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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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12 February 2006
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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.
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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
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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.
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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.
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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.
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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).
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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.
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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.
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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
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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
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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.
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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
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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.
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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).
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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
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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.
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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)
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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
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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/.
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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
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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.
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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
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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.
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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
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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.
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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
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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
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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
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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.
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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/.
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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.
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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.
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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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
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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
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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.
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68 February 2006
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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
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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
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Appendix A
Biosafety Guidelines for Laboratories Analyzing Environmental
Samples for Cryptosporidium
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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.
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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
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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
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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
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Appendix B
Method 1622: Cryptosporidium in Water by Filtration/IMS/FA
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&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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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).
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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
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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
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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
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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.
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Appendix C
Method 1623: Cryptosporidium and Giardia in Water by
Filtration/IMS/FA
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&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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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).
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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:
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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.
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Method 1623 - Cryptosporidium and Giardia
Figure 4. Methods for Scanning a Well Slide
65
December 2005
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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
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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
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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
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Appendix D
Abbreviated Checklist for Cryptosporidium Sample Results
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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
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Appendix E
Cryptosporidium Laboratory Quality Assurance Program
Application
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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
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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
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What method does laboratory currently use to estimate spike doses:i
well-slide-counted spikes, (C) hemacytometer-counted spikes, or (D)
spikes
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With what filter type did the laboratory demonstrate their spiking proc
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instructions? [Section 1 1 .4.3]
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Other than the issues noted for items 1.2 through 7 6 (if any) was sai
successfully?
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8.1 .1 Are all components required for sample filtration present a
[Section 6.2]
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8.1 .2 Is the filter assembly set up correctly? [Figure 3a, pg 63]
c
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8.1 .3 Is the pump adequate for needs? [Section 6.3.3]
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Section 12.3 1.5.2]
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s the filter assembly set up correctly? [Fig. 3b, pg. 64]
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Section 6.2.3]
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Dther than the issues noted for items 8.2.1 through 8.2.7 (if any) was Env
slution demonstrated successfully?
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Does the laboratory have an acceptable SOP for Envirochek capsule filtei
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\re the samples shaken three times for 5 minutes each time, and each in
irientation? [Section 12.2.6.2]
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\re the samples shaken at an appropriate speed? [Section 12.2.6.2.3]
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Does the eluting solution cover the membrane? [Section 12.2.6.2.2]
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s the wrist-shaker assembly set up correctly? [Section 12.2.6 1.1]
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s the elution buffer prepared as per Method 1622/1623? [Section 7.4.1]
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s the system well maintained and cleaned appropriately following use?
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12.2.4.2]
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s the appropriate flow rate maintained (approximately 2 L/min)? [Section
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12.3.1.3]
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Is an automatic or manual wash station used9
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Is the filter wash station set up correctly9 [Section 12 3.2.1]
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Is the plunger moved up and down gently to avoid generating excess foat
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Is the wash station cleaned adequately between samples? [Section 123'
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Does the laboratory have an acceptable SOP for Filta-Max elution with th
station?
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Other than the issues noted for items 10.2.2 through 10.2.10 (if any) was
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Is an appropriate amount of PBST used for each wash? (approx. 600 mL
12.3.2.3]
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Are two washes performed for 5 minutes each9 [ Section 12.3.2.3]
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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?
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Is residual suspension rinsed from all containers and gloves?
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Is there a set of standards for comparison of pellet size?
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Is the pellet volume determined? [Section 13.2.1]
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Is the sample centrifuged for 15 minutes? [Section 13 2.1]
Method
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Other than the issues noted in items 10.4.1 through 10.4.12 (if any)
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Method
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Was 5 ml of PBST used each time? [Section 12.3 3.2.3]
Method
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Was the filter membrane washed twice? [Section 12.3.3.2.3]
Method
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Are the stir bar and concentration tube rinsed after each concentrati
added to the concentrate? [Section 12.3 3 2.1 c.]
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Other than the issues noted in items 11.1.1 through 11.1.8 (il
concentration demonstrated successfully?
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Are all reagents clearly labeled with date of preparation, tech
expiration date?
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t? [Section 13.2.2]
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the lab have an appropriate SOP for dividing pellets greater
amples and analyzing?
11
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more than 0.5 mL of pellet used per IMS? [Section 13.2.4]
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Are positive and negative staining controls performed? [Section 14.1]
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Is FITC stain applied according to manufacturer's directions? [Section 1
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Other than the issues noted in items 13.1 through 13.21 (if any) were si
slide preparation performed successfully?
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Is slide dried at a) room temperature or b) 35 to 42 C? [Section 13.3.3.'
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13.3.3.7]
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When the second dissociation is performed, does the laboratory: (A) us
(B) add the additional volume to the original slide?
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Is sample vortexed vigorously for 50 seconds immediately after the add
seconds after the sample has set for 10 minutes at room temperature?
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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
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Is Leighton tube correctly placed in magnet and rocked through 90 degi
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Are the IMS beads thoroughly resuspended prior to addition to the Leig
[Section 13.3.22]
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Procedure
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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]
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Are the labeling reagents rinsed away properly after incubation, without disturb
sample? [Section 14.5]
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73
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14.10 2 Are all the edges of the cover slip sealed well with clear fingernail po
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Procedure
XI
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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]
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Does the laboratory have an acceptable SOP for sample staining?
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Other than the issues noted in items 14 2 through 14.13 (if any) was sample st;
demonstrated successfully?
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Icroscope and Examination
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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]
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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
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CT
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Does the microscope have HMO or DIG, objectives? [Section 6.9.1]
CO
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Is microscope operation easily changed from epifluorescence to DIC/HMO?
^~
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Does the microscope have a 20 X scanning objective? [Section 6.9.1]
m
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-------
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
-------
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Examine and
Record
laracteristics
'hree Oocysts
2.
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1
-------
Appendix G
Method 1622/1623 Bench Sheet
-------
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-------
[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
-------
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-------
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
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