified monthly





Key Sample Requirements
Location/Schedule
Temperature
Volume analyzed
Staining Controls
Description of sampling location and schedule submitted to the State
The sample temperature upon receipt was <20°C, and not frozen
The volume analyzed for the sample was at least 10 L OR 2 mL of packed pellet
volume OR as much volume as can be filtered by 2 EPA-approved filters
Positive staining control for this sample'had appropriate fluorescence and
characterization of FITC, size, shape, DIG and DAPI characteristics for 3 oocysts
was recorded. The negative staining control for this sample did not contain any
oocysts or potentially interfering materials




Holding Time Requirements
Sample collection
Sample processing
Sample drying
Sample examination
The elution step for the sample was initiated within 96 hours of sample collection (if
shipped to the laboratory as a bulk sample) or filtration (if filtered in the field)
The sample was processed (eluted, concentrated, purified, and applied to the
slide) in 1 working day
The slide(s) for the sample were stained within 72 hours of application of the
sample to the slide
The slide(s) for the sample were examined within 168 hours (7 days) of staining




Matrix Spike (MS) Sample Requirements
Sample volume
MS Frequency
Method version
Spike for MS
The same sample volume (within 10%) was analyzed for the MS sample and the
associated, unspiked field sample
MS sample analyzed with first field sample and at least every 20 samples
thereafter
The same method version (type of filter, IMS kit, and staining kit) was used for the
MS sample and the associated, unspiked field sample
The MS sample was spiked with no more than 500 oocysts as counted by flow-
cytometry




                         D-l
February 2006

-------
                 Appendix E

Cryptosporidium Laboratory Quality Assurance Program
                   Application

-------
This page intentionally left blank

-------
                                                                OMB Control Number: 2040-0246
                          United States Environmental Protection Agency
                            Office of Ground Water and Drinking Water
                             Standards and Risk Management Division

                                        February 21,2006

Dear Laboratory Manager:

       Thank you for your interest in the U.S. EPA's Laboratory Quality Assurance Evaluation Program
for Analysis of Cryptosporidium under the Safe Drinking Water Act (Lab QA Program). This is a
voluntary program open to laboratories analyzing Cryptosporidium in water using EPA Method 1622 and
EPA Method 1623. To increase the likelihood that laboratories analyzing water samples for
Cryptosporidium generate reliable data, EPA has established the following process for evaluating
laboratory performance and quality assurance practices:

Step 1. Application. Laboratories must first submit the Lab QA Program application. The application
       forms are enclosed with this letter, and the application process is described in detail below. EPA
       will evaluate laboratory applications to confirm the following: (1) the laboratory has the
       equipment required in EPA Method 1622 and/or EPA Method 1623 (December 2005 version), (2)
       laboratory personnel have the recommended experience to analyze samples, and (3) the
       laboratory has successfully completed the initial precision and recovery and matrix spike/matrix
       spike duplicate tests specified in the method. Laboratories will be requested  to correct any
       deficiencies in these areas before proceeding to the next step in the evaluation process.

Step 2. Proficiency testing. After an application has been accepted, the laboratory will be sent a set of
       eight initial proficiency testing (IPT) samples consisting of a suspension of oocysts in a
       concentrated matrix. Laboratories will resuspend these spikes in reagent water to produce
       simulated source water samples, and analyze the samples using the 2005 version of Method
       1622/1623 that the laboratory plans to use for routine Cryptosporidium analyses. If a laboratory
       wishes to be evaluated for more than one variation of the method, the laboratory will  receive a set
       of eight proficiency test (PT) samples for each variation. Laboratory IPT data will be evaluated
       against the mean recovery and precision (as relative standard deviation) for the IPT samples from
       other laboratories.

       Laboratories already participating in the EPA Cryptosporidium PT Program, may use the initial
       round of samples from the PT program to meet the IPT sample requirement.

Step 3. On-site evaluation. After a laboratory completes the IPT, an on-site evaluation of the laboratory
       will be scheduled. The on-site evaluation will include two separate but concurrent assessments:
       (1) assessment of the laboratory's sample processing and analysis procedures, including
       microscopic examination, and (2) evaluation of the laboratory's  personnel qualifications, quality
       control program, equipment, and  record keeping procedures.
    Each laboratory will receive an audit report, which will document deficiencies, if any, that should be
corrected by the laboratory. After a laboratory has corrected any deficiencies noted in the audit report,
EPA will confirm that the laboratory is approved under the Lab QA Program.
                                              E-1                                  February 2006

-------
       Laboratories that meet the program performance criteria will also receive a set of three ongoing
proficiency testing (OPT) samples approximately every four months that must be analyzed in the same
manner as the IPT samples. EPA will evaluate the precision and recovery data for OPT samples to
determine if the laboratory continues to meet the performance criteria of the Laboratory QA Program.
Application Requirements

       The first step in the laboratory evaluation process is submission of a laboratory application
package. The following materials should be submitted for each laboratory application package:

1.   Signed, completed application form (attached).

2.   Completed self-audit checklist (attached). This checklist is similar to the checklist that will be used to
    audit your laboratory during the on-site evaluation.

3.   Resumes detailing qualifications of your laboratory's proposed principal analyst/supervisor and each
    analyst and technician listed on the application form and documentation of the training, including the
    list of samples analyzed by each and the time period during which the samples were performed (the
    list for each analyst and technician should include at a minimum the number of samples specified
    below for personnel prerequisites).

    The recommended personnel prerequisites for the laboratory evaluation program are as follows:

       Principal Analyst/Supervisor (one required per laboratory)
       •   BS/BA in microbiology or closely related field
       •   A minimum of 1 year of continuous bench experience with Cryptosporidium and IF A
           microscopy
       •   A minimum of 6 months experience using EPA Method 1622 and/or EPA Method 1623
       •   A minimum of 100  samples analyzed using EPA Method 1622 and/or EPA Method  1623
           (minimum 50 samples if the person was an approved analyst for Cryptosporidium under the
           Information Collection Rule(ICR))

       Other Analysts (no minimum requirement per laboratory)
       •   Two years of college in microbiology or equivalent or closely related field
       •   A minimum of 6 months of continuous bench experience with Cryptosporidium and IFA
           microscopy
       •   A minimum of 3 months experience using EPA Method 1622 and/or EPA Method 1623
       •   A minimum of 50 samples analyzed using EPA Method 1622 and/or EPA Method 1623
           (minimum 25 samples if the person was an ICR-approved analyst)

       Technician (no minimum requirement per laboratory)
       •   Three months experience with the specific parts of the procedure he/she will be performing
       •   A minimum of 50 samples analyzed using EPA Method 1622 and/or EPA Method 1623
           (minimum 25 samples if the person was an ICR-approved technician) for the specific
           analytical procedures they will be using.
                                             E-2                                February 2006

-------
4.   Detailed laboratory standard operating procedures (SOP) for each variation of the method your
    laboratory plans on using for routine Cryptosporidium analyses. SOP's for the following should be
    included:

    •   Performance of each method step including, sample spiking, filtration, elution, concentration,
        purification, slide preparation, sample staining and examination
    •   Dividing pellets greater than 0.5mL
    •   Preparation of reagents
    •   Dishwashing
    •   Staff training
    •   Corrective action procedures for failing to meet OPR, method blank, staining controls, sample
        acceptance, and performance verification criteria
    •   Sampling procedures to be followed by field or utility personnel
    •   Procedures for data recording, checking manual calculations, and checking accuracy of all data
        transcriptions

5.   EPA Method 1622/1623 initial demonstration of capability (IDC) data which include initial precision
    and recovery (IPR) test results and matrix spike and matrix spike duplicate (MS/MSD) test results for
    Cryptosporidium. The IPR test consists of four reagent water samples spiked with between 100 - 500
    oocysts and one method blank. The MS/MSD test consists of one unspiked and two spiked source
    water samples. These tests are described in Section 9 of EPA Method 1622/1623 and the results
    should meet the criteria in the method (December 2005 version). The following data should be
    submitted:

    •   Completed EPA Method 1622/1623 bench sheets and report forms for each of the eight samples
        (attached)
    •   Initial demonstration of capability summary form (attached)
    •   Spiking suspension preparation data. This should include completed flow-cytorneter calibration
        forms.

    Laboratories wishing to be evaluated for more than one variation of the method (different volumes,
    filters, elution and concentration procedures, and immunomagnetic separation kits) should submit a
    complete set of IDC data for each variation.

    If your laboratory currently participates in the EPA PT sample program and the required IDC data
    have already been submitted, the data do not need to be resubmitted. Please indicate this is the case
    on the initial demonstration of capability summary form.

6.   Table of contents from your laboratory's quality assurance plan. The quality assurance plan should
    specifically address the requirements of Cryptosporidium analysis under the Lab QA Program.

7.   An example of the data reporting form used to submit Cryptosporidium results to your clients.

8.   A statistical summary of percent recoveries for all OPR and MS samples analyzed at your laboratory
    for the past six months.
                                              E-3                                  February 2006

-------
Application materials should be submitted to the following address:

           Cryptosporidium Laboratory QA Program Coordinator
           CSC Water Programs
           6101 Stevenson Avenue
           Alexandria, VA 22304

       Send comments on the Agency's need for this information, the accuracy of the provided burden
estimates, and any suggested methods for minimizing respondent burden, including through the use of
automated collection techniques to the Director, Collection Strategies Division, U.S. Environmental
Protection Agency (2822T), 1200 Pennsylvania Ave., NW, Washington, D.C. 20460. Include the OMB
control number in any correspondence. Do not send the completed form to this address.

       When your application package has been received and reviewed, you will be notified whether it is
complete or has any deficiencies. After your application has been accepted, you will be notified of when
you should expect your initial set of PT samples. If you have any questions about the laboratory
application materials or evaluation process, please feel free to contact either me at
moulton.carrie@epamail.epa.gov or Jennifer Scheller atjscheller@csc.com.
                                     Sincerely,
                                    Carrie Moulton
                                    Manager, Cryptosporidium Laboratory Approval Program
                                    Technical Support Center
                                    26 West Martin Luther King Drive
                                    Cincinnati, OH 45268
Attachments
                                              E-4                                February 2006

-------
Burden Statement: The public reporting and recordkeeping burden for this collection of
information is estimated to average 18 hours per response or 72 hours per respondent annually.
Burden means the total time, effort, or financial resources expended by persons to generate,
maintain, retain, or disclose or provide information to or for a Federal agency. This includes the
time needed to review instructions; develop, acquire, install, and utilize technology and systems for
the purposes of collecting, validating, and verifying information, processing and maintaining
information, and disclosing and providing information; adjust the existing ways to comply with any
previously applicable instructions and requirements; train personnel to be able to respond to a
collection of information; search data sources; complete and review the collection of information;
and transmit or otherwise disclose the information. An agency may not conduct or sponsor, and a
person is not required to respond to, a collection of information unless it displays a currently valid
OMB control number.
                                            E-5                                 February 2006

-------
                   Appendix F

Checklist for the Laboratory Quality Assurance Evaluation
 Program for Analysis of Cryptosporidium under the Safe
                 Drinking Water Act

-------
This page intentionally left blank

-------
 re
 c
 0)  i. ^
 O  0) w
•S  c?
 tO •£ C
 a? vS o>
 II  O D
 3 .C 0-
 (A *- UJ

     <0) in
    •n o>
 O  O  o

 2|S
 O 5 Q.
•O  n
 re

 o
 o

o
                 re
                 Q.
                  O
                 "re

                 "re

                 ui

                 "o
                  c


                 "re


                 1
                 TJ
                  C
                  re
                  0)

                  re
                  c
                  o

                 73

                 "re

                 ui
                 re
                 0
                 o>

                 re
re
O

re












0)
c
TO
V.
^^

tf\
^
^^r

^i

rt A: Facilities, Equipment, and Quali
re
Q.


Z
O
c
o
c
c
^>
6
Z
0>
ssification
re
O











Item to be Evaluated




























tory Equipment and Supplies

0
3
*.
























[-grade water testing
c
0)
cc
2










1
6






m

^_

!E
o
3

Is reagent water tested monthly for these minimum parameters: con*
chlorine residual; and annually for metals-Pb, Cd, Cr, Cu, Ni, Zn?

T-
^











1
O


CD
ts ,_
ff S
roU


"CD o
•3 c

0 CD
*~ F
Were the results for the above parameters acceptable, total chlorine
than 0. 1 mg/L, conductivity not greater than 2 umhos/cm, and each i
than 0.05 mg/L and collectively not greater than 0.1 mg/L?

CM
^











1
O













Is reagent water tested monthly for heterotrophic plate count?

co
^











1
6








fv
1
.E
5
Li-
Are the results for the heterotrophic plate count acceptable, < 500 C

Tt
^

























I
a
o
S
tut
3
2










1
o













Accuracy ± 0.1 units, scale graduations, 0.1 units'

T-
CM











i
6













Is a record maintained for pH measurements and calibrations used'

CM
CN











8
o


c
g
"o
CD
CD
3,

k_
&
fc
D
f?

Is pH meter standardized each use period with pH 7, 4 or 10 standai
dependant upon desired pH)?

CO
2











CD
O
0


c
.g
2

X
CD
CD


'S
•o

All pH buffers are dated when received and opened and are discardi
date?

Tt
CN

























s (top loader or pan balance):
i
j
i
n










1
6





, .
CD
.Q
CD
CD
O
£
in
t~
Are balances calibrated monthly using Class S/S-1 weights, or weigl
Class S/S-1 weights?

T-
co











CD
O
6













Is correction data available with S/S-1 weights?

CM
CO











commendation
CD
cc












Is preventative maintenance conducted yearly at a minimum?

CO
CO

                                                                                                                        to
                                                                                                                        o
                                                                                                                        o
                                                                                                                        CM
                                                                                                                        o
                                                                                                                        o
                                                                                                                        CM
                                                                                                                         l_
                                                                                                                         0)
                                                                                                                        .a

                                                                                                                         E
                                                                                                                         0)
                                                                                                                        Q

                                                                                                                        CO
                                                                                                                        CM
                                                                                                                        CO
                                                                                                                         o
                                                                                                                        .c
                                                                                                                        "53
                                                                                                                         CO
                                                                                                                         0)
                                                                                                                         o
                                                                                                                         c
                                                                                                                         g

                                                                                                                         "o
                                                                                                                         Q)
                                                                                                                         co
                                                                                                                         0)
                                                                                                                         o

-------
CD


^


01
CD
O


5'



(S

nT

CD


O
CD
cn
CD

CD
§.


Ol

CO

D
CD

8


CT
CD

ro
o
o
cn
Tl

CD
CT
—i
C

01
N3
O
O
C35
M
cn
2j*
e employee training records available and up to date? [Section 9.











7)
5
C
CD
CD












Nl
if*.
8 »
the laboratory performing analyst verification of examination moni
rrective action procedures in place if criteria are not met? [Sectio
3 =?
ily and does
10.6]
£j
CD
oT
cr
01

CD



73
CD
— .
CD
3
CD












N
CO
vT
a training protocol for new employees present? [Section 9.1]











73
CD
JD
— .
CD
CD












to
k>
2f
e employee resumes present and complete? [Section 9.1]











;Q
5
_ .
CD
CD












N
— k
, — . O
rri o
ies the laboratory have a formal QA laboratory plan prepared and
Section 9.1]

•eady for exa
3

o'
•o





73
CD
.a
— .
CD
CD












ro

O
jality Assurance






























r^
).1 Are calibration and maintenance records complete and w
CD
II organized?
, — ,
CO
CD
6'
3
CO
_l



1

3
CD
13
Q_
9i
5'











_»
to
»
3






























r^
5.2 Are RPM and RCF calibrated yearly?












O
13-
1
















i.1 Is a maintenance contract in place, or internal maintenanc
9.1]
m
CD
T3
0
O
O
01
01
oT
cr
CD
'cn
CD


O


O
3^
o'
SL













_»
bo
O
«
ID
«






























.
r.1 Have micropipetters been calibrated within the past year?

CO
CD
G
o
co
KJ
, 	 ,








73
CD
J2
— .
S
CD












_»
""*
S
croplpetters:






























r^1
3 1 Are calibration of glass/mercury thermometers checked a
quarterly) at the temperature used against a reference Nl
[Section 8.1.4]
CO 3
nually (dial tt
T thermomel
CD 
o 3
" 3
O -J
c 2.
<' CD
01 ^
CD


73
CD
.a
— .
i
CD












_»
at
»
mperature recording device:






























r^1
5.2 Is temperature recorded once daily for days in use?












o
13.
o'
52.















r^1
01
oT
1
CO
CD
S
01
cr
CD
cT
Q)
sT.
c?
1
£.
s
o
o
0
cn
O
•o











O
2.
I













_,
in
3
ifrigerator/Freezer:































t 5 Are spore strips or ampules used monthly to confirm steri

N
S.
o'
3
•0










O
13.
o'
9L
















1.4 Is automatic timing mechanism checked with stopwatch q
c
01
•o










o
g.
o'
CD.
















1.3 Is a maximum registering thermometer or continuous mor
each autoclave cycle?

o
CD
Q.
CD
o'
CD
c

Q.
Q.
C

CQ




O
3.
O
SL
















\.2 Are date, contents, sterilization time and temperature reci
u
3.
CD
Q.
o1
-i
CD
01
O
o
•*
CD
•O







O
2.
1
















i.1 Is unit equipped with a temperature gauge/operational sa
m
sty valve?










O
13.
1













_»
*

itoclave:































Item to be Evaluated










o
o>
v>
••t!
o"
0)
r+
5"
CD
JO
Z
p
c
3
PT
3
O
O
"1
Z

-------
O
*
O
c
c
=3
o"
z
CO
0




c

u^£
•^^
re
_o
'to
(0

o




Item to be Evaluated



















1
6




~o
o
J
0)
c
"en
i
Q.
ra
2.5.1 Have technicians/analysts analyzed the required number of s;
1622/1 623' [Section 22.2J



















1
6







Are all relevant SOPs present and current?
CD
04

















ro
o
6





i>-
~o
CD
V=
CD
£
_C
in
CD
Q.
Are sampling instructions present for clients collecting and/or filtering sa

CN
















"E
CD
CD
"5
CT
CD





O-
in
action procedure
Does the laboratory have criteria for sample acceptance and corrective
[Section 8. 1.4]
00


















ro
•"E
o







Are data recording procedures present'

CM

















ro
o
O






Iculations?
2.9.1 Does the laboratory have an SOP for checking all manual cal


















"E
0
2?
3
CT
0)







Are corrective action contingencies present?
o
CN
















"^
CD
£
3
CT
0
CL






2.10.1 For OPR failures? [Section 9.7.4]


















"£j
CD
£
'3
CT
CU
K.






2.10.2 For method blank contamination? [9.6.2.2]



















1
O







2.10.3 For positive/negative staining control failures?



















1
6




0
C
3
-
ter (if applicable]
When did the laboratory begin processing samples with the Filta-Max fil'
CD
CN










~~~













o-
ter (if applicable!
When did the laboratory begin processing samples with the CrypTest fill
r-
CM






CO
•^

"5.
E
ro
"53
L_









.Q
_ro
CD
.c
622/1 623 since'
Approximately how many field samples were analyzed using methods 1
CO
CN


























•>
g
3
g
g
3
I
\
',
?
5
3
3
>
j
0


















"E
0)
£
D
CT
CD
ce


0
£
"o
c
i for each versio
Have acceptable initial precision and recovery analyses been performec
method the laboratory is using? [Section 9.1.2.1.1]
O>
CM















^
°E
0
CD
"5
c?
o:



• — •
CO
m
eriod? [Section !
Were method blanks run once per week or per 20 samples during this p
0
CM























T3
Q)
E
CD
Q.
1
ro
jQ
T3
O
.c
2.20.1 If the answer to 2.20 is no, then at what frequency where met



























mtamination?
2.20.2 What percentage of method blanks evaluated were without cc


CD
O
O
CM
 _
_Q
 CD
LL
    oo
    li.
in
o
o
CM

 CD
-Q
 E
 CD
Q
cr>
CN
CD
T—
"D
 O
CD
O
C
CD
 g
"o
CD
0>
O

-------




Note: All section references in [ ] refer to Method 1623 December 2005






















CD
cr
01
o
0
to
io
CD
Q. ^
Si CL
ita is stored electronically, does the laboratory have an SOP for checking the accuracy
) entry into an electronic system?
0
~*






o

o"
8L
















to
io
00
"~* m
ita are stored electronically, are files backed up on more than one disk to ensure data i
lost in the eventuality of some hardware failure?
UJ
CD






o

I
















to
io

CD
results from each sample maintained electronically?



























to
io
CD
f°
O m
is the laboratory have an adequate record system for tracking samples from collection
ugh log-in, analysis, and data reporting?








p

o'
















to
to
en
m
is the laboratory have records of all QC checks available for inspection? [Section 9.1]






73
CD
.a

CD
CD















to
Ko

c/> t»
CD =;
CD CD
the laboratory personnel performing the QC analyses representative of the personnel
king approval under this program?








O

o
9L



















IO
io
5.1 If the answer to 2.23 is no, then at what level were samples spiked?



























to
to
CO
1
re OPR and MS samples spiked with 100 - 500 organisms? [Section 9.7]






^0
CD
.Q
C
3'
CD
"*


















the past six months?














73
CO
D













10
k>
K >
>.4 What is the mean and relative standard deviation of the MS samples analyzed d
c
5'
a











2
CD
CO













io
1.3 How many MS samples were analyzed during the past six months?






























10
to
1.2 How many MS samples were evaluated?






























to
io
'.1 If the answer to 2.22 is no, then at what frequency were MS samples analyzed?



























IO
io
to
CO
•e matrix spike (MS) samples analyzed at the method -specified frequency? [Section 9.
^
00




73
CD
_Q
c
CD
=J



















samples analyzed during the past six months?














73
C/5
D













to
to
1 .7 What is the mean and relative standard deviation (RSD) of the recoveries of the
O
Tl
73











^
CD
01













to
io
1 6 How many OPR samples were analyzed during the past six months?






























to
io
1 5 How many OPR samples were evaluated?






























to
io
1 .4 Was an acceptable OPR associated with each field sample examined? [Section
CO
~g
^.
jo


73
CD


Ct>
CD
~

















to
to
1 .3 Does the laboratory maintain control charts of OPR results? [Section 9.7.6]





73
CD
O
o
3

CD
^
Si
o'

















IO
io
1 .2 What percentage of OPR samples evaluated met the recovery criteria? [Table 3
Section 9.7.3]






























to
io
1.1 If the answer to 2.21 is no, then at what frequency where OPR samples perform
8.
•o

























to
to
— *.
If
re ongoing precision and recovery (OPR) samples run once per week or per 20 sample
ng this period? [Section 9.7]
en





73
CD
.a
c
3
CD


















to
Ko
).4 How many method blanks were evaluated?






























NJ
k>
13 Was an acceptable method blank associated with each field sample examined?
9.6.2.2]
in
CD
o^
o'


73
CD
-Q
C
3'
CD



















Item to be Evaluated




o
W"~


(A

O'
Q)
5"
3



CD
z
p
c
3
O
*
o
z
^


-------
O
C
o
c
c
o"
z
tff
Pication
'55
CD

o









Item to be Evaluated








rement
CT
CD
K.


an
1 1


(>•
in
CD
Q.
E
CO
CO
CN
I—
m
Is the laboratory using the December 2005 version of Method 1622/1623 for
40 Part 141.704]
o
CO





















Data Recording Procedures

«






rement
cr
CD
o:


c
o
'•p
T3
a >
Q. CD
C CJ
CO *-
"> CO
j-T -
CO
E
o

"C
o
Q.
CD
ff\
Do sample numbers on the shipping forms match the sample numbers on th<

«






lendation
c
o
o
CD
o:





O"
CO
15
T3
D>
C
Are current Method 1622/1623 bench sheets used to record sample process
CO
CO






rement
CT
0






c
-Q
0
CO
3
JO
15
Are all primary measurements during each step recorded, including all raw d;
calculations? [Section 11 0, 12.0, 13.0]

CO






I
O












Name of analyst or technician performing the elution is recorded?
ID
CO






rement
cr
CD
o:











Date and time of elution is recorded9 [Section 12.2.6.2.1]
CD
CO






CO
O
•c
O












Name of analyst or technician performing the concentration is recorded?

co






rement
3
f£











Date and time of concentration is recorded9 [Section12.3.3.3.2]
CO







8
O








Cv-
"O
0)
E
o
Are batch and lot numbers of reagents used in the analysis of the sample rec
O)
CO






8
o












Lot number for the IMS kit is recorded?
o
ro






rement
cr
CD



JO
"3
CO
CD
C
o
15
c
E'
CD
x
CD
Are Method 1622/1623 Cryptosporidium report forms used to record sample
[Section 15.2]
*-
co






rement
3
CT
$.











Name of examining analyst is recorded? [Section 15.2.6]
CN
co






rement
S"
DC











Date and time of sample examination is recorded9 [Section 15.2.4]
CO
co






rement
CT
CD
cr











Are calculations of final concentrations and recoveries complete and correct'
•<*
cri






rement
3
CT
CD
o:











Do values recorded on the data sheets match the reported values?
10
co
'





"CD
o
O












Are mistakes on all forms crossed out with a single line, initialed, and dated?
CD
CO






8
o












o~
1
CD
"2
8
CO
"ro
a
15
•o
CD
t—
CO






8
o












Are hardcopy records well organized, complete, and easily accessible?
00
co






lendation
t
o
o
CD
(£


JO

c
CD
£
3
CT
CD
O
O
Does the laboratory include a disclaimer on the report to the client if method
were not met?
O5
co






1
O












Is the manually recorded data legible?
o
CN
CO






rement
'5
cc



CD

'to
a
E
Q

CO
>,
Do records demonstrate each analyst's characterization of 3 oocysts and 3 c
control for each microscopy session? [Section 15.2.1.1]
i-
co






rement
CT
0
o:








I~T'
csi
CO
c
g
0
W
CD
Q.
0
CO
CD
0
I
'o
_J
m
0
c
CO
.c
£
o
E
o
c
15
.c
o
to
15
D
CN
«
(O
o
o
CN

 £•
 CD
 0)
LL
LO
O
o
CN
 0}
 O
 CU
Q

CO
CM
CD
 o

 oS
 CO
 0}
 o
 c
 CD
"o
 0)
I

-------
o
5T
CD
a
o
3

I
%
cn
o
Q.
CO

D
CD
O
CD

cr
CD
-1
to
o
o
01
•
cn f <
1=1*
w «"
CL 3
CD fl>
thod does the laboratory currently use to estimate spike doses:(A) flow-sorted spikes, (B)
-counted spikes, (C) hemacytometer-counted spikes, or (D) membrane-filter-counted
M
cn

cn
•o
1

d)
numeration procedures

'
^^^^Jt^m^^fitf-';"""\ • I'iW^^^m c •••••illBIB
3
3


	


























*












CO












N












-

























wm








~~




^m
o
CD"
0
CD
>
03
O
O





















to
*•


Are stained slides read and confirmed within 7 days of staining? [Section 8.2 4]

73
CD
C
a!
3
CD
~
















ho
GO


Are slides stained within 72 hours of application of the purified sample to the slide?
[Section 8 2.3]

73
c
CD
3
CD
~
















Kj
Ko


Are sample elution, concentration, and purification steps completed in one work day?
[Section 8 2.2]

73
CD

CD
3
CD

















S
^


Is sample elution initiated within 96 hours of sample collection or field filtration? [Section
82.1]

73
CD
C.

3
CD
~














^
to
fl>
V
«r
ffi
> analyzed according to the April 2001 , 2003, or December 2005 version of Method 16
«
1





















£
to


3
O)
§f.
CD
CL
5.
CD
Cfl
CD
0)
Q.
0)
CL
8
CD
CL
§.
3;
o
c
O
1
CD
•O

C?
-Q
C

3
CD

















£
fO


Concentrate is held no longer than 24 hours between IMS and staining?

73
CD
C

3
CD
~
















£
'-*


Is time from initiation of sample collection to completion of concentration 72 hours or
less?

73
CD
C
CD
3
CD
~














^
i*
S



s analyzed according to December 1999 version of Method 1622/1623





















^

' o
a
3
to
ID
ID



























Item to be Evaluated
o
Q)
V>
to

5
o
B)
0
3




(D
W
O
C
3
3
0
*
O

-------
         CQ

         r
          re
         Q.
          O
         1-1
          re


         "re

         UJ
         w-
          O

          C
          o

         "re
          C
          re
          o>


          re
 C
 o
'•S
 ra
 c


 (0
 X
LJLJ

T3
 C
 re

 D)
 c

"55
 (0
 o
 o

 2
CL

JD

 Q.

 E
 re
(O

m
 re
Q.
            .
          o -43
          m  re
   UJ
 0)


 re
 o
4-1
 re

 o
^
 re




c
*>
o
c
c
D
O
z
o"
to"
Q)
^™




C
.0


re
o

**••
'55
(0
re
O









Item to be evaluated








































Laboratory Facilities and Laboratory Safety

to























8
^
o












Are laboratory coats and gloves worn in the laboratory? [Section 5.3]
,-
CD






































No other safety or facility issues were observed?
CN
CD




































(0
]5
£
U
H
D>
5
"5.
w
*
Q.
i

*•














.. o
CO
c 0
o
0 <











S*"^
" — " 2*
tfi c

!•£
"D "*"'
co «=
||
||
< E
What method does laboratory currently use to estimate spike doses:i
well-slide-counted spikes, (C) hemacytometer-counted spikes, or (D)
spikes
T—
f-'





































1
•a
9J
With what filter type did the laboratory demonstrate their spiking proc
CNj
"•























8
]^
o



'o
>*
0
CO
o
"
0
-5
0
o
0
a>
Is the carboy used for negative control randomly selected from carbc
cleaning system?
CO
N-'



















CO
k_
•u
CD
0
2
0.
T3
0
^
2

CO
^
•t^

'S
CO

co
a.
o
in
T3
8
CO
If flow-sorted spikes are used, was suspension vial vortexed for 30 s<
instructions? [Section 1 1 .4.3]
,j.
i--'



















CD

•o

0
ol
•D
O
£
CO










Was the suspension vial adequately rinsed? [Section 1 1.4.3.1]
in
f~-























0!
O
V^
6












Does the laboratory have an acceptable SOP for sample spiking?
co
i-.'





























-D
£
OJ

10
CO

O)
c
'5.
CO
Q.
Other than the issues noted for items 1.2 through 7 6 (if any) was sai
successfully?
^
N'






































Envtrochek (Complete Sections that apply)

co

































e
1
e
o

e
il
0
£
111
•r-
ni





















"c
CO
CO

CT
CO
ir






c
_o
c
8
E
o
CD
C
-o
c
8.1 .1 Are all components required for sample filtration present a
[Section 6.2]





















CD

•D
CO
8
OL
T3
O
£
CO










8.1 .2 Is the filter assembly set up correctly? [Figure 3a, pg 63]























c

1
CO

'5
CT
CO












8.1 .3 Is the pump adequate for needs? [Section 6.3.3]


to
o
o
CN
CD
-D
CD
LL




























LO
0
O
CN
.Q
E
S
Note: All section references in [ ] refer to Method 1623 De



-------
     C/l
     CD
     o
     u


     CD

     CD*


     §
     O
     CD
     CO
     CD

     CD"
     2.


     ef


     en

     co

     D
     CD

     8

     cr
     g>

     N>
     o
     o
     Ul
00
     -ri
     CD
       -
     c:
     01
     10
     o
     o
     O)


p

en

s system able to maintain seal during use with no leaks?





73
equireme

r"*'











p

en

s system well maintained and cleaned appropriately following use? [Sect
o'

ro
CO


"33
equireme
3
l~f











O

*•

s the volume filtered measured correctly using a flow meter or calibrated
Section 12.3 1.5.2]
o
01
1
•o


73
equireme

*""*"











o

CO

s appropriate flow rate maintained of <4 L per minute? [Section 12.3.1 .1 .
CO



£-
5
o
Q.
3
8
Q.
C
3










p

ro

s the filter assembly set up correctly? [Fig. 3b, pg. 64]




2

I
T3
0
8
0.
c
3










0
7^
— ^

\re all components required for sample filtration present and in good com
Section 6.2.3]
l_L

5'
3
•O


7J
equireme
3
"*









_,
p
3
»
2
0)
X
SB
o"
3_
ST

&>
3















_^
O
"ft
Tl
•"?
3

01
X



















(O

3;
0)

a
o
1
ST
o
O
(Q
CD
to
•v
•o
s
cr
i"
n>
a
3"
W
8
3
O.
a
I




















CO
ro



Dther than the issues noted for items 8.2.1 through 8.2.7 (if any) was Env
slution demonstrated successfully?

3
o
3"
CD
3T
=31
5T
















Co
ro
cn


Does the laboratory have an acceptable SOP for Envirochek capsule filtei
'
cp_
6'
3
•O



O
I













io
en


\re the samples shaken three times for 5 minutes each time, and each in
irientation? [Section 12.2.6.2]
Ql

differen

2-

1
3

c
3










CO
ro



\re the samples shaken at an appropriate speed? [Section 12.2.6.2.3]




2

o
Q.
3
8
Q.
C
3










Co
ro
CO


Does the eluting solution cover the membrane? [Section 12.2.6.2.2]




CD

o
Q.
CD
Q.
C
3










CO
ro
fo


s the wrist-shaker assembly set up correctly? [Section 12.2.6 1.1]




CD

lod Proce
Q.
C
3










k>



s the elution buffer prepared as per Method 1622/1623? [Section 7.4.1]




2.

0
Q.
O

c:
3








00
K>
S1
£.
3
o



s the system well maintained and cleaned appropriately following use?






o
1













CO
en


Is the volume filtered measured using a flow totalizer or calibrated carboy
12.2.4.2]
•o

175
CD
a
o


TJ
equireme

'"*"











oo
ji.


s the appropriate flow rate maintained (approximately 2 L/min)? [Section
«
ro
ro
ro

2.

0
Q.
Tl
O
8
Q.
^
CD














Item to be evaluated




o
Q)
(A
V)
5
o
Q>

o
D
CD
V)
z
p
0
c
3
3
O

-------
C
O
c
c
o
o"
tn
0)
c
JO
73
J^
'55
w
re
O






Item to be evaluated













ro
o
^
6









Does the laboratory have an acceptable SOP for Filta-Max filtration9
^
•*-
d










8
±±
6



c

"o
Oi
(/)
5
0
Does the laboratory indicate on the filter housing the correct direction of fl
12.3.1.3]
CO
T-
d



















X
CO
*S
Other than the issues noted in items 10.1.1 through 1 0.1 .8 (if any) was Fi
filtration demonstrated successfully?
en
T-
d




















1
O
"S
JC
o
£
1
"3
c
o,
1
•g
ti
1
£
i
3
sm
rM
e>




















Is an automatic or manual wash station used9
^
c\i
d










"c
CD
CD

cr
CD
CC








Is the filter wash station set up correctly9 [Section 12 3.2.1]
CM
CN
d










rocedure
0.
T3
O
£
CD







Is PBST used to elute the filter? [Section 7.4 2 4]
CO
CM
d










rocedure
0.
"§
f.
CD




2
CD
1 — '
Is an appropriate amount of PBST used for each wash? (approx. 600 mi;
12.3.2.2.1]
^
CM
d










rocedure
Q.
o
.c
n

~

CM
CO
CM
O
During the first wash, is the plunger moved up and down 20 times? [Secti
in
c\i
d










rocedure
0.
o
£
CD






t=
Is the plunger moved up and down gently to avoid generating excess foat
(0
CM
d










rocedure
a
T>
o
-C
ID





c
g
CD
During the second wash, is the plunger moved up and down 10 times? [S
12.3.2.22]
^
CM
d










CO
,ir
CD
o:




CM
CO
CN
C
0
If the automatic washer is used, is the machine operating properly? [Secti
CO
CM
d










"E
CD
CO

IT
CD
OL






CM
•&
Is the wash station cleaned adequately between samples? [Section 123'
o>
CM
d










8
±?
6







ro
CD
Does the laboratory have an acceptable SOP for Filta-Max elution with th
station?
o
CM
d
















CO

*-*
o
.0
"3
CD
Other than the issues noted for items 10.2.2 through 10.2.10 (if any) was
Filta-max filter using the wash station demonstrated successfully?
-
CM
d




















c
ra
5
'E
1
o
ys
"5
1
o
(8
1
i
X
IB
£
EL
<*>
o








rocedure
0.
o
£
ID







Is PBST used to elute the filter9 [Section 7.4 3 4]
r
CO
d










rocedure
n.
E
£
Q)



^
o
'o
CO
1 '
Is an appropriate amount of PBST used for each wash? (approx. 600 mL
12.3.2.3]
CM
CO
d










rocedure
£L
T3
O
-fr
J







Are two washes performed for 5 minutes each9 [ Section 12.3.2.3]
CO
CO
d










'c
(D
0)

cr
CD









Is the stomacher in good condition and operating properly?
^
CO
d










8
.t;
d





S
o
to
o
"w

Does the laboratory have an acceptable SOP for Filta-Max elution using i
m
CO
d

















CD
£
"5
C
.0
J5
Other than the issues noted for items 10.3 1 through 10.3 5 (if any) was e
Filta-Max filter using the stomacher demonstrated successfully?
CD
CO
d




















c
3
o
"S
I
T"
i
1
i
c
(0
<0
ra
c
1
1
1
ra
in
I
IB
i
jf

^
o
CD
O
O
CM
in
o
o
CM

 CD
-Q
 E
 CD
Q

CO
CM
CD

-------
    o

    CD
    0)
    o
    o'
    s
    CO
    O
    Q.
    O>
    IV)
    to

    D
    CD
    N)
    O
    O
    01
 I


O
    (D
    O"
    —%
    C
    01

    •2
    ro
    o
    o


00

Is residual suspension rinsed from all containers and gloves?










o
I













Uj

Does the laboratory have an acceptable SOP for sample concentral
o'
•0









O
g.
o'
9-L













CD

Is there a set of standards for comparison of pellet size?










Recom
CD
CL
01

5'











cn

Is the pellet volume determined? [Section 13.2.1]










73
CD

^*











Ip.

Is the centrifuge slowly decelerated at the end without the brake? [
to
CD
a
o
13

CO
ro

• — >



Method
-o
5
o
CD
CL
c.
CD










CO

Is the sample centrifuged for 15 minutes? [Section 13 2.1]










Method
3
o
CD
CL
c
3










ro

Are the centrifuge tubes properly balanced prior to centrifugation?










p
1













1,.

Is the sample centrifuged at 1500 x G using a swinging bucket rotor
CO
CD
a
o'
13
— *
CO
ro
li.


Method
Tl
CD
Q.

CD








it
3
«T
o
iek, CrypTest, and Filta-Max fitter sample centrifugation



»*
A
-T

1
sr















0
o
CD
i
o
























o
li
00
Other than the issues noted in items 10.4.1 through 10.4.12 (if any)
concentration using the Filta-Max concentrator demonstrated succe
W <
rn <
%, 0>
C. cn
s< cn
•o 01
3

CD"



















o
L»
ro
Does the laboratory have an acceptable SOP for concentration usin
concentrator?
CO
CD
Tl
TY
01
^
cu
X



O
o'
CB-












O
li
~*
Is there a set of standards for comparison of pellet size?










Recom
CD
a.

o'










o
:_!
0
Is the pellet volume determined? [Section 12 3.3.3]










I
3
CD

*""*"










0
CD

Is the membrane adequately washed to remove oocysts from filter?










Method
T)
CL

3









o
CO

Was 5 ml of PBST used each time? [Section 12.3 3.2.3]










Method
T)
8
CD
CL
C
3









p
^

Was the filter membrane washed twice? [Section 12.3.3.2.3]










Method
Tl
o
CL

3









0
0>

Are the stir bar and concentration tube rinsed after each concentrati
added to the concentrate? [Section 12.3 3 2.1 c.]
o
01
CL
r-r
CD
—
.d'
C
CL


I
3
CD
13
~"










0
on

Is the sample concentrated so that some liquid remains above the fi
cover the stirbar about naif-way)? [Section 12.3.3.2.1]
CD'
CD
3
O
c
CQ

^f
O



Method
TJ
CD
D.
c.
3









o
"rv


Is the concentrate from the first wash added to the 600 ml of eluate
wash?
3
«.
CD

CD
O
o
CL


Method
Tl
CD
CL

3









p
CO

Is concentration performed after each of the washes?










Method
3
8
CL
C
CD









p
"r^


Is the force of the vacuum maintained below 30 cm Hg? [note, pg. 4
CO









Method
T)
CD
CL

3









O
^

Is concentrator set up correctly? [Section 12.3.3.2.1 b.]










I
3
CD

"*













Item to be evaluated









o
a>
V)
o"

o
D
(D
y
z
p
o
c
3
D
O

-------
c
O
c
c
o
o"
eo
0)
fication
'35
(A

CO
o








Item to be evaluated





















CD
D.
E
55
ro
5
c
CO
Other than the issues noted in items 11.1.1 through 11.1.8 (il
concentration demonstrated successfully?
o>
^1























s, equipment and clean-up
I
&

N
T-





















r reagent-grade water:
S
1
I
^>
N
T"







CD
O
6









^
CO
c
0
"o
"GO
"o
•§
c
CO
ro
c
CO
73
CD
C
•i
'co
.•t;
c
Q
0
"co
01
-
CM









c.
CD
CD
!T
CD
Qi







co"
c
0
'•£
0)
W
o
CO
Is reagent grade water used to prepare all media and reagen
CN
c\i























ffl
ill
1
»
U
fM
(V







Jirement
CD
n:










, 	 ,
Does centrifuge have a swinging bucket rotor? [Section 6.8.
CM
CN









CO
O
O






o
05
fugal foro
l"i
c;
0
Does lab have easily accessible method for determining relal
centrifuges?
CN
CM
CN























Reagents
£
in
&
CO
£







CD
O
O








reagents'
-D
C
CO
CO
CO
o
Are SOP's available for the preparation of all essential chemi
CO
CN









mendation
o
o
CD
OL









Are SOP's posted or easily accessible at the bench?
CN
CO
CN









i
•E
o








c
CO
lo
"c
c
CO
0
'c
Are all reagents clearly labeled with date of preparation, tech
expiration date?
CO
CO
CN
























a
i
w
o
^
?







CO
o
O




CO
0
CO
3
between
>,
&
CD
a.
o
Is all glassware and plasticware washed well and stored appi
T_
CN









S
"E
O












Is distilled or deionized water used for final rinse?
CM
c\i









CO
o
6












Is an SOP available for glassware washing?
CO
CN


















* .
:hnician
v
V
H

ion and Slide Preparation
1
•c
,3
Q.

?





















IMS kit/manufacturer is used?
CO

Y-
co







Jirement
cr
CD
CC





CD
£:
iL above t
t
10
CO
i supernatant from the centrifuged sample aspirated no lower
t? [Section 13.2.2]
1 8.

CN
CO







Procedure
E
.^
'CD








co"
CN
CO
i pellet vortexed a sufficient time for resuspension? [Section '
cu
_co

CO
CO







1
o








0
c;
'E
in
d>
c
ra
the lab have an appropriate SOP for dividing pellets greater
amples and analyzing?
11
Q lo

^-
CO







Procedure
•o
o
^
'E










more than 0.5 mL of pellet used per IMS? [Section 13.2.4]
o
c
JO

10
cri







Procedure
"8
-pr
CD


^
CO
CD
CO
CD
JD
c
o
D)
'CD
i resuspended pellet volume quantitatively transferred to the 1
ion 13.3.2.1]
jo W_

CD
CO
O
O
CN
 CO
 :3

i
 cu
in
o
o
CM
 t_
 CD
JO


 i
 CD
Q

n
CN
CO
 o

"53
(fl
0)
o
 g
"o
 0)

-------
o
Q.


O)
N)
CO

a
CD
O
CD

3
cr
CD
o
o
01
CD
cr
—
O
O
O5

Ji
00



Are positive and negative staining controls performed? [Section 14.1]






73
CD
C
liremen

















£
Ko



Is FITC stain applied according to manufacturer's directions? [Section 1
to





1
o
Q.
T)
3
§L
c
CD















^
Lk



What staining kit/manufacturer is used? [Section 14.2]

























jj

tfi
01
3
•o
*
0)
&
5.
to
Technician
























CO
k>



Other than the issues noted in items 13.1 through 13.21 (if any) were si
slide preparation performed successfully?
LU
"0
CD
•o
3
s

o
01
Q.




















GO
Ko
-^


Does the laboratory have an acceptable SOP for sample purification?






O
1

















CO
KJ
0


If the slide is warmed, is incubator or slide tray calibrated and labeled?






O
I

















00
.
CD


Is slide dried at a) room temperature or b) 35 to 42 C? [Section 13.3.3.'










O
CD"
o
CD

>

CD







00
V
00


What type of slides are used?

























00
.
-1


Are the slides clearly labeled so they can be associated with the correc
13.3.3.7]
t sample? [Se
0
5



73
CD
.a
iremen

















00
.
CD


When the second dissociation is performed, does the laboratory: (A) us
(B) add the additional volume to the original slide?
CD
cu
en
CD
o
O
§.
CL
CD
O






O
d
CD
O
CD

>

CO







00
l_k
CJl


Is a second dissociation performed? [Section 13.3.3.10]






CD.
0
CL
Tl
3
8
Q.

3















CO
.
*•


Is sample vortexed vigorously for 50 seconds immediately after the add
seconds after the sample has set for 10 minutes at room temperature?
Is.
3 01
-i O
00 CL
CO 01

• — • CL
CO
o


CD
Er
o
CL
Proced
c
CD















00
' X
CO


Is standard NaOH (5/4., 1N) and standard HCI (50 //L, 0.1 N) used? [Si
IU
CD
3
O
CD
O
•o
CQ

ep



73
CD
n
c
liremen

















CO
' .
M


Are extra rinses to minimize debris performed appropriately when need
laboratory rinse A) IMS beads in the Leighton tube prior to transfer, B) I
beads, prior to transfer, C) IMS beads in microcentrifuge tube prior to d
ed? Does the
.eighton tube,
issociation?

o
—
GO

CD
O
Q.
Tl
3
8
CL
c
CD
O
9_
CD
O
CD

>

CO

o





CO
' t
-*


Is the sample quantitatively transferred from the Leighton tube to the m
rinses)? [Section 13.3.2.13]
icrocentrifuge

c
cr
CD
To

CD
o
CL
Tl
3
s
CL

CD















00
' V
o


Is all the liquid removed when decanting is performed with the magnet i
13.3.2.11]
t_
"c/j
CD
a
o
13





CD.
T>
c
CD















00
CD



Is Leighton tube correctly placed in magnet and rocked through 90 degi
second? [Section 13.3.2.9]
rees about on
o
CD
CD


CD
O
CL
Tl
3
CL

CD















CO
00



en"
CD
CD"
CQ'
0
ET
cr
CD
o
&
m
CL
SJ.
00
•3
31
o
c
s.
CD
T3
CD
3
£
CD
'cn
CD
a
o'
CO
ro

CT)



I
T)
CD
Q.
c
01















CJ
l^j



Are the IMS beads thoroughly resuspended prior to addition to the Leig
[Section 13.3.22]
0
1





CD.
O
a.
Tl
o
c
CD




















Item to be evaluated





o
fl>
V)
(fl
o"
s*
6"





(D
W
Z
o

z
o
c
PT
o
1

-------
c
o
c
c
o
o"
«f
0)
o
f3
o
'35
w

O






Item to be evaluated











Procedure
•o
o
£
2






n
Are the slides incubated in a humid chamber in the dark at room temperature f<
approximately 30 minutes or per manufacturer's directions9 [Section 14.3]

*t
^r








Procedure
T3
o
f
^




£
D)

Are the labeling reagents rinsed away properly after incubation, without disturb
sample? [Section 14.5]

in
•
Q.
IS
i
C)











Procedure
73
o
.c
"53
^


in
(fi
=>
CO

14.10 2 Are all the edges of the cover slip sealed well with clear fingernail po
Elvenol is used? [Section 14 9]











Procedure
XI
o
.c
"5
S


CD
_Q
E
CO
-o

Are the finished slides stored in a humid chamber in the dark at 1 to 10°C (hurr
not required for Evenol)? [Section 14.10]
^_
T—
-fr








CO
o
O








Does the laboratory have an acceptable SOP for sample staining?
CM

•*















D)
C
"cn
Other than the issues noted in items 14 2 through 14.13 (if any) was sample st;
demonstrated successfully?
m

•G


















Icroscope and Examination
£

in








Jirement
CT
CD
a:


o
t
D>E"
c c
'E °
1 ^

Is microscope equipped with appropriate excitation and band pass filters fore:
labeled specimens? (Exciter filter - 450-490 nm, dichroic beam-splitting mirror
barrier or suppression filter 515-520 nm)? [Section 6.9 2]

^~
in








Jirement
cr
CD



D>§

1 *
CD t_
x .t
m p
UJ p
Is microscope is equipped with appropriate excitation and band pass filters for
DAPI labeled specimens? (Exciter filter - 340-380 nm, dichroic beam-splitting
nm, barrier or suppression filter - 420 nm) [Section 6.9.3]

CM
in








Jirement
CT
cu








Does the microscope have HMO or DIG, objectives? [Section 6.9.1]

CO
in








mendation
I
CD
o:







Is microscope operation easily changed from epifluorescence to DIC/HMO?

^~
in"








Jirement
cr
CD
OL








Does the microscope have a 20 X scanning objective? [Section 6.9.1]

m
in








Jirement
ir








Does the microscope have a 100 X oil immersion objective? [Section 6.9.1]

CO
in








Jirement
cr
CD
OL








Is the microscope equipped with an ocular micrometer? [Section 6.9.1]

h^
in








Jirement
CT
CD
cr








Is a stage micrometer available to laboratory? [Section 10 3.5]

00
in

CD
O
O
CM
_
 CD
Li.
    CO
in
o
o
CM
 0>
Q

CO
CM
CD

T3
 O
 
-------
o
CD
CO
CD
5'


CD

CO*


§
O
CD
CO
o

(D
O
Q.
N)
W

D
CD
K)
O
O
Ul
CD
CT

C
01
O
O
C3)
Ul
_*
OJ
Q) 3"
3 S>
than the issues noted f
nation issues acceptab
CD" q
CD
CO
Ul
3
S
CQ
01
w
, — .
^
0)
Z3
* — -
CD
CD
CD
3
o'
S
CO
o
o
CD
O













cn
l_k
NJ
O
O
CD
the laboratory have an
acceptable SOP for sampi
CD
CD
£
3
3
£U
o"










73
CD
—
emen







Ol
i^.
•"*•
5' D
^- o
3, CD
GO W
the positive control con
ity for both FITC and D
tain Cryptosporidium oocy
API? [Section 15.2.1.3]
CO
c/r
s.
3
CD
HI
T3
•o
O
T3
S'
(D*
E"
o
S
CO
2
CD
O
CD


73
CD
.d
—
CD
CD
~






cn
l_i
o
D) Q)
ie mercury bulb been u
facturer? [Section 10.3
sed less than the maximui
.2.11]
=j
^-
o
c
c^
-t
scomme
Q.
CD
Q.
cr

**•
CD



73
CD

3
CD
Q.
m
o'






ul
CD

cT
0)
o
jlibration table for each
objective located close to
>-*
CD
3
o
S
GO
O
CD
1ft'
^O
c/5
CD
£^.
o'
13
O
oo
iji

73
CD
— •
CD
CD











n
•**
:o be evaluated















o
vt
w
^*
o
6'
3
CD
O
O
C
UMOU)

-------
16  Evaluation of sample processing
Method Step
(filtration, elution, concentration, purification
or staining)













Name













Position













Demonstrated
Technique
Successfully
yes/no













Recorded data as
sample was
analyzed
yes/no











i

        Note: All section references in [ ] refer to Method 1623 December 2005




                                                      F-15
February 2006

-------
O
3T
en
(D
CD

O
(D
(n
CD

CD"
O
Q.


0>
a
CD
cr
CD
O
O
01
O
O



(D

r*
(A
 (D
 D-


 Q>

-5

 N3
 O
 O
 C3




























































































































































































































































































































































































z
D>
3
(D





TJ
O
2.
5'
3




Adjust Inter;
Distan
c
"2.
—
01
.3

"5. (A
(D
Establish
Kohler
Illuminatio
3
O
_l ?T
Examine and
Record
laracteristics
'hree Oocysts
2.
00
II


,. s
o 5  -a
Q.
A rn
X
.b 0)

? — •
*> g
*v (D
* 52
^ 5*
t< 
$



5"
y

30
1
f
3
<5
a



Requirer
[Section 1
o 3
o> §
^ ^*


r-. ®
Pg* =
" S 3
JS§ |
Requireme
[Section
10.3.6]
3
7n
Requirement
ection 15.2.1.
—
'w 1
« m S.
° O -i
•25- 1
3 0
3
f^
TO

oi'w -0

^ o ^'
^A ** *0
I* o 3
2.
ww 1
Si 3
**
(0
0
o
3
3
(0
o.
s
5'
^

f
5!

B>
3
SL
3
o'
5
10
8
•o
9
ffi
X
0)
g
lination acce
•o
sr
O"
5"
f
1






























-------
      Appendix G




Method 1622/1623 Bench Sheet

-------
This page intentionally left blank

-------
[Laboratory Name:
1    |Laboratory ID:
                                  Method 1622/1623 Bench Sheet
Sample Identification Information
* Lab Sample ID
* PWS ID:
* Facility ID'
* Sample Collection Point ID:
*
Sample type (circle one):
Person Receiving Sample.
*Sample collection date and time:
Turbidity (NTU)'
Temperature, date and time @ sample receipt:
Initial precision and recovery (IPR) Method blank Field (monitoring) sample
Ongoing precision and recovery (OPR) Matrix spike (MS) Proficiency testing (PT)
Sample Spiking Information (for IPR, OPR, MS, and PT samples only)
* Estimated number spiked:
* Sample volume spiked (L):
Crypto Giardia

Spiking suspension ID:
Spiking time:
Spiking date:
Spiking analyst:
Sample Filtration
Filter type (circle one):
Did filter clog? (circle one):
Envirochek Envirochek HV Filta-Max CrypTest Other (specify)
Yes No
* Number of filter(s) used?:
* Volume filtered (L) to nearest 1/4L'
Filtration time: Filter lot number:
Filtration date:
Filtration analyst:
Sample Elution (must be initiated within 96 hours of sample collection/filtration)
Elution procedure (circle one).
Wrist shaker Filta-Max wash station Stomacher Backflush/sonication
Type of Elution buffer:
Elution buffer lot number:
Elution buffer expiration date:
Elution time'
Elution date'
Elution analyst:
Concentration, IMS, and Slide Preparation
(must be completed on same working day that samples are eluted)
Procedure (circle one):
* Pellet volume after concentration
Centrifugation Fi!ta-Max concentrator Other (specify)
(mL) to nearest 0.1 ml: Concentration analyst:
* Total volume of resuspended concentrate (ml): IMS analyst:
* Volume of resuspended concentrate transferred to IMS (ml): Slide preparation time:
Number of subsamples processed through entire method. Slide preparation date'
IMS lot number:
IMS system (circle one):
Slide preparation analyst:
Dynal GC-Combo Dynal anti-Crypto Other (specify)
Slides (circle one): Meridian Dynal Waterborne Other (specify)
Slide Staining (must be completed within 72 hours of application of sample to the slide)
Detection kit (circle one):
BTF EasyStam Merifluor Crypt-a-glo Giardi-a-glo Aqua-glo Other (specify)
Detection kit lot number.
Number of slides for this sample:
Staining date and time
Staining analyst:
I* Examination Results as Total FA number from all slides for sample Cryptosporidium:	Giardia:
 Comments:
 * = Data entered into LT2/Stage2 Data Collection and Tracking System
                                                  G-1
                                           February 2006

-------
           Appendix H



Method 1622/1623 Slide Examination Form

-------
This page intentionally left blank

-------
Laboratory name:
Laboratory ID:
                       Method 1622/1623 Slide Examination Form
Sample ID:
Examination/verification completion date and time •
(must be completed within 168 hours (7 days) of staining)
Positive staining control acceptable Q YES a NO
Analyst:
Slide number-
Total number of slides for this sample:
Negative staining control acceptable n YES
D NO
FITC, Size, Shape, DIG and DAPI Characteristics of 3 Oocysts Recorded n YES a NO
                                Cryptosporidium Results
Object
located
by FA
No.
1
2
3
4
5
6
7
8
9
10
Shape
(oval
or
round)










Size
LxW
(urn)










DAPI-
Light blue internal
staining, no distinct
nuclei, green rim
(A)










DAPI +
Intense
blue
internal
staining
(B)










Total FA number from this slide:
DAPI -: Total number (A):
DAPI +: Total number (B):
DAPI +: Total number (C):
Number of
nuclei stained
sky blue
(C)










D.I.C.
Empty
oocysts
(D)










Oocysts with
amorphous
structure
(E)










Oocysts with internal
structure (F)
Number of sporozoites










D.I.C. - Total number of empty oocysts (D):
D.I.C. - Total number of oocysts with amorphous structure (E):
D.I.C. - Total number of oocysts with internal structure (F).
Total count DAPI + (C) that show structure by D.I.C. (F):
                                    Giardia Results
Object
located
by FA
No.
1
2
3
4
5
6
7
8
9
10
Shape
(oval
or
round)










Size
LxW
(Mm)










DAPI-
Light blue internal
staining, no distinct
nuclei, green rim
(A)










DAPI +
Intense
blue
internal
staining
(B)










Total FA number from this slide:
DAPI-: Total number (A):
DAPI+: Total number (B):
DAPI+: Total number (C):

Number of
nuclei
stained sky
blue
(C)










D.I.C.
Empty
cysts
(D)










Cysts with
amorphous
structure
(E)










Cysts with internal structure (F)
Number
of nuclei










Median
body










Axonemes










D.I.C.: Total number of empty cysts (D):
D.I.C.: Total number of cysts with amorphous structure (E):
D.I.C.: Total number of cysts with one internal structure (F):
D.I.C.: Total number of cysts with >one internal structure (F):
Total number DAPI + (C) that show structure by D.
C. (F):
                                          H-l
                                                                          February 2006

-------