vvEPA
United States
Environmental Protection
Agency
Microbial Laboratory Guidance Manual
for the Long Term 2 Enhanced Surface
Water Treatment Rule (LT2 Rule)
June 2003
Draft
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Office of Water (4607)
EPA815-D-03-006
http://www.epa.gov/safewater/lt2/index.html
June 2003
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.
Questions concerning this document or its application should be addressed to:
Mary Ann Feige
U.S. EPA Office of Ground Water and Drinking Water
Technical Support Center
Room 127
26 West Martin Luther King Drive
Cincinnati, OH 45268-1320
(513)569-7944
(513) 569-7191 (facsimile)
feige .maryann@epa.gov
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TABLE OF CONTENTS
Section 1: Introduction 1
1.1 Background 1
1.2 LT2 Rule Microbial Monitoring Requirements 2
1.3 Use of Cryptosporidium Data 3
1.3.1 Cryptosporidium Field Sample Data 3
1.3.2 Cryptosporidium Matrix Spike Data 4
1.4 Use of E. coli Data 4
Section 2: General MicrobialLaboratory Quality Assurance 6
2.1 Quality Assurance Plans 6
2.2 Sample Temperature Monitoring 8
Section 3: Guidance for Cryptosporidium Laboratories 10
3.1 LT2 Rule Cryptosporidium Sample Analysis Requirements 10
3. .1 Approved Laboratories 10
3. .2 Revised Cryptosporidium Method 10
3. .3 Minimum Sample Volume Analysis Requirements 11
3. .4 Spiking Suspensions Requirements for Spiked Quality Control Samples .... 12
3. .5 Acceptable Sample Results 12
3. .6 Cryptosporidium Oocyst Counts to Report 12
3.2 Laboratory Quality Assurance Evaluation Program 13
3.2.1 Application 13
3.2.2 Personnel Qualifications and Training 14
3.2.3 Initial Proficiency Testing 15
3.2.4 On-Site Evaluation 15
3.2.5 Ongoing Proficiency Testing 15
3.2.6 Changing Status 16
3.2.7 Notifying Utilities of Laboratory Status 17
3.3 Cryptosporidium Method Quality Control 17
3.3.1 Cryptosporidium Spiking Materials 19
3.3.2 Initial Precision and Recovery Test 19
3.3.3 Method Blank Test 20
3.3.4 Ongoing Precision and Recovery Test 20
3.3.5 Ongoing Precision and Recovery Control Charts 20
3.3.6 Quality Control Batches 21
3.3.7 Holding Time Requirements 21
3.3.8 Staining Controls 23
3.3.9 Examination Preparation 24
3.3.10 Ongoing Analyst Verification 24
3.3.11 Proficiency Testing Samples 24
3.3.12 Acceptance Criteria for Receipt of Field Samples 24
3.3.13 Matrix Spike Samples 25
3.3.14 QC Guidance for Method Modifications and Use of Multiple Method
Variations 26
3.3.15 Guidance on QC Requirements for Different Sample Volumes 28
Draft
June 2003
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3.4 Sample Collection Procedures 29
3.5 Sample Processing and Analysis Procedures 29
3.6 Recordkeeping 29
3.7 Calculations for EPA Methods 1622/1623 32
3.7.1 Field Sample Calculations 32
3.7.2 Matrix Spike Sample Calculations 34
3.7.3 OPR Sample Calculations 35
3.8 Electronic Data Reporting 35
3.8.1 Data Entry/Upload 36
3.8.2 PWS Data Review 37
3.8.3 EPA/State Review 37
3.9 Data Archiving 37
3.9.1 Hardcopy Data 38
3.9.2 Slides 38
3.10 Equipment, Supplies, Reagents, and Standards 39
3.11 Vendor Contact List 39
3.11.1 Sample Collection and Filtration Supplies 39
3.11.2 Sample Concentration and Purification Supplies 40
3.11.3 Slides and Related Supplies 41
3.11.4 Antibody Stains 41
3.11.5 Microscope Equipment 42
3.11.6 Spiking Suspensions and Positive Staining Control Materials 42
3.11.7 Other Laboratory Supplies for EPA Method 1622/1623 43
Section 4: Guidance forE1. coli Laboratories 44
4.1 Laboratory Certification Program 44
4.2 Quality Assurance/Quality Control for E. coli Analyses 45
4.2.1 Quality Control Specifications Applicable to All E. coli Methods 46
4.2.2 Quality Control Specifications for Most Probable Number Methods 48
4.2.3 Quality Control Specifications for Membrane Filtration Methods 49
4.3 Sample Collection Procedures 50
4.3.1 Acceptance Criteria for Receipt of Field Samples 51
4.4 Sample Volume and Dilution Guidance 51
4.4.1 Sample Volume and Dilution Guidance for Multiple-Well Methods 51
4.4.2 Sample Volume and Dilution Guidance for Membrane Filtration 51
4.4.3 Sample Volume and Dilution Guidance for Multiple-Tube Methods 52
4.5 E. coli Data Recording and Calculations 52
4.5.1 Multiple-Well Data 53
4.5.2 Membrane Filtration Data 55
4.5.3 Multiple-Tube Data 59
4.6 Electronic Data Reporting 62
4.6.1 Data Entry/Upload 63
4.6.2 PWS Data Review 63
4.6.3 EPA/State Review 64
4.7 Vendor Contact List for E. coli Methods 64
Section 5: References 66
Section 6: Acronyms 67
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TABLES
Table 1-1. LT2 Rule Monitoring Requirements 3
Table 1-2. Bin Classifications 3
Table 3-1. Laboratory QA Program Personnel Qualifications 14
Table 3-2. Cryptosporidium Holding Times for EPA Method 1622/1623 23
Table 3-3. Sample Receipt Data Elements to Record in the Laboratory for EPA
Method 1622/1623 Cryptosporidium Analysis 31
Table 3-4. Primary Data Elements to Record in the Laboratory for EPA Method 1622/1623
Cryptosporidium Analysis 31
Table 3-5. QC Data Elements to Record in the Laboratory for EPA Method 1622/1623
Cryptosporidium Analysis 32
Table 4-1. Approved E. coli Methods for LT2 Rule and Corresponding Drinking Water
Certification Program Techniques 45
Table 4-2. Quality Control Procedures for E. coli Methods 46
Table 4-3. Positive and Negative Control Cultures 48
Table 4-4. Incubation Time and Temperature Specifications for MPN Methods 49
Table 4-5. Incubation Time and Temperature Specifications for Membrane Filter Methods 50
Table 4-6. Minimum Sample ID Information and General Sample Data to Record 53
Table 4-7. Minimum Data to Record for Quanti-tray 2000® Colilert® and Colilert-18®
Analyses 53
Table 4-8. Minimum Data to Record for Quanti-Tray® Colilert® and Colilert-18® Analyses ... 55
Table 4-9. Minimum Data Elements for Record for Membrane Filtration Analyses 56
Table 4-10. Minimum Data Elements to Record for 15-Tube MPN Methods 59
Table 4-11. Minimum Data Elements to Record for 15-Tube Fermentation Methods 60
Table 4-12. Examples of Different Combinations of Positive Tubes 61
FIGURES
Figure 3-1. Process for Laboratory Approval and Changes in Laboratory Approval Status 18
Figure 3-2. Ongoing Precision and Recovery Control Chart Example 21
Draft
June 2003
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APPENDICES
Appendix A Laboratory Biosafety Guidelines
Appendix B EPA Method 1622 for Cryptosporidium (June 2003)
Appendix C EPA Method 1623 for Cryptosporidium and Giardia (June 2003)
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 Examination Results Form
Appendix I Standard Methods 9223B: Colilert®
Appendix J Standard Methods 9223B: Colilert-18®
Appendix K Standard Methods mEndo/LES-Endo-NA-MUG and mFC-NA-MUG
Appendix L EPA Method 1103.1
Appendix M EPA Method 1603: Modified mTEC
Appendix N EPA Method 1604: MI Medium
Appendix O m-ColiBlue24® Broth
Appendix P Standard Methods 9221B/9221F: LTB -EC-MUG
Appendix Q . . . E. coll Most Probable Number Sample Results Acceptability Checklist for the LT2 Rule
Appendix R E. coll Membrane Filtration Sample Results Acceptability Checklist for the LT2 Rule
Appendix S E. coll Report Forms for Colilert™ 97-Well Procedures
Appendix T E. coll Report Form for Colilert™ 51-Well Procedures
Appendix U E. coll Report Form for Stamdard Methods 9222B/9222G
Appendix V E. coll Report Form for Standard Methods 9222D/9222G
Appendix W E. coll Report Form for Standard Methods 9213D mTEC
Appendix X E. coll Report Form for EPA Method 1603 modified mTEC
Appendix Y E. coll Report Form for EPA Method 1604 MI Medium
Appendix Z E. coll Report Form for mColiBlue24® Broth
Appendix AA E. coll Report Form for Multiple-Tube Fermentation Methods
Appendix AB E. coll Report Form for Multiple-Tube Most Probable Number Methods
Draft June 2003
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SECTION 1: INTRODUCTION
The Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR or LT2 rule) 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) for Cryptosporidium, E. coli, and turbidity
for a limited period [40 CFRpart 141.701(a)-( 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:
1. 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 advice.
2. 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.
3. User's Manual for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule) Data
Collection System. This manual provides PWSs and laboratories with instructions on the entry,
review, and approval of electronic data using the LT2 Data Collection System, and for generating
reports using the system.
All of these manuals are available at http://www.epa. gov/safewater/lt2/index.html. Responses to
frequently asked questions (FAQs) on sampling, analysis, and data reporting questions for the LT2 rule
also are available on this website.
777/5 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.
1.1 Background
The LT2 rule is a National Primary Drinking Water Regulation that requires monitoring, reporting, and
public notification requirements for all PWSs that use surface water sources. The rule requires additional
treatment techniques for some systems, based on Cryptosporidium monitoring results (40 CFR part
141.720 - 141.721). The LT2 rule was developed to improve control of microbial pathogens, including
specifically the protozoan Cryptosporidium, in drinking water and to address risk trade-offs with
disinfection byproducts.
Draft 1 June 2003
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Section 1: Introduction
The LT2 rule provides for increased protection against microbial pathogens in public water systems that
use surface water sources. The rule focuses on Cryptosporidium, a protozoan pathogen that is widespread
in surface waters. EPA is particularly concerned about Cryptosporidium because it is highly resistant to
inactivation by standard disinfection practices. Ingestion of Cryptosporidium oocysts can cause acute
gastrointestinal illness, and symptoms in sensitive subpopulations may be severe, including
risk of mortality. Cryptosporidium has been identified as the pathogenic agent in a number of waterborne
disease outbreaks.
EPA convened a Federal Advisory Committee to develop recommendations for both the Stage 2
Disinfectants and Disinfection Byproducts Rule and the LT2 rule. As recommended by the Federal
Advisory Committee, the LT2 rule requires public water systems that use surface water or ground water
under the direct influence of surface water to monitor their source water (influent water prior to treatment
plant) for Cryptosporidium, E. coll and turbidity [40 CFR part 141.701 (a)-(h)]. These data would be used
to determine whether additional treatment is needed at PWSs and to assess whether a relationship could
be established between the Cryptosporidium and E. coll levels in source water. A summary of the LT2
rule monitoring requirements is provided in Section 1.2. The use of Cryptosporidium data is discussed
Section 1.3 and the use of E. coll data is discussed in Section 1.4.
1.2 LT2 Rule Microbial Monitoring Requirements
Large systems (PWSs serve a population of at least 10,000 people) monitoring under the LT2 rule are
required to collect and analyze source water samples for Cryptosporidium, E. coll and turbidity for a
minimum of 2 years [40 CFR part 141.701 (b)]. Small systems (PWSs that serve fewer than 10,000
people) are required to monitor their source water for E. coll for a minimum of 1 year. A subset of small
systems would then be required to conduct Cryptosporidium analyses over a 1-year period if they exceed
E. coll trigger levels [40 CFR part 141.701 (c)].
Monitoring requirements for each system size, and the schedule for each stage of monitoring, is 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 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/safewater/lt2/index.html). Cryptosporidium samples must be analyzed by a
laboratory approved for analysis under the Laboratory Quality Assurance Evaluation Program for the
Analysis of Cryptosporidium Under the Safe Drinking Water Act (Section 3.2, below) using EPA Method
1622/1623 [40 CFR part 141.706 (a)].
E. coll samples must be analyzed using methods approved under the LT2 rule for surface water
monitoring (Section 4, below) [40 CFR part 141.705 (b)]. Source water turbidity must be measured when
Cryptosporidium and E. coll samples are collected using methods approved under the LT2 rule [40 CFR
part 141.705 (c)].
Draft 2 June 2003
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Section 1: Introduction
Table 1-1. LT2 Rule Monitoring Requirements
Public water
system size
Large systems
(serving 10,000 or
more people)
Small systems
(serving fewer than
10,000 people)
Monitoring begins
6 months after
promulgation of LT2
rule
30 months (2 1/2
years) after
promulgation of LT2
rule
Monitoring
duration
2 years3
1 year3'"
Monitoring parameters and sample
frequency requirements
Cryptosporidium
minimum
1 sample/month0
see below5
£. co//
minimum
1 sample/month11
1 every 2 weeks
§ Possible additional monitoring requirement for Cryptosporidium
If small systems exceed £ co// trigger levels, then. . .
Small systems
(serving fewer than
10,000 people)
48 months (4 years)
after promulgation
of LT2ESWTR
1 year
2 sample/month
N/A
a PWSs may be eligible to use historical (grandfathered) data in lieu of these requirements if certain quality
assurance and quality control criteria are met
b Small systems may be required to monitor for Cryptosporidium for one year, beginning 6 months after completion of
£ co// monitoring; Cryptosporidium monitoring required if the £. co// annual mean concentrations exceed 10/100
ml_ for systems using lakes/reservoirs or exceed 50/100 ml_ for systems using flowing streams
c PWSs monitoring for Cryptosporidium may collect more than 1 sample per month if sampling is evenly spaced over
the monitoring period
d Not applicable to large, unfiltered systems because these systems are not required to monitor for £ co// or turbidity
N/A = Not applicable; no monitoring required
1.3 Use of Cryptosporidium Data
Two types of Cryptosporidium data are collected under the LT2 rule: Cryptosporidium 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 Field 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 CFRpart 141.709). 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
Cryptosporidium Bin Concentration
Cryptosporidium < 0.075/L
0.075/L < Cryptosporidium < 1.0/L
1.0/L < Cryptosporidium < 3.0/L
Cryptosporidium > 3.0/L
Bin Classification
Bin 1
Bin 2
Bin 3
Bin 4
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June 2003
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Section 1: Introduction
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 PWS serving at
least 10,000 people bin classification would be based on the following:
• For PWSs that collect at least 48 samples during the required monitoring period, the Cryptosporidium
bin calculation is equal to the mean of all sample concentrations
• For PWSs that collect at least 24 samples, but not more than 47 samples, during the required monitoring
period, the Cryptosporidium bin calculation is equal to the highest average of all sample concentrations
in any 12 consecutive months in the monitoring period
For PWSs serving fewer than 10,000 people, bin classification would be based on the highest running six
month average because these systems collect samples twice a month for 1 year.
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 [40 CFRpart 141.705 (a) (2)]. Details on MS
sample requirements are provided in Section 3.3.13.
For PWSs in the following categories, two MS samples will need to be analyzed during LT2 rule
monitoring:
• PWSs serving more than 10,000 people, that perform monthly monitoring for 2 years, and that collect
24 field samples
• PWSs serving fewer than 10,000 people, that are triggered into monitoring for 1 year, and that collect
24 field samples
For PWSs serving more than 10,000 people and that perform semi-monthly monitoring for 2 years and
collect 48 or more samples, 3 MS samples will 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. The descriptive statistics of the MS sample results will be compared
to the performance of the methods during the Information Collection Rule Supplemental Surveys to
verify the assumptions on method performance upon which the LT2 rule was based.
When considering the method performance that could be achieved for analysis of Cryptosporidium under
the LT2 rule, the Federal Advisory Committee evaluated the results of EPA Methods 1622/1623 in the
ICRSS, which involved 87 PWSs sampling twice per month over 1 year for Cryptosporidium and other
parameters. During the ICRSS, the mean Cryptosporidium recovery and mean relative standard deviation
of the MS samples were 43% and 49%, respectively (Reference 5.1).
1.4 Use of E. coll Data
E. coli data are being collected by large systems during LT2 rule monitoring to assess whether a
relationship can be established between the Cryptosporidium and E. coli levels in source water and a
microbial index developed to establish trigger levels for E. coli that would indicate high Cryptosporidium
concentrations in a source water (40 CFRpart 141.701). If a relationship can be established, small
systems initially will be permitted to monitor for E. coli, rather than more expensive Cryptosporidium
analyses. Only those systems with E. coli levels above the trigger level established in the microbial index
would then be required to monitor for Cryptosporidium to determine bin placement (40 CFR part
141.702).
Draft 4 June 2003
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A preliminary index was developed during LT2 rule development using data from the Information
Collection Rule (ICR) and ICRSS. These data were evaluated for parameters that could indicate the
likelihood that a source water mean Cryptosporidium level would be above the Bin 2 threshold
concentration of 0.075 oocysts/L. Initially, fecal coliforms, total coliforms, E. coli, viruses (ICR only),
and turbidity were assessed for development of the microbial index. Data analyses placed greater
emphasis on E. coli and fecal coliforms because of the direct relationship between these parameters and
fecal contamination. E. coli was determined to provide the best performance as a Cryptosporidium
indicator with the available data. After E. coli levels were developed as a screening tool, turbidity was
considered in an effort to enhance the screening tool. However, turbidity was not found to improve
accuracy.
Based on the data from the ICR and ICRSS, the preliminary E. coli trigger levels were set at a mean of 50
E. colil 100 mL for flowing stream-type source waters and WE. coli/WO mL for reservoir/lake source
waters.
Draft 5 June 2003
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SECTION 2: GENERAL MICROBIAL
LABORATORY QUALITY ASSURANCE
All laboratories analyzing Cryptosporidium and E. coll 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://www.epa. gov/safewater/lt2/cla final .html and Section 3.2 of this manual) and the Laboratory
Certification Manual (Chapter III, page III-4) for E. coli laboratories, each laboratory should operate a
formal QA program and should 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). For E. coli laboratories, this QA plan should be available for review during recertification
audits as part of the National Primary Drinking Water Regulations.
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
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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, 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 and/or E. coll analyses
• Calibration procedures, frequency, standards for each piece of equipment used for Cryptosporidium
and/or E. coll analyses
• Quality control records for each piece of equipment used for Cryptosporidium and/or E. coll analyses
• Preventative maintenance and schedules, documentation for each piece of equipment used for
Cryptosporidium and/or E. coll analyses
7. Supplies
• Laboratory glassware and plasticware acceptance conditions
• Chemicals, reagents, dyes and culture media acceptance conditions
• Chemicals, reagents, dyes, and culture media storage conditions
• Filters acceptance conditions
8.Laboratory practices (may reference SOP)
• Preparation of reagent-grade water
• Glassware washing and preparation
• Sterilization procedures
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
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Section 2: General Microbial Laboratory Quality Assurance
• 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. coli/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 how long records are to be kept
• 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 (G-5),"
(EPA/600/R-98/018, February 1998), which is available as a download from
http://www.epa.gov/qualitv/qa docs.html#noneparqt.
However, the goals of a lab QA plan in general are different from the goals of a 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, because the LT2 rule is a finite study on
Cryptosporidium and E. coli levels in specific source waters, 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.
2.2 Sample Temperature Monitoring
Source water samples are dynamic environments and, depending on sample constituents and
environmental conditions, Cryptosporidium 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 that are not analyzed the same day they are collected must be maintained below 10°C to
reduce biological activity. This is specified in Section 8.0 of the June 2003 versions of EPA Method
1622/1623 for Cryptosporidium samples and at 40 CFR part 705 (b) (1) and Chapter V, Section 6.3, of
the Laboratory Certification Manual (Reference 5.2) 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 June 2003 versions of EPA Method 1622/1623. Although not a significant concern for 10-L water
samples, this is a real 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. E. coli holding time studies
performed in support of the LT2 rule (Reference 5.4) demonstrated that E. coli samples can freeze under
these conditions if samples are not packed properly.
Draft 8 June 2003
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Section 2: General Microbial Laboratory Quality Assurance
The sample collection protocols procedures 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. coll and Cryptosporidium samples. Utility personnel should
follow these procedures to ensure that samples remain at acceptable temperatures during shipment.
Because Cryptosporidium samples collected for the LT2 rule must meet the QC criteria in the methods
[40 CFRpart 705 (a) (3)], and because these QC criteria include receipt of samples at <10°C and not
frozen, laboratories must reject LT2 Cryptosporidium samples that were not collected the same day they
were received, and are received at >10°C or frozen (this is discussed further in Section 3.3.12 in this
manual). In these cases, the PWS must re-collect and re-ship the sample.
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. coll 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 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.
• 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 reads the thermometer. Example product:
Eagle-Picher Sentry Temperature Vial 3TR-40CS-F or 3TR-40CS.
• 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. 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/.
• 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 still mitigates the risk of sample contamination while
providing an indication of sample temperature to verify that the sample temperature is acceptable.
Example product: Cole Farmer cat. no. U-90316-00.
As with other laboratory equipment, all temperature measurement devices should be calibrated routinely
to ensure accurate measurements. See the EPA Lab Certification Manual (Reference 5.2) for more
information.
Draft 9 June 2003
-------
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 CFRpart 141.705 (a)]. Guidance on the use of these methods during the
LT2 rule are provided in this section of the manual.
3.1 LT2 Rule Cryptosporidium Sample Analysis Requirements
LT2 rule requirements of particular significance to Cryptosporidium laboratories are summarized in
Sections 3.1.1 through 3.1.6, below, and discussed in more detail 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 or a laboratory that has been certified for Cryptosporidium analysis by an
equivalent State laboratory certification program [40 CFRpart 141.706 (a)]. Details on the elements of
the Lab QA Program QA evaluation are provided in Section 3.2.
3.1.2 Revised Cryptosporidium Method
EPA has proposed the use of the April 2001 versions of EPA Methods 1622/1623 in the LT2 rule.
However EPA has requested comment on the use of updated versions (dated June 2003) to consolidate
several method-related changes EPA believes are necessary to address LT2 rule monitoring requirements.
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 of methanol during slide staining in Section 14.2 is per manufacturer's instructions.
• 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 and clarifies that any positive sample in a
batch associated with an unacceptable method blank is unacceptable and that any sample in a batch
associated with an unacceptable ongoing precision and recovery (OPR) sample is unacceptable.
• A change in the sample storage and shipping temperature to "< 10°C, and not frozen," for samples that
are not processed the day they are collected, and provides additional guidance on sample storage and
Draft 10 June 2003
-------
Section 3: Guidance for Cryptosporidium Laboratories
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 of examination using differential interference contrast (DIG) microscopy to
the analyst verification procedure.
• Addition of an approved method modification using the Pall Gelman Envirochek HV filter. This
approval was 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 Giardia, however, individual laboratories are permitted to demonstrate
acceptable performance in their laboratory).
• Incorporation of detailed procedures for concentrating samples using and IDEXX Filta-Max™ foam
filter. (A method modifications using this filter already is 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.
The changes in the June 2003 draft revisions of EPA Methods 1622 and 1623 reflect method-related
clarifications, modifications, and additions that EPA believes should be addressed for LT2 rule
Cryptosporidium monitoring. Alternatively, these issues could be addressed through regulatory
requirements in the final LT2 rule, however, EPA believes that addressing these issues through a single
source in updated versions of EPA Methods 1622 and 1623 (which could be approved in the final rule)
may be more straightforward and easier for systems and laboratories to follow than addressing them in
multiple sources (i.e., existing methods, the final rule, and laboratory guidance).
This draft manual assumes that the June 2003 versions of EPA Methods 1622 and 1623 will be used for
LT2 rule monitoring. The June 2003 versions of these methods are included as Appendix B and
Appendix C of this guidance manual. If EPA determines that the updated methods will not be used,
based on comments to the proposed rule, then the rule may be updated to include critical clarifications as
requirements and this guidance document will be updated to include non-critical clarifications as
guidance.
3.1.3 Minimum Sample Volume Analysis Requirements
Under LT2 rule Cryptosporidium sample volume requirements [40 CFRpart 141.705 (a) (1)], 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 FiltaMax™ foam filter)
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. Sample volumes should be rounded to the
Draft 11 June 2003
-------
Section 3: Guidance for Cryptosporidium Laboratories
nearest whole liter to determine compliance (e.g., 9.5 L would be rounded to 10 L, and would meet rule
requirements).
Systems may analyze larger volumes than 10 L, and larger volumes analyzed should increase analytical
sensitivity (detection limit), provided method performance is acceptable. EPA encourages systems to
analyze similar sample volumes throughout the monitoring period. However, data sets including different
samples volumes will be accepted, provided the system analyzes the minimum sample volume
requirements noted above.
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.epa.gov/safewater/lt2/index.html.
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 CFRpart 141.705 (a)(3)]. The use of flow cytometer-counted spiking
suspensions is a recommendation in EPA Method 1622/1623, but a requirement for 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 meet the quality control (QC)
requirements specified in EPA Method 1622/1623 [40 CFRpart 141.705 (a)(3)]. 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. These requirements are based on the June 2003 versions
of EPA Methods 1622/1623, the use of which EPA has requested comment on in the LT2 rule proposal.
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 (DIC) to detect internal
structures [40 CFRpart 141.705 (a)(4)]. Cryptosporidium oocysts to be reported using EPA Method
1622/1623 are defined as the following:
1. Those determined by brilliant apple green fluorescence under UV light, size (4 to 6 (im), and shape
(round to oval)
2.Excluding atypical organisms specifically identified as other microbial organisms by FITC and DIC
(for example, 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 the sample volume analyzed, based on
the calculations specified in Section 3.7, will be used to calculate the oocyst concentration for each
sample during the LT2 rule.
Draft 12 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
3.2 Laboratory Quality Assurance Evaluation Program
To improve Cryptosporidium data quality and consistency during LT2 rule monitoring, EPA requires that
only qualified laboratories analyze Cryptosporidium samples. A laboratory's qualifications are
determined through the Laboratory Quality Assurance Evaluation Program for Analysis of
Cryptosporidium Under the Safe Drinking Water Act (Lab QA Program) (67 FR 9731, March 4, 2002)
[40 CFRpart 141.706 (a)]. If laboratories are certified for Cryptosporidium analyses under equivalent
State laboratory certification programs, EPA plans to include these laboratories in the list of approved
laboratories posted at http://www.epa.gov/safewater/lt2/aprvlabs.html.
The objectives of the Lab QA Program are to evaluate laboratories' capacity and 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)
• 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/lt2/index.html
and 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 and also may be requested from
the following address:
EPA's Laboratory Quality Assurance Evaluation Program Coordinator
c/o DynCorp/CSC Biology Studies Group
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
• Initial demonstration of capability (IDC) data, which consist of the following:
• Acceptable initial precision and recovery (IPR) test results
• Acceptable method blank result run with IPR test
• Acceptable matrix spike/matrix spike duplicate (MS/MSD) results with results from the unspiked
matrix sample analyzed at the same time
• Table of contents from the laboratory's quality assurance plan
• Documentation of personnel training and number of samples analyzed and duration of time using the
method
• Example of client data reporting form
Draft 13 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
Completed applications should be submitted to EPA's Laboratory Quality Assurance Evaluation Program
Coordinator, c/o DynCorp/CSC Biology Studies Group, at the address listed above.
3.2.2 Personnel Qualifications and Training
Laboratory personnel must meet the qualifications of the Lab QA Program in order to analyze
Cryptosporidium samples for LT2 rule monitoring. Personnel qualifications for the Lab QA Program are
provided in Table 3-1. Each laboratory must have at least one principal analyst.
Table 3-1. 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 with
specific parts of
procedure
performing
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), laboratory records will be evaluated to verify that the
personnel performing EPA Method 1622/1623 analyses meet the requirements of the Lab QA Program.
For new staff that are added after the on-site evaluation, the laboratory should send a letter to EPA
providing the following information on the new staff member:
• 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
In addition to meeting the qualifications of the Lab QA Program, the following steps should be completed
by new personnel as part of their training prior to analyzing samples for LT2 (this should be specified 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
If EPA determines that a laboratory is using personnel to analyze LT2 samples who do not meet the
qualifications established in the Lab QA Program, the laboratory will be put on conditional status
(Section 3.2.6). The laboratory must discontinue the use of the personnel in question until they have met
the qualifications of the Lab QA Program, and must submit a signed letter to EPA confirming that this
action has been taken, to be removed from conditional status.
Draft
14
June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
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 (IPX) 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. If a laboratory wishes to be evaluated for
more than one version of the method, the laboratory will receive a set of eight PT samples for each
version.
Laboratory IPX data will be evaluated against the mean recovery and precision (as relative standard
deviation [RSD]) criteria that EPA has established for IPX samples. Laboratories have two opportunities
to pass the IPX test. If a laboratory fails two times, it will not be eligible for another set until after the
laboratory staff has received additional training in performing the method and reapplied to the program
(discussed further in Section 3.2.6).
3.2.4 On-Site Evaluation
Each laboratory that passes the IPX will next participate in an on-site evaluation, 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. Xhe 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
Xo 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.
Xo prepare for the on-site evaluation, the laboratory should 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. Xhe laboratory will be assessed on its capabilities including the following:
• Sample processing and analyses
• Microscopy
Xo 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 Ongoing Proficiency Testing
Laboratories that meet the program performance criteria will also receive a set of three ongoing
proficiency testing (OPX) samples approximately every four months that must be analyzed in the same
Draft 15 June 2003
-------
Section 3: Guidance for Cryptosporidium Laboratories
manner as the IPX 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.
3.2.6 Changing Status
Laboratories will be approved after they have submitted an acceptable application, passed the IPX and
passed the on-site evaluation. Laboratories must be approved in order to analyze samples under LT2. To
maintain approval the laboratory must successfully analyze a set of OPT samples once every fourth
month. Details are provided below on what happens when a laboratory fails to meet the program
requirements. The process for laboratory approval and changes in approval status is outlined in Figure
3-1.
Laboratories that fail two rounds of IPT samples must do the following to be eligible to receive a third set
of IPT samples:
• Receive additional training
• Analyze 25 additional practice samples
• Repeat their IPR analysis and submit the results to EPA
The additional practice samples should include method blanks, OPR samples, and at least 50% matrix
samples including multiple matrix spike and matrix spike duplicate samples. Additional training can be
received at EPA's Technical Support Center in Cincinnati or another approved laboratory. Laboratories
that only have a few analysts/technicians may want to send all their staff involved in the method for
training. For laboratories that cannot send their entire staff for training, they may elect to send the analysts
normally responsible for training of other analysts/technicians in their laboratory. These analysts can then
pass along the information they received during training at EPA to all of their staff.
If the laboratory does not pass the third IPT following additional training, they will be removed from
consideration for the program for 6 months, during which time they must analyze an additional 50
practice samples and repeat their IPR and submit the results to EPA. The additional practice samples
should include method blanks, OPR samples, and at least 50% matrix samples including multiple matrix
spike and matrix spike duplicate samples. After satisfying these requirements the laboratory may re-apply
for ONE more set of IPT samples. If the laboratory fails this final IPT, then they will not be allowed to
participate in analysis of samples for Cryptosporidium using EPA Method 1622/1623 under the LT2 rule.
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. The laboratory's status changes to "conditional" but they are still
permitted to analyze samples under LT2.
• If the laboratory's next set of OPT data are acceptable, the laboratory will be returned to "approved"
status.
• If a laboratory fails the next set of OPT samples (two sets of OPTs in a row), the laboratory will be
disapproved for analysis of LT2 samples and must get additional training. The laboratory must consult
with EPA regarding the level and type of training required, and must submit to EPA proof of this
training upon completion. If the laboratory's training is acceptable, the laboratory must repeat the IPT
test before being re-approved.
If the laboratory is disapproved a second time, the laboratory will no longer be allowed to participate in
the program. Actions taken at this point are discussed in Section 3.2.7.
Draft 16 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
3.2.7 Notifying Utilities of Laboratory Status
Two actions are taken when a laboratory's status changes:
• The laboratories must notify clients
• EPA will post updates on http://www.epa.gov/safewater/lt2/index.html
When a laboratory receives notice that they have been "disapproved," the laboratory must notify their
clients of their status change within 2 weeks and work with their clients to finish up analysis of samples
that they have in house. After the 2-week period the laboratory will no longer be able to analyze samples
under LT2 until they have been reapproved. Laboratories should have a plan in place to divert samples to
another laboratory in the event that this situation arises.
All samples being processed by the laboratory at the time of the disapproval are considered acceptable,
provided all QC and holding time requirements (detailed in EPA Methods 1622 and 1623 and Section 3.3
and summarized in Appendix D) are met. Analysis of these samples should be completed by the
laboratory. However, no new LT2 samples may be processed; this includes samples that have been
received by the laboratory, but that have not been filtered (for bulk samples) or eluted (for field-filtered
samples). Any new samples processed after the laboratory has been disapproved will not be considered
acceptable for monitoring under the LT2 rule.
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 [40 CFRpart 141.705 (a) (3)]. The requirements discussed in this guidance
manual are based on the June 2003 versions of EPA Methods 1622/1623, the use of which EPA has
requested comment on in the LT2 rule proposal (see Section 3.1.2 for details).
Sections 3.3.1 through 3.3.14 provide guidance on the implementation of the QC requirements specified
in the June 2003 version of EPA Method 1622/1623. Routine QC requirements that must be verified
internally by the laboratory before reporting LT2 rule monitoring results are called out below, and
included in checklist format as Appendix D. This guidance is provided to help implement the QC
requirements in the methods and does not substitute for, or alter, the method requirements.
Draft 17 June 2003
-------
Section 3: Guidance for Cryptosporidium Laboratories
Figure 3-1. Process for Laboratory Approval and Changes in Laboratory Approval Status
Submit
Program
Application
Submit missing or incomplete
information
• Yes
Lab must do the following:
1. Receive training on deficient areas
2. Analyze 25 samples
3. Repeat I PR analysis
Yes
Analyze
Initial
Performance
Testing Samples
(IPTs)
Lab must wait 6 months to receive 4th
set of IPTs and do the following:
1. Analyze 50 additional samples
2. Repeat I PR analysis
Yes
Laboratory can no longer participate
in Lab QA Program
Respond to any deficiencies cited in
EPA audit report
On-Site
Evaluation
Provide additional information to EPA
Lab is "Disapproved," and must
receive additional training and analyze
IPT samples
Analyze Ongoing
Performance
Testing Samples
(OPTs)
Laboratory can no longer participate
in Lab QA Program
Draft
18
June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
3.3.1 Cryptosporidium Spiking Materials
During LT2 Cryptosporidium monitoring, laboratories must analyze samples spiked with
Cryptosporidium parvum oocysts to assess ongoing laboratory and method performance, per method QC
requirements. These ongoing spiked sample analyses include ongoing precision and recovery samples
(OPRs) (Section 3.3.4) and matrix spike samples (Section 3.3.13). Flow cytometer-counted spiking
suspensions must be used for these QC samples [40 CFRpart 141.705 (a) (3)].
Sources of flow cytometer-counted Cryptosporidium spiking suspensions for use with routine, spiked
Cryptosporidium QC samples include the following:
I.Wisconsin State Laboratory of Hygiene
Flow Cytometry Unit
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)
www.biotechnologyfrontiers.com
Unit 1 35-41 Waterloo Road
North Ryde NSW
Australia
Fax: +61 2 9889 1805
Email: contact@biotechnologyfrontiers.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. Per
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.
Flow-cytometer-counted spiking suspensions used for spiked quality control (QC) samples must be used
within the expiration date noted on the suspension. 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.
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 initial 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 3.3.14).
The results of the four analyses are used to calculate the average percent recovery and the relative
standard deviation (RSD) of the recoveries for Cryptosporidium (Section 3.7). For EPA Method
1622/1623, the mean Cryptosporidium recovery must be in the range of 24% to 100% and the RSD of the
four recoveries must be less than 55%.
Draft 19 June 2003
-------
Section 3: Guidance for Cryptosporidium Laboratories
3.3.3 Method Blank Test
The method blank test required by EPA Method 1622/1623 consists of analysis of an unspiked reagent
water sample to demonstrate freedom from contamination. Per the method, one method blank sample
must be analyzed each week or every 20 field and matrix spike samples, whichever is more frequent. If
more than one method variation will be used for filtration and/or separation of samples, a separate method
blank may be required for each variation (see Section 3.3.14).
If one or more Cryptosporidium oocysts (as defined in Section 3.1.6) are found in a blank, analysis of
additional samples is halted until the source of contamination is eliminated and a blank shows no
evidence of contamination.
,•'
L T2 rule requirement: For each method blank, oocysts must not be detected [40 CFRpart
141.705 (a) (3)].
part \
3.3.4 Ongoing Precision and Recovery Test
The ongoing precision and recovery (OPR) in 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
must be analyzed each week or every 20 field and MS samples, whichever is more frequent. If more than
one method variation will be used for filtration and/or separation of samples, a separate method blank
may be required for each variation (see Section 3.3.14).
OPR samples should be analyzed before any field samples in a batch are processed to verify acceptable
performance. OPR Cryptosporidium recovery must be in the range of 11% to 100% to be considered
acceptable.
/*
L T2 rule requirement: Ongoing precision and recovery results must be 11% to 100% [40
CFK part 141.705 (a) (3)].
40 \
3.3.5 Ongoing Precision and Recovery Control Charts
As noted in Section 9.7.5 of the June 2003 version of EPA Method 1622/1623, laboratories should
maintain a control 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. Based on the
mean of the recoveries 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 an OPR control chart is provided in Figure 3-2. After each 5 to 10 new recovery
measurements, new control limits should be recalculated using the most recent 20 to 30 data points. These
calculated control limits should not exceed the OPR criteria of 11% to 100%. Control charts can be used
to track the laboratories performance and determine if any trends in recovery are occurring. If recovery
measurements fall below the lower control limit or above 100%, laboratories should take corrective
Draft 20 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
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 also is useful for investigating the cause of
acceptable, but outlying, OPR measurements identified through the use of control charts.
3.3.6 Quality Control Batches
As noted in Appendix D and Sections 3.3.3 and 3.3.4, all LT2 Cryptosporidium samples must be
associated with an acceptable OPR and method blank sample [40 CFR part 141.705 (a) (3)]. LT2 samples
are associated with QC samples through a "QC batch." A QC batch consists of an OPR and method blank
and the maximum of 20 field and MS samples that are eluted, concentrated, and purified in the same
week as the OPR and method blank samples. 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 eluted,
concentrated, and purified in the same time period.
Figure 3-2. Ongoing Precision and Recovery Control Chart Example
100%
90%-
Recovery
Mean + 2 Standard Deviations
Mean - 2 Standard Deviations
0%
01/04/2001
02/01/2001
03/01/2001
03/29/2001
04/26/2001
05/24/2001
06/21/2001
07/19/2001
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 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.
Draft
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June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
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 (if shipped to the laboratory as a bulk sample) or filtration (if 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.
• Staining. The sample must be stained within 72 hours of application of the purified sample to the slide.
• Examination. Although immunofluorescence assay (FA) and 4',6-diamidino-2-phenylindole (DAPI)
and differential interference contrast (DIG) microscopy examination and confirmation ideally should be
performed immediately after staining is complete, laboratories have up to 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 must reduce this holding time. In addition, the
laboratory may adjust the concentration of the DAPI staining solution so that fading/diffusion does not
occur. The laboratory also should evaluate the use of another mounting medium (one alternative is
provided in Section 3.9.2, below).
/
L T2 rule requirement: Each sample must meet the QC criteria for the methods [40 CFK
part 141.705 (a) (3)]. Per EPA Method 1622/1623, samples must be
processed or examined within each of the holding times specif led by
A breakdown of the holding times for each set of steps is provided in Table 3-2.
Draft 22 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
Table 3-2. Cryptosporidium Holding Times for EPA Method 1622/1623
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
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 positive antigen or 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. 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 must 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 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 must indicate on each sample examination results form whether the positive staining control
and negative staining control were acceptable.
L T2 rule requirement:
Each sample must meet the QC criteria for the methods [40 CFK
part 141.705(a) (3)]. Per EPA Method1622/1623, positive and
negative staining controls must be acceptable (Section 15.2.1)
Draft
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June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
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 DIC examination and
size measurements must be conducted at a minimum of 1000X.
Size, shape, and DIC and DAPI characteristics of the three Cryptosporidium oocysts must be recorded by
the analyst in a microscope log.
3.3.10 Ongoing Analyst Verification
Analyst verifications are ongoing comparisons of slide counts among multiple analysts in a laboratory to
assess and maintain consistency in slide examination among analysts. At least monthly when microscopic
examinations are being performed, the laboratory shall prepare a slide containing 40 to 100 oocysts. More
than 50% of the oocysts must be DAPI positive and undamaged under DIC.
For laboratories with multiple analysts, each analyst shall determine the total number of oocysts and cysts
and the number that are DAPI positive and DAPI negative for the entire slide. For 10 oocysts and 10
cysts, each analyst shall determine the number of nuclei by DAPI and the DIC category (empty,
containing amorphous structures, or containing identifiable internal structures) of each. The total number
and the number of DAPI positive and DAPI negative oocysts and cysts determined by each analyst
(Section 10.5.2.) 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. Differences in the number of nuclei by DAPI and in DIC
categorizations among analysts must be discussed and resolved, and these resolutions must be
documented.
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 also should perform repetitive counts of a
single verification slide for FITC and DAPI. These laboratories should also coordinate with other
laboratories to share slides and compare counts.
3.3.11 Proficiency Testing Samples
As part of the Laboratory QA Program, laboratories must successfully analyze IPT samples initially, and
OPT samples approximately every 4 months when provided by EPA. Proficiency testing samples required
as part of the Laboratory QA Program are described in Sections 3.2.2 and 3.2.5.
3.3.12 Acceptance Criteria for Receipt of Field Samples
Cryptosporidium samples for LT2 rule monitoring that are not analyzed the same day they are collected
must be maintained below 10°C and not allowed to freeze to reduce biological activity. This is specified
in Section 8.0 of the June 2003 versions of EPA Method 1622/1623. Because Cryptosporidium samples
collected for the LT2 rule must meet the QC criteria in the methods [40 CFR part 705 (a) (3)], and
because these QC criteria include receipt of samples at <10°C and not frozen, laboratories must reject
LT2 Cryptosporidium samples received at >10°C or frozen. In these cases, the PWS must re-collect and
re-ship the sample.
Draft 24 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
Several options are available to measure sample temperature upon receipt at the laboratory and, in some
cases, during shipment, are provided in Section 2.2.
X
L T2 rule requirement: Each sample must meet the QC criteria for the methods [40 CFK
part 141.705 (a) (3)]. Per EPA Method 1622/1623, samples not
processed on the day of collection must be received at the
laboratory at < 10°C and not frozen (Section 8.1)
3.3.13 Matrix Spike Samples
The matrix spike (MS) in EPA Method 1622/1623 (Section 9.5 of the June 2003 version) entails analysis
of a separate field aliquot spiked with 100 to 500 oocysts to determine the effect of the matrix on the
method's oocyst recovery. During LT2 Cryptosporidium monitoring, PWSs are required, at a minimum,
to analyze one MS sample for every 20 field samples from their source water. However, matrix spike
samples may be analyzed more frequently 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:
• PWSs serving more than 10,000 people that perform monthly monitoring for 2 years and collect 24
field samples
• PWSs serving fewer than 10,000 people that are triggered into monitoring for 1 year and collect 24
field samples
For PWSs serving greater than 10,000 people that perform semi-monthly or more frequent monitoring for
2 years and collect 48 or more samples, a minimum three MS samples must be analyzed.
S
L T2 rule requirements: (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% of the volume of the field sample analyzed. (3)
The MS and field sample must be analyzed by the same procedure
[40 CFRpart 141.705 (a) (2) (i)].
3.3.13.1 Matrix Spike Frequency
For all PWSs, the first MS sample should be collected and analyzed during the first sampling event under
the monitoring program and at least 12 months must elapse between the first and last MS sample, per
EPA Method 1622/1623 (Section 9.1.8). The PWS should evaluate the MS recoveries, as well as other
attributes of sample processing and examination, and work with the laboratory to determine whether
sample filtration and processing procedures are working acceptably, or need to be re-evaluated.
If it is not possible to analyze an MS sample for the first sampling event due to laboratory sample
processing burden or other reasons, the first MS sample should be analyzed as soon as possible to identify
potential method performance issues with the matrix. The requirement that at least 12 months must elapse
between the first and last MS sample still applies. For example, if a PWS that is monitoring monthly for
24 months is unable to process an MS sample until the 8th sampling event, due to laboratory sample
processing load, the second MS sample can be processed no earlier than the 20th sampling event.
Draft 25 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
3.3.13.2 Matrix Spikes for Field-Filtered Samples
Matrix spike samples must be spiked and filtered in the laboratory. PWSs that field-filter 10-L sample
may field filter the monitoring sample for a scheduled sampling event during which an MS is collected,
but must collect and ship the MS sample in bulk to the laboratory for spiking, filtering, and analysis.
PWSs that field-filter >10-L samples should follow the same procedure. As noted above, the volume of
the MS sample analyzed must be within 10% of the volume of the associated field sample. However, if
shipping the entire bulk sample is cost-prohibitive, the PWS is permitted to filter all but 10 L of the MS
sample in the field, and ship the filtered sample and the remaining 10 L of source water to the laboratory
to have the laboratory spike the remaining 10 L of water and filter it through the filter used to collect the
balance of the sample in the field [40 CFRpart 141.705 (a)(2)(ii)].
3.3.13.3 Matrix Spikes Across Multiple Method Variations Used for the Same Public Water
System
During the LT2 rule, each PWS must perform MS analyses at a frequency of 1 MS per 20 field samples,
regardless of whether the analytical procedure is changed during the course of the analysis of the 20 field
samples (such as changing from the use of one approved filter to another approved filter). EPA
recommends, but does not require, that a PWS evaluate method performance using an additional MS
sample if the PWS changes the filter used.
3.3.14 QC Guidance for Method Modifications and Use of Multiple Method
Variations
EPA Methods 1622/1623 are performance-based methods and, therefore, allow method modifications and
the use of different method variations (including the original method and modifications of this method) if
a laboratory can meet applicable QC criteria (EPA Method 1622/1623 [Section 9.1.2]) . 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 bulk water sample for examination. The use of different sample processing components (such as
different filters) or substantively different sample processing procedures (such as additional rinses and
transfers to reduce carryover after IMS) are considered to be different method variations. The following
are considered to be different method variations of EPA Method 1622/1623; this list is not exhaustive,
and serves only to provide examples of different method variations:
• Standard Envirochek™ filtration procedure using elution, centrifugation, IMS, and IFA
• Envirochek™ HV filtration procedure using elution, centrifugation, IMS, and IFA
• Standard Filta-Max™ procedure using elution tube and concentrator tube, IMS, and IFA
• Filta-Max™ procedure using stomacher, concentrator tube, IMS, and IFA
• Filta-Max™ procedure using stomacher, centrifugation, IMS, and IFA
• Filta-Max™ procedure using elution tube and centrifugation, IMS, and IFA
• Direct centrifugation, IMS, and IFA
• The use of different IMS kits
• The use of different antibody staining kits
• The use of additional rinses and transfers to reduce carryover from IMS to the slide
• The use of multiple filter membranes in the Filta-Max™ concentrator tube
Draft 26 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
Guidance on requirements for initial and ongoing demonstrations of acceptable laboratory performance
for different method variations is provided in Sections 3.3.14.1 through 3.1.14.3, below.
3.3.14.1 Using Only One Method Variation
Per EPA Method 1622/1623 (Section 9.1.2) and the Lab QA Program, if a laboratory uses the same
method variation for all samples, then the laboratory is only required to demonstrate acceptable initial
laboratory performance (through the IPR and IPX tests) once and demonstrate acceptable ongoing
laboratory performance (through the OPR, method blank, and OPT tests) using that method variation.
3.3.14.2 Switching from One Method Variation to Another
Per EPA Method 1622/1623 (Section 9.1.2) and the Lab QA Program, if a laboratory intends to switch
completely from one method variation to another, then the laboratory must demonstrate acceptable initial
laboratory performance (through the IPR and IPX tests) using the new method variation before
implementing this procedure for the analysis of field samples. If the laboratory demonstrates 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.
3.3.14.3 Using Multiple Method Variations
Per EPA Method 1622/1623 (Section 9.1.2) and the Lab QA Program, if a laboratory intends to use
multiple method variations concurrently, then the laboratory must demonstrate acceptable initial
laboratory performance (through the IPR and IPX tests) using each method variation before implementing
this procedure for the analysis of LT2 rule samples. If the laboratory demonstrates acceptable initial
laboratory performance and implements multiple method variations, then the laboratory must demonstrate
acceptable ongoing laboratory performance (through the OPR, method blank, and OPT tests) using each
method variation if the method variations differ through the use of different filters or IMS systems.
The laboratory is not required to demonstrate acceptable ongoing laboratory performance using each
method variation if the method variations differ through the use of different antibody stains, the use of
additional rinses and transfers for some samples to reduce carryover from IMS onto the slide, or multiple
membrane filters in the Filta-Max™ particle concentrator for some samples.
• If a laboratory alternates among more than one antibody staining kit, the laboratory must perform
positive and negative staining controls for each antibody kit for each batch of slides to which the kit is
used and must 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.
• If the laboratory uses additional rinses and transfers for some samples to reduce carryover from IMS
onto the slide, the laboratory must use this procedure (which may reduce recoveries) to demonstrate
acceptable ongoing 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.
• If the laboratory uses multiple membrane filters in the Filta-Max™ particle concentrator for some
samples, the laboratory must use this procedure (which may reduce recoveries) to demonstrate
acceptable ongoing 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.
Draft 27 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
3.3.15 Guidance on QC Requirements 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
3.3.15.1 through 3.3.15.3.
3.3.15.1 Guidance on 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 (IPX) 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 (however,
demonstrating acceptable performance at spikes of 100 - 200 is preferable).
3.3.15.2 Guidance on Ongoing Precision and Recovery Tests and Method Blank Tests for
Different Sample Volumes
A laboratory with multiple PWS clients representing a range of sample volumes is not responsible for
performing ongoing precision and recovery (OPR) tests for each of the volumes. The following
approaches should be used, depending on whether the laboratory analyzes more than 20 PWS samples per
week:
• A laboratory that processes different sample volumes and more than 20 PWS samples per week—and
that would necessarily be required to process more than one set of OPR and method blank samples each
week—should demonstrate ongoing acceptable performance at both extremes of the volume spectrum
by performing one OPR and method blank at a volume consistent with the highest sample volume
submitted by clients (e.g. 50 L) and performing a second OPR and method blank at a volume consistent
with the lowest sample volume submitted by clients (but not less than 10 L). If more than 40 field
samples are processed in a week, and more than two QC batches are required, the laboratory may use
the volume at either extreme or a volume in between the two extremes. The laboratory 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.
• Laboratories that process fewer than 20 PWS samples per week only need to analyze one set of QC
samples per week, even if the laboratory processes samples of various volumes. However, the
laboratory should perform the OPR and method blank test using the volume most representative of the
anticipated sample volumes of the LT2 rule samples to be analyzed that week. The laboratory also
should work with their PWS clients to schedule clients with different sample volume sizes for different
weeks, so the field samples can be batched with QC samples of comparable volume.
3.3.15.3 Guidance on 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.
Draft 28 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
3.4 Sample Collection Procedures
Several options are available to the PWS in collecting untreated surface water samples for
Cryptosporidium analysis, including the following.
• Collection of bulk water samples for shipment to the laboratory for filtration and analysis.
• On-site filtration of water samples from pressurized or unpressurized sources using the Pall Gelman
Envirochek or Envirochek HV capsule filter.
• On-site filtration of water samples from pressurized or unpressurized sources using the IDEXX Filta-
Max foam filter.
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).
As noted in the PWS guidance manual, EPA strongly 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 should conduct at least one sampling event prior to monitoring to identify and resolve any
problems.
3.5 Sample Processing and Analysis Procedures
Although EPA has proposed the use of the April 2001 versions of EPA Methods 1622/1623 in the LT2
rule, EPA has requested comment on the use of updated versions (dated June 2003) to consolidate several
method-related changes EPA believes are necessary to address LT2 rule monitoring requirements. These
changes are summarized in Section 3.1.2.
This draft manual assumes that the June 2003 versions of EPA Methods 1622 and 1623 will be used for
LT2 rule monitoring. The June 2003 versions of these methods are included as Appendix B and
Appendix C of this guidance manual.
3.6 Recordkeeping
An acceptable 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 and 1623, data should be provided on bench sheets
(Appendix G) and slide examination results forms (Appendix H). Original data, including microscope
examination counts and notes, must be recorded on these forms.
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. Microbiological analyses records must be kept for at
least 5 years.
Draft 29 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
As laboratories perform Cryptosporidium analyses during the LT2 rule, the following data recording
practices should be followed:
• Maintain 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 examination results forms are available for download on the
LT2 website http://www.epa.gov/safewater/lt2/index.html.
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*
• Date and start/stop times of collection*
• Sampler's name and phone number (or alternate contact for laboratory if problems are encountered)
• Start and stop times of collection (if the sample was filtered in the field)
• 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: The combination of these three elements is the unique sample number used to identify the LT2
sample from 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.epa. gov/safewater/lt2/index.html.
Upon receipt of the sample at the laboratory, laboratory personnel should record, at a minimum, the
information in Table 3-3.
Draft 30 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
Table 3-3. Sample Receipt Data Elements to Record in the Laboratory for EPA Method 1622/1623
Cryptosporidium Analysis
Public water system name and ID
Facility name and ID
Sample collection point name and ID
Date of sample collection
Date and time of sample receipt by laboratory
Name of laboratory person receiving the sample
Temperature of sample upon receipt
Any deficiencies (deficiencies may include but are not limited to:
temperature exceeded 10°C, or sample leaked during transport)
exceeded sample holding time, 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 and ongoing precision and recovery samples. These primary data elements are
provided in Table 3-4, and should be recorded on the EPA Method 1622/1623 bench sheet (Appendix
G).
Table 3-4. Primary Data Elements to Record in the Laboratory for EPA Method 1622/1623
Cryptosporidium Analysis
Estimated number of oocysts spiked (MS and OPR samples), based on information provided by the flow-cytometry
laboratory with the spiking suspension
Samples volume spike, in L (MS and OPT 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 was 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 Table 3-5.
Draft 31 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
Table 3-5. QC Data Elements to Record in the Laboratory for EPA Method 1622/1623
Cryptosporidium Analysis
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 7 days
of sample staining)
Analyst 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 could include 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 examination
results form
This information should be recorded on the EPA Method 1622/1623 bench sheet (Appendix G) and slide
examination results form (Appendix H), as appropriate.
Size, shape, and DIG and DAPI characteristics of the three Cryptosporidium oocysts for ongoing analyst
verification (Section 3.3.10) must be recorded by the analyst on a microscope log.
3.7 Calculations for EPA Methods 1622/1623
During LT2 rule monitoring, field sample results must be reported in oocysts/L and MS recoveries must
be calculated and reported. In addition, laboratories must calculate recoveries for other QC samples [40
CFRpart 141.705 (a)]. Calculations for EPA Method 1622/1623 field and QC samples are provided
below, in Sections 3.7.1 through 3.7.3.
3.7.1 Field Sample Calculations
To calculate the concentration of Cryptosporidium in your field sample, reported as oocysts/L, the
following information is needed:
• Number of oocysts detected in the sample (recorded as a primary measurement from the examination
results form)
• Volume analyzed
Draft 32 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
Using these two data elements, the final concentration is 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 3.7.1.1.
3.7.1.1 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 must be determined using the following equations to determine the percentage of the
sample that was examined.
total volume of resuspended concentrate transferred to IMS (mL) (see Section 3.7.1.2)
percent examined =
total volume of resuspended concentrate produced (mL)
volume analyzed (L) = percent examined * sample volume filtered (L)
3.7.1.2 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. 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
3.7.1.3 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 to IMS and examination.
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 = x 5 mL = 27 mL
0.5 mL
20 mL
percent examined = = 0.74 (74%)
27 mL
Draft 33 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
volume analyzed (L) = 0.74 x 10 L = 7.4 L
20 oocysts
final concentration (oocysts/L) = = 2.7 oocysts/L
7.4 L
3.7.2 Matrix Spike Sample Calculations
To determine the percent recovery for a matrix spike (MS) sample the following information is needed:
• The number of oocysts detected in the MS sample
• The estimated number of oocysts spiked into the MS sample
• The number of oocysts detected in the unspiked field sample (to correct for background concentration)
oocysts counted in MS sample - oocysts counted in unspiked field sample
percent recovery = x 100%
oocysts spiked into MS sample
If both a matrix spike (MS) and a matrix spike duplicate (MSB) are analyzed, then the mean recovery and
relative percent difference (RPD) should be calculated and compared to the acceptance criteria in Section
21.0, Tables 3 and 4 of the June 2003 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, calculate the percent recovery for each sample, as described
above, then use the following formula:
percent recovery of MS sample + percent recovery of MSD sample
mean percent recovery =
To calculate the RPD, the absolute value (without sign) of the difference between the number of oocysts
detected in the MS and MSD is divided by the mean of the oocysts detected in both samples to yield a
percentage of the difference.
oocysts detected in MS - oocysts detected in MSD
RPD= xioo%
((oocysts detected in MS + oocysts detected in MSD)/2)
Example. The laboratory prepared both the MS and MSD by spiking two 10-L samples with 100 oocysts
each. For both the MS and MSD, the entire 10-L sample was filtered and 100% of the sample
was examined. The laboratory detected 45 oocysts in the MS sample and 50 oocysts in the
MSD. In the 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.
45 oocysts - 2 oocysts
MS percent recovery = x 100% = 43%
100 oocysts
Draft 34 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
50 oocysts - 2 oocysts
MSD percent recovery = x 100% = 48%
100 oocysts
43% + 48%
mean recovery = x 100% = 45.5%
45 oocysts - 50 oocysts
RPD = x 100% = 10.5%
((45 oocysts + 50 oocysts) / 2)
3.7.3 OPR Sample Calculations
The percent recovery of an OPR sample is calculated using the following formula:
oocysts detected
percent recovery = x 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-|00% = 50.7%
150 oocysts
OPR recoveries are compared to the limits for ongoing recovery in Table 3 and 4 in Section 21.0 of the
June 2003 version of EPA Method 1623. These recoveries are tracked overtime using control charts to
assess precision, as discussed in Section 3.3.5, above.
3.8 Electronic Data Reporting
During the LT2 rule, laboratories will report Cryptosporidium data to their PWS clients electronically
through EPA's LT2 Data Collection System. The LT2 Data Collection System 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 LT2 Data Collection System 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 LT2 Data
Collection System is provided in Table 3-6, below.
Draft 35 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
Table 3-6. LT2 Data Collection System Data Entry, Review, and Transfer Process
Laboratory actions
• Laboratory posts analytical results to the LT2 Data Collection System
• LT2 Data Collection System reduces data and checks data for completeness and compliance with LT2
rule requirements
• Laboratory Principal Analyst confirms that data meet quality control requirements
• Laboratory "releases" results electronically to the PWS for review
• Laboratory user cannot edit data after it is released to the PWS
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PWS actions
• PWS cannot edit data - only review data and either return to laboratory to resolve errors or submit to EPA
• PWS reviews electronic data through LT2 Data Collection System
• PWS "releases" data back to the laboratory if 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 are not valid for use in LT2 binning)
EPA and State actions
• EPA and State users cannot edit data - only review data
• EPA and State review data through LT2 Data Collection System
The data reporting process is discussed in more detail below, in Sections 7.2.1 through 7.2.3, and
discussed in detail in the Users' Manual for the Long Term 2 Enhanced Surface Water Treatment Rule
(LT2 Rule) Data Collection System. The LT2 data system users' guide also provides detailed information
on the PWS user registration process. Information on the LT2 Data Collection System, as well as a
downloadable users' manual, is available at http://www.epa.gov/safewater/lt2/index.html.
3.8.1 Data Entry/Upload
The analyst or another laboratory staff member enters a subset of the data recorded at the bench (Section
7.1) into the LT2 Data Collection System, either by entering the data using web forms or by uploading
data in XML format. Per 40 CFRpart 141.707(e)(l), this information includes the following:
• PWS ID
• Facility ID
• Sample collection point
• Sample collection date
• Sample type (field or MS)
• Sample volume filtered (L), to nearest % L
• 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
Draft 36 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
• 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; these data are not required for field samples
The laboratory must verify that all holding times and other QC requirements were met.
After the information has been entered or uploaded into the 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 Primary Analyst under the Lab QA Program is responsible for verifying the quality and
accuracy of all sample results in the laboratory, and is required to review and approve the results before
they are submitted to the PWS for review. If inaccuracies or other problems are identified, the primary
analyst discusses the sample information with the analyst or data entry staff and resolves the issues before
the data are approved for PWS review.
If no inaccuracies or other issues are identified, the Primary Analyst 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.
3.8.2 PWS Data Review
After the laboratory has released Cryptosporidium data electronically to the PWS using the LT2 Data
Collection System, 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 LT2 Data Collection System for the return of the data to the
laboratory, you also 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 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 you contend are not
valid for use in LT2 binning, and have submitted to EPA for evaluation.
3.8.3 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 LT2 Data Collection System. EPA and State users cannot edit the data.
3.9 Data Archiving
he PWS is required to archive all original, hardcopy quality control data associated with LT2 sample
analyses for 36 months after the end of the second round of Cryptosporidium monitoring (which is
scheduled to take place 6 years after the start of the first round [40 CFRpart 141.731(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 designate this responsibility to the laboratory.
Draft 37 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
3.9.1 Hardcopy Data
The following data should be archived:
• Bench sheets and examination results forms for all LT2 monitoring samples, including both field
samples and MS samples
• Bench sheets and examination results 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 examination results forms for all OPT samples
As part of the Lab QA Program, the laboratory also must maintain the same documentation for their IPR
and IPT data for each method variation used for LT2 samples.
3.9.2 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 a humid chamber in the dark at 0°C to 10°C.
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. Reagents for the mounting medium include the following:
• 8.0 g elvanol—ICN Biomedicals cat. no. 151937, Aurora, Ohio, or equivalent
• 48.0g(40mL)glycerol
• 10%NaN3
• DABCO—Sigma Chemical Co. cat no. D-2522, or equivalent
• Tris buffer—Dissolve 1.2 g Tris (Fisher cat. no. BP152) in 95 mL reagent water, adjust pH to 8.5 with
1NHC1
To prepare the medium, 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.
Draft 38 June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
3.10 Equipment, Supplies, Reagents, and Standards
See Sections 6 and 7 of EPA Method 1622 (Appendix B) or EPA Method 1623 (Appendix C) for details
on the materials needed to perform the Cryptosporidium analyses specified in the methods. Detailed
information on vendors for these materials is provided in Section 3.11.
3.11 Vendor Contact List
3.11.1 Sample Collection and Filtration Supplies
Alamo Water Treatment
13 700 Highway 90 West
San Antonio, TX 78245
Phone: (800) 659-8400 ext. 2
Fax: (800) 659-8402
E-mail: scsales@alamowater.com
BDH/Merck
www.merckeurolab.ltd.uk
Merck Eurolab Ltd.
UK Headquarters
Laboratory Supplies
Merck House, Poole, Dorset
BH15 1TD
Sales:
Phone: 01202669700
Fax: 01202665599
E-mail: info@merckeurolab.ltd.uk
Technical Support:
Phone: 01202669700
Fax: 01202665599
E-mail: info@merckeurolab.ltd.uk
Bertram Controls Corporation LLC
5 Eastview Drive
Farmington, CT 06032
Sales and Technical Support:
Phone: (800) 243-2340
Fax: (800)243-7166
E-mail: techsupport@BertramContols .com
Cole-Parmer
www. coleparmer. com
625 East Bunker Court
Vernon Hills, IL 60061
Sales:
Phone: (800) 323-4340
Fax: (847) 247-2929
E-mail: sales@coleparmer.com
Technical Support:
Phone: (800) 323-4340 ext. 122
Fax: (847) 327-2987
E-mail: techinfo@coleparmer.com
E. Clark and Associates
www. devicelink.com/company9 8
10 Brent Drive
Hudson, MA 01749
Sales and Technical Support:
Phone: (800) 253-2497
Fax:(978)568-0060
Email: mail@clarksol.com
Grainger
www.grainger.com
Contact sales rep. for local address and more
contact info.
Sales:
Phone: (888) 361-8649
Technical Support:
Phone: (800) 535-5400
Draft
39
June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
Sample Collection and Filtration Supplies (Continued)
IDEXX
www.idexx.com
Sales:
Phone: (800) 321-0207 ext.l
Fax: (207)856-0630
Technical Support:
Phone: (800) 321-0207 ext.2
Fax: (207)856-0630
E-mail: water(g),idexx.com
Pall Gelman Sciences
www .pall. com/gelman
600 South Wagner Road
Ann Arbor, MI 48103
Sales:
Phone: (800) 521-1520 ext.2
Fax:(734)913-6495
Technical Support:
Phone: (800) 521-1520 ext.3
Fax:(734)913-6495
PPG Industries
www.ppg.com
One PPG Place
Pittsburgh, PA 15272
Sales and Technical Support:
Phone: (412)434-3131
Tygon
www.tygon.com
Saint-Gobain Performance Plastics Corp.
Distributor:
Ryan Herco. Products
1501 Waco St.
Richmond, VA 23244
Sales:
Phone: (804)672-1158
Fax: (804) 672-0878
Technical Support: Saint-Gobain
Phone: (800) 798-1539
Fax:(800) 488-9466
Email: linda.d.anderson@,saint-gobain.com
3.11.2 Sample Concentration and Purification Supplies
Dynal, Inc.
www.dynal.net
5 Delaware Drive
Lake Success, NY 11042
Sales:
Phone: (800) 638-9416 ext.l
Fax: (516)326-3298
E-mail: uscustserv@dvnalbiotech.com
Technical Support:
Phone: (800) 638-9416 ext.2
Fax: (516)326-3298
E-mail: ustechserv@dvnalbiotech.com
International Equipment Company
www. labcentrifuge. com
300 Second Avenue
Needham Heights, MA 02492
Sales and Technical Support:
Phone: (800) 841-1113 ext.9723
Fax: (781)444-6743
E-mail: pat.webb@thermoiec.com
Draft
40
June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
3.11.3 Slides and Related Supplies
Dynal, Inc.
www. dynal .net
5 Delaware Drive
Lake Success, NY 11042
Sales:
Phone: (800) 638-9416 ext.l
Fax: (516)326-3298
E-mail: uscustserv(g),dynalbiotech.com
Technical Support:
Phone: (800) 638-9416 ext.2
Fax: (516)326-3298
E-mail: ustechserv(g),dynalbiotech.com
Mauser Scientific
www.deltasci.com
Distributed by Delta Scientific
1979 Stout Drive, Bldg. 5
Ivyland, PA 38974
Sales and Technical Support:
Phone: (800) 428-9400
Fax: (800)428-3271
E-mail: dsirep@aol.com
Meridian Diagnostics, Inc.
3471 River Hills Drive
Cincinnati, OH 45244
Sales:
Phone: (800) 543-1980
Fax: (513)271-0124
Technical Support:
Phone: (800) 343-3858
Fax: (513)271-0124
PGC Scientifics
www.pgcscientifics.com
P.O. Box 7277
Gaithersburg, MD 20898-7277
Sales and Technical Support:
Phone: (800) 424-3300
Fax: (800)662-1112
E-mail: cs(g),pgcscientifics.com
Waterborne, Inc.
www.waterborneinc.com
6047 Hurst Street
New Orleans, LA 70118-6129
Sales and Technical Support:
Phone: (504) 895-3338
Fax: (504) 895-3338
E-mail: custserv@waterborneinc.com
3.11.4 Antibody Stains
BioTechnology Frontiers (BTF)
www.biotechnologyfrontiers.com
Unit 1 35-41 Waterloo Road
North Ryde NSW
Australia
Fax: +61 2 9889 1805
Email:contact(gibiotechnologyfrontiers.com
Meridian Diagnostics, Inc.
3471 River Hills Drive
Cincinnati, OH 45244
Sales:
Phone: (800)543-1980
Fax: (513)271-0124
Technical Support:
Phone: (800) 343-3858
Fax: (513)271-0124
Waterborne, Inc.
www.waterborneinc.com
6047 Hurst Street
New Orleans, LA 70118-6129
Sales and Technical Support:
Phone: (504) 895-3338
Fax: (504) 895-3338
E-mail: custserv@waterborneinc.com
Draft
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June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
3.11.5 Microscope Equipment
Chroma Technology Corp.
www.chroma.com
74 Cotton Mill Hill
Brattleboro, VT 05301
Sales:
Phone: (800)824-7662
Fax: (802) 257-9400
Technical Support:
Phone: (800)824-7662
Fax: (802) 257-9400
Nikon
www .nikonusa. com
NIKON
1300 Walt Whitman Road
Melville, NY 11747
Customer Information:
Phone: (800) 52-NIKON
Email: Available through website
Olympus
www.olympusamerica.com
Olympus America INC.
2 Corporate Center Drive, Melville, NY 11747-
3157, U.S.A.
Product Support:
Phone: (800) 446-5967
Email: Available through website
Zeiss
www.zeiss.com
Carl Zeiss Microimaging, Inc.
One Zeiss Drive
Thornwood, N.Y. 10594
Sales and Technical Support:
Phone: (800) 233-2343
Fax: (914) 684-7446
E-mail: micro(a),zeiss.com
3.11.6 Spiking Suspensions and Positive Staining Control Materials
BioTechnology Frontiers (BTF)
www.biotechnologyfrontiers.com
Unit 1 35-41 Waterloo Road
North Ryde NSW
Australia
Fax: +61 2 9889 1805
Email: contact(g),biotechnologyfrontiers.com
Hyperion Research, Ltd.
1008 Allowance Avenue, SE
Medicine Hat, Alberta TIA 3G8
Canada
Sales and Technical Support:
Peter Wallis
Phone: (403) 529-0847
Fax: (403) 529-0852
E-mail: hvperion(g),telusplanet.net
Waterborne, Inc.
www.waterborneinc.com
6047 Hurst Street
New Orleans, LA 70118-6129
Sales and Technical Support:
Phone: (504) 895-3338
Fax: (504) 895-3338
E-mail: custserv@waterborneinc.com
Sterling Parasitology Laboratory,
University of Arizona, Tucson
Building 90, Rm. 202
Tucson, AZ 85721
Sales and Technical Support:
Marilyn Marshall
Phone: (520) 621-4433
Fax: (520) 621-3588
E-mail: marshalm(g),u.arizona.edu
Wisconsin State Laboratory of Hygiene,
Flow Cytometry Unit
2601 Agriculture Drive
Madison, WI 53718
Sales and Technical Support:
Phone: (608) 224-6260
Fax: (608)224-6213
Draft
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June 2003
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Section 3: Guidance for Cryptosporidium Laboratories
3.11.7 Other Laboratory Supplies for EPA Method 1622/1623
Fisher Scientific VWR
www.fishersci.com www.vwr.com
9999 Veterans Memorial Drive 05 Heron Drive
Houston, TX 77038 Bridgeport, NJ 08014
Sales: Sales:
Phone: (800) 766-7000 Phone: (800) 932-5000
Fax: (800) 926-1166 Fax: (856) 467-3336
Technical Support: Technical Support:
Phone: (800) 766-7000 Phone: (800) 932-5000
Fax: (800) 926-1166 Fax: (856) 467-3336
Sigma
www. sigma-aldrich. com
P.O. Box 14508
St. Louis, MO 63178
Sales:
Phone: (800)325-3010
Fax: (800) 325-8070
E-mail: custserv@sial.com
Technical Support:
Phone: (800)325-5832
Fax: (314)286-7828
E-mail: techserv@sial.com
Draft 43 June 2003
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SECTION 4: GUIDANCE FOR E. cou LABORATORIES
Although public water system (PWS) treatment requirements will be determined from Cryptosporidium
monitoring results, the Long-Term 2 Enhanced Surface Water Treatment (LT2) rule also requires that
large PWSs (those serving > 10,000) perform E. coll and turbidity analyses on source water samples [40
CFR part 141.701 (b)]. The Cryptosporidium, E. coli, and turbidity data will be analyzed to evaluate
whether E. coli and/or turbidity levels can be used as reliable indicators of Cryptosporidium occurrence in
surface waters used as source waters for drinking water facilities. Depending upon the results of these
analyses, EPA may permit small PWSs to use E. coli monitoring, or a combination of E. coli and turbidity
monitoring, to determine the need for implementing more expensive Cryptosporidium monitoring or
improved treatment.
Cryptosporidium analysis requirements and recommendations are discussed in Section 3 of this guidance
manual. 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) (available for download from http://www.epa. gov/safewater/lt2/index.html).
E. coli analysis requirements and recommendations for the LT2 rule, including laboratory certification,
quality assurance/quality control (QA/QC), and the procedures for each of the methods approved for use
during LT2 E. coli monitoring, are presented below.
All E. coli sample analyses performed under the LT2 rule must be quantitative [40 CFR 141.705 (b)].
Presence/absence data cannot be used to assess whether E. coli concentration can be used to indicate
Cryptosporidium occurrence levels.
4.1 Laboratory Certification Program
PWS laboratories and other laboratories performing LT2 rule E. coli analyses must be certified for
coliform analyses under the drinking water certification program for a similar method type (e.g.,
membrane filtration, multiple-tube, multiple-well) [40 CFR part 141.706 (b)]. The drinking water
certification program was established to fulfill requirements stipulated by the Safe Drinking Water Act
(SDWA) and the requirements of the National Primary Drinking Water Regulations (NPDWR) (40 CFR
141.28). The certification program requires that all laboratories analyzing drinking water must be certified
by EPA, including EPA's Regional labs, laboratories on Federal Indian Lands, principal state
laboratories, and laboratories that perform analyses under the SDWA in states without primacy. States
that have primary enforcement responsibility (primacy) are required to have laboratory facilities available
which have been certified by EPA (see 40 CFR 142.10) or National Environmental Laboratory
Accreditation Conference (NELAC). All labs that perform testing for compliance purposes must be
certified by the state or EPA. Public water systems serving at least 25 persons or having at least 15 service
connections must comply with the SDWA and the requirements of the NPDWR.
The certification program consists of annual performance evaluation samples and on-site lab audits every
three years by EPA or the state. During laboratory audits EPA or the state will evaluate the laboratory's
Quality Assurance Plan (QAP), performance on routine water samples, and chain-of-custody procedures.
The certification process and detailed specifications for certification are described in the EPA Manual for
the Certification of Laboratories Analyzing Drinking Water Certification Manual) (Reference 5.2)
(http://www.epa.gov/safewater/certlab/labindex.html)
Draft 44 June 2003
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Section 4: E. coli Laboratory Guidance
Laboratories analyzing E. coli samples for the LT2 rule must use an E. coli method approved for use
under the rule and must be certified under the drinking water certification program for a similar method
type (e.g., membrane filtration) corresponding to the method the laboratory plans to use for LT2 rule
monitoring [40CFRpart 141.705 (b) and 141.706 (b)]. Approved E. coli methods and their
corresponding drinking water certification program coliform techniques are provided in Table 4-1.
Table 4-1. Approved E. coli Methods for LT2 Rule and Corresponding Drinking Water Certification
Program Techniques
E. coli Methods Approved for LT2 Rule
Standard Methods 9223B (Colilert)®
Standard Methods 9223B (Colilert-18)®
Standard Methods 9222B/9222G1
(mEndo/LES-Endo-NA-MUG)
Standard Methods 9222D/9222G (mFONA-MUG)
Standard Methods 921 3D/
EPA Method 1103.1 (mTEC)
EPA Method 1603 Modified mTEC
EPA Method 1604 Ml medium 1
m-ColiBlue24® Broth 1
Standard Methods 9221B/9221F (LTB-EC-MUG)
Appendix References
for Methods Included
in this Manual
Appendix I
Appendix J
Appendix K
Appendix L
Appendix M
Appendix N
Appendix O
Appendix P
Corresponding Drinking Water
Certification Program Coliform
Analysis Technique
Most probable number (MPN):
Multiple-tube and multiple-well
techniques
Membrane filtration technique
MPN: Multiple-tube fermentation
technique
1 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, SM9221B/9221F)
4.2 Quality Assurance/Quality Control for E. coli Analyses
Laboratory QA for E. coli analyses during LT2 monitoring should meet the specifications in the
Certification Manual (Reference 5.2) and Standard Methods for the Examination of Water and
Wastewater, 20th Edition (1998) (Standard Methods) (Reference 5.3) for facilities, personnel, laboratory
equipment, instrumentation, and supplies used for LT2 E. coli analyses. In addition, each laboratory
should have implemented a written QA plan describing the QA program and QC activities necessary to
meet the laboratory's specific needs. The program 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.
E. coli sample results reported under the LT2 rule should meet the quality control (QC) specifications set
forth in the approved versions of the methods listed in Table 4.1 above. Section 4.2.1 describes overall
quality control specifications for E. coli analyses performed under the LT2 rule. E. coli QC specifications
specific to membrane filtration methods and MPN methods are provided in Sections 4.2.2 to 4.2.3. This
guidance is provided to help implement the QC specifications in the methods and does not substitute for
or alter the method specifications. Sample results that do not meet these specifications are not considered
valid, and cannot be reported under the LT2 rule. A checklist for these specifications is provided as
Appendices Q and R.
Draft
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June 2003
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Section 4: E. coli Laboratory Guidance
Note: E. coli samples that do not meet the QCspecifications set forth in these sections are
considered to be invalid, and may not be reported under the L T2 rule.
Table 4-2. Quality Control Procedures for E. coli Methods
are ^
Quality Control Procedure
Reference
All E. coli Methods
Holding time and temperature requirements
(Section 4. 2. 1.1)
Dilution/rinse water sterility check
(Section 4.2.1. 2)
Media sterility check
(Section 4.2.1. 3)
Positive/negative controls
(Section 4. 2. 1.4)
Media storage
(Section 4. 2. 1.5)
LT2 Rule: Section IV.K.2
Certification Manual: Section
4.4.3
Certification Manual: Section
5.1.6.4
Certification Manual:
Section 5.1.6.4
Certification Manual:
Section 5.3.2.4
MPN Methods (includes multiple-tube and multiple-well techniques)
Incubation time and temperature specifications
(Section 4.2.2.1)
MPN preparation blank
(Section 4.2.2.2)
Verification
(Section 4.2.2.3)
Specified in Method
Recommended
Specified in Method
Membrane Filtration Methods
Incubation time and temperature specifications
(Section 4.2.3.1)
Filtration unit sterilization
(Section 4.2.3.2)
Membrane filter preparation blank
(Section 4.2.3.3)
Verification
(Section 4.2.3.4)
Specified in Method
Certification Manual:
Section 4.1
Certification Manual:
Section 5.2.1.3
Specified in Method
4.2.1 Quality Control Specifications Applicable to All E. coli Methods
Independent of QC specifications for the type of method used for analysis, there are QC measures that
should be taken by all laboratories performing E. coli analyses under the LT2 rule to ensure the laboratory
is in control during the analysis of LT2 monitoring samples. Quality assurance/quality control procedures
for all E. coli methods are summarized in Sections 4.2.1.1 through 4.2.1.5.
4.2.1.1 Holding Time and Temperature Requirements for Field Samples
EPA strongly encourages laboratories to analyze samples as soon as possible after collection. However,
due to the need by some utilities to ship samples overnight to an off-site laboratory for analysis, the
holding time for E. coli sample analyses has been extended to 24 hours. Based on studies to assess the
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effect of increased sample holding time in E. coli analysis results, EPA has concluded that E. coli samples
can be held for up to 24 hours prior to analysis without compromising the data quality objectives of the
LT2 rule (Reference 5.4).
Samples must be maintained between 0°C and 10°C during transit [CFRpart 141.705 (b) (1)] and should
not be allowed to freeze. For samples shipped off-site, utilities should consider including a temperature
blank along with the sample so that laboratories can measure the temperature of the sample upon receipt.
Alternately, utilities could use Thermochron iButtons® or other measuring devices to record the
temperature throughout transport (discussed in Section 2.2 in this manual). Additional information
pertaining to temperature monitoring 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).
4.2.1.2 Dilution/Rinse Water Sterility Check
QC specifications for dilution/rinse water checks are as follows:
• Each batch (or lot, if commercially prepared) of dilution/rinse water should be checked for sterility by
adding 50 mL of water to 50 mL of a double-strength non-selective broth (e.g., tryptic soy, trypticase
soy, or tryptose broth). Incubate at 35°C ± 0.5°C, check for growth after 24 hours and 48 hours (or for
the longest incubation time specified in the method), and record results. Discard batch if growth is
detected.
Note: These QC specif ications are meant to be performed routinely for all samples, at the
frequency specif led in the Certification Manual. The QC specifications are not
additional analyses specifically for LT2 E. coli samples.
4.2.1.3 Media Sterility Check
To test sterility of newly prepared media prior to the analysis of field samples incubate one plate per each
media batch at the appropriate temperature for 24 and 48 hours (or for the longest incubation time
specified in the method) and observe for growth. If any contamination is observed, determine the cause,
correct, and reject any data from samples tested with the media.
Note: These QC specif ications are meant to be performed routinely for all samples, at the
frequency specif led in the Certification Manual. The QC specif ications are not
additional analyses specifically for LT2 E. coli samples. If any contamination is
observed, determine the cause, correct, and reject any data from samples associated
with: (1) dilution or rinse water, or (2) media.
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4.2.1.4 Positive/Negative Controls
For each new lot or batch of medium, check the analytical procedures and integrity of the medium before
use by testing with known positive and negative control cultures. See Table 4-3 for examples of test
cultures.
Laboratories using commercially-prepared media with manufacturer shelf-lives of greater than 90 days
should run positive and negative controls each quarter in addition to running the batch/lot-specific
controls and sterility checks. Laboratories are encouraged to perform positive and negative control tests
each day that field samples are analyzed.
Table 4-3. Positive and Negative Control Cultures
Positive Control Culture
E. coli
Negative Control Culture
Enterobacter aerogenes (ATCC # 13048)1
Klebsiella pneumoniae (ATCC # 31488)2
1 E. aerogenes normally does not grow at 44.5°C; if growth occurs, the waterbath or incubator is not holding the
44.5°C temperature. The recommended strain is MUG negative and is notthermotolerant.
2 K. pneumoniae is MUG negative and should not fluoresce when transferred to NA-MUG. In addition, this strain is
thermotolerant.
Positive and negative controls should be chosen based on the method-specific requirements. For example
if a 44.5°C waterbath is not required by the method, it is not necessary to include Enterobacter aerogenes
as a negative control.
Note: Additional QC analyses are not required for the analysis of E. coli samples under L T2,
provided that laboratories perform required QC analyses at the frequency specified in
the Certification Manual.
4.2.1.5 Media Storage
The following media storage specifications should be met for E. coll analyses:
Agar plates may be held for up to 2 weeks at 1°C to 5°C in plastic bags or containers. Protect media
containing dyes from exposure to light.
Broth in loose fitting caps (e.g., snap caps) should be stored at 1°C to <30°C for no more than 2
weeks
Broth in tight fitting caps (e.g., screw caps) should be stored at 1°C to <30°C for no longer than 3
months
All media should be at room temperature prior to use
• Media exhibiting growth or gas should be discarded
4.2.2 Quality Control Specifications for Most Probable Number Methods
In addition to the overall QC specifications set forth in Section 4.2.1, laboratories using MPN methods
(e.g., multiple-tube, multiple-well) for LT2 rule E. coli analyses should meet method-specific incubation
time and temperature specifications and method-specific E. coll verification specifications.
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4.2.2.1 Incubation Time/Temperature Specifications for MPN Methods
The required incubation times and temperatures for MPN methods are provided in Table 4-4.
Table 4-4. Incubation Time and Temperature Specifications for MPN Methods
Method
Standard Methods 9223B
Standard Methods 9221B/9221F
Media
Colilert®
Coliert-18®
LIB
EC-MUG
Incubation Time/Temperature
24-28hoursat35°C±0.5°C
18-22hoursat35°C±0.5°C
22-51 hoursat35±0.5°C
22 - 26 hours at 44.5°C ± 0.2°C
4.2.2.2 Most Probable Number Preparation Blank
EPA recommends that a volume of sterilized, buffered water be analyzed exactly like a field sample each
day samples are analyzed. The preparation blank should be incubated with the sample batch and observed
for growth of the target organism. If the control indicates contamination with the target organism, all data
from affected samples should be rejected.
4.2.2.3 Verification
Verification specifications are detailed in the Certification Manual (Reference 5.2), Standard Methods
(Reference 5.3), and Appendices I and J of this document. Verifications should be performed in
accordance with method-specific requirements.
Note: Additional QC analyses are not required for the analysis of E. coli samples under L T2,
provided that laboratories perform the verifications at the frequency specif led in //?<
Certification Manual.
4.2.3 Quality Control Specifications for Membrane Filtration Methods
In addition to the overall QC specification in Section 4.2.1, laboratories using membrane filtration
methods should meet method-specific incubation times and temperatures, and should perform additional
QC steps to demonstrate that membrane filtration equipment is free from contamination and cross-over.
Note: Additional QC analyses are not required for the analysis ofE. coli samples under LT2, provided
that laboratories perform required QC analyses at the frequency specified in the Certification Manual.
4.2.3.1 Incubation Time and Temperature
The required incubation times and temperatures for membrane filtration methods are provided in Table 4-
5.
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Table 4-5. Incubation Time and Temperature Specifications for Membrane Filter Methods
Method
Standard Methods 9222B/9222G
Standard Methods 921 3D
EPA 1603
EPA 1604
Other Membrane Filter Methods
Media
mENDO-NA-MUG
Les-ENDO-NA-MUG
mFONA-MUG
mTEC agar
Modified mTEC
Ml agar
m-ColiBlue24 Broth
Incubation Time/Temperature
22 - 26 hours at 35°C ± 0.5°O
4 hours at 35°C ± 0.5°C
22 - 26 hours at 35°C ± 0.5°O
4 hours at 35°C ± 0.5°C
22 - 26 hours at 44.5°C ± 0.2°O
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
2 hours at 35°C ± 0.5°C -
22 - 24 hours at 44.5°C ± 0.2°C
24 hours at 35°C ± 0.5°C
24 hours at 35°C ± 0.5°C
4.2.3.2 Filtration Unit Sterilization
Membrane filter equipment should be autoclaved before the beginning of a filtration series. A filtration
series ends when 30 minutes or longer elapses after a sample is filtered. Ultraviolet (UV) light (254 nm)
may be used to sanitize equipment (after initial autoclaving for sterilization), if all supplies are pre-
sterilized. UV light can also be used to reduce bacterial carry-over between samples during a filtration
series. The UV lamp should be tested quarterly with a UV light meter or an agar plate. Appropriate
corrective actions should be taken, if necessary.
4.2.3.3 Membrane Filter Preparation Blank
If membrane filtration is used, an MF preparation blank is performed at the beginning and the end of each
filtration series by filtering 20-30 mL of dilution water through the membrane filter and testing for
growth. If the control indicates contamination with the target organism, all data from affected samples
should be rejected. A filtration series ends when 30 minutes or more elapse between sample filtrations.
4.2.3.4 Verification
Verification specifications are detailed in the Certification Manual (Reference 5.2), Standard Methods
(Reference 5.3), and in Appendices K through O of this document. Verifications should be done in
accordance with method-specific requirements.
Note: Additional verifications are not required for the analysis ofE. coli samples under LT2, provided
that laboratories perform verifications at the frequency specified in the Certification Manual.
4.3 Sample Collection Procedures
During the LT2 rule, PWSs must take source water samples at a location prior to any treatment and where
the water is no longer subject to surface runoff [40 CFR part 141.704 (a)]. Guidance on sample collection
locations and detailed procedures for collecting 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/safewater/lt2/index.html.
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4.3.1 Acceptance Criteria for Receipt of 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 141.705 (b)(l)]. Samples should not be allowed to freeze.
Given the importance of maintaining sample temperatures for E. coli, laboratories approved for
performing E. coli analyses during the rule 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 are provided in Section 2.2.
4.4 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 necessary to assess high E. coli levels. Initially all PWSs will be
required to analyze four sample volumes (100, 10, 1.0, and 0.1 mL) for all methods except MTF. For
MTF methods the PWS will be initially required to analyze 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. However, if the PWS decides to eliminate any of the required volumes due to
historical data, it should be noted that changes in conditions (e.g., increased precipitation, seasonality,
etc.) may impact water quality. As a result, sample volumes may need to be adjusted on a per sampling
event basis. For example, if there has been a substantial rainfall in the 24 hours prior to sample collection
causing runoff, sample volumes would 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 lower than anticipated, forcing 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.
4.4.1 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). 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 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).
4.4.2 Sample Volume and Dilution Guidance for Membrane Filtration
The analysis of water samples under LT2 requires four different aliquots of sample (100, 10, 1.0, and 0.1
mL) for membrane filtration methods. Iternate 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
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analzyed). 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.
4.4.3 Sample Volume and Dilution Guidance for Multiple-Tube Methods
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 requires the use of a 15-tube most probable number format (i.e., 5
tubes at each of three dilutions, 10.0 mL, 1.0 mL, and 0.1 mL). Five tubes must be inoculated for each
sample volume. Initially, the PWS will be required to analyze four sample volumes (10.0, 1.0, 0.1, and
0.01 mL). Additional guidance on selection of dilutions for multiple-tube methods is available in
Standard Methods 9221 (Reference 5.3).
4.5 E. coli Data Recording and Calculations
Laboratories performing E. coli analyses during the LT2 rule must be certified for the analytical
technique used for LT2 E. coli analyses under the drinking water certification program, and therefore the
data recording practices required under the drinking water certification program should be followed [40
CFRpart 141.706 (b)]. These practices include the following:
Maintaining sample identification information (e.g., including sample collection and receipt dates,
times, and conditions)
Recording all raw data (i.e., primary measurements) used to calculate final sample concentration for
each sample
• Recording the incubation/read times for each method to verify that method specifications were met
Recording the name of the analyst performing the sample analysis
At a minimum, the data elements discussed in detail below should be recorded for E. coli samples during
LT2 monitoring. These data elements are critical to ensure that final sample results can be verified using
primary data, if necessary, and to demonstrate that sample analyses were performed within method-
specified holding times and incubation times. Standardized bench sheets are available for download on
the LT2 website (http://www.epa. gov/safewater/lt2/index.html). Please note that, while E. coli
laboratories should record the data elements discussed in this section to comply with certification manual
specifications, not all of the recorded data will be reported in the LT2 data system. E. coli data reporting
under the LT2 rule is discussed in Section 4.6.
In addition to data elements, 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 critical that laboratories follow the
same procedures for determining E. coli sample concentrations to reduce variability in reporting.
Table 4-6 specifies the minimum sample ID information that must be recorded for each E. coli sample,
either 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 (examples of all forms are provided Appendices S through
AB) [40 CFRpart 141.707 (2)].
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Table 4-6. Minimum Sample ID Information and General Sample Data to Record
PWS ID
Facility ID
Sample collection point
Sample collection date
Analytical method number
Method type
Source type
E. co///100mL
Turbidity
Method-specific data to record for each of the individual method types, as well as standardized
calculations for each method type, are discussed in Sections 4.5.1 to 4.5.3.
4.5.1 Multiple-Well Data
In addition to the data elements listed in Table 4-6, 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 4-7; data to be recorded for the 51-well format (Quanti-Tray®) are noted in Table 4-8.
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-we 11) formats are provided after each table.
4.5.1.1 Data Elements for Quanti-Tray 2000® (97-well) Analyses
Table 4-7. Minimum Data to 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/read date
Incubation start/read time
Incubation start/read temperature
Additional incubation start/read date
Additional incubation start/read time
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4.5.1.2 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% to 80% 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 Data Collection System.
See Section 4.6 for additional information.
B. Determine MPN. Using the number 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 10 and 2 in the table.
MPN = 81.3
100
Analytical result = 81.3 x = 813 £. coli MPN/100 mL
10
4.5.1.3 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-
8.
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Table 4-8. Minimum 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/read date
Incubation start/read time
Incubation start/read temperature
Additional incubation start/read date
Additional incubation start/read time
Additional incubation start/read temperature
4.5.1.4 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% positive wells (41
positive wells) should be used to determine MPN value.
Note: The analytical result can be automatically calculated using the LT2 Data Collection System.
See Section 4.6 for additional information.
B. Determine MPN. Using the number of positive wells from the appropriate dilution, identify the
corresponding MPN/100 mL in the 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/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
MPN value x = £. coli MPN/100 mL
mL 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
83.1 x = 831 E. coli MPN/100 mL
10
4.5.2 Membrane Filtration Data
In addition to the general sample data listed in Table 4-6, 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 4-9, and include the primary measurements needed to calculate the E.
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coll 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.
4.5.2.1 Data Elements for Membrane Filtration Analyses
Table 4-9. Minimum 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_)
CFUon Filter 2
Filter 3 volume (ml_) (e.g., 1.0 ml_)
CFUon Filters
Filter 4 volume (ml_) (e.g., 0.1 ml_)
CFUon Filter4
Holding Time / Incubation Time Information
Primary isolation medium (e.g., mENDO, mFC) incubation start/read date
Primary isolation medium (e.g., mENDO, mFC) incubation start/read time
Primary isolation medium (e.g., mENDO, mFC) incubation start/read temperature
Secondary isolation medium (e.g, NA-MUG) incubation start/read date
Secondary isolation medium (e.g, NA-MUG) incubation start/read time
Secondary isolation medium (e.g, NA-MUG) incubation start/read temperature
4.5.2.2 Determining E. coli Concentrations Using Membrane Filter Data1:
A. E. coll counts should be determined from the volume(s) filtered that yielded 20 to 80 E. coll 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)
Note: The analytical result can be automatically calculated using the LT2 Data Collection System.
See Section 4.6 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 cannot 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.
Adapted from Standard Methods for the Examination of Water and Wastewater, APHA, AWWA,
WEF (20th Edition)
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Note: Results that are TNTC or CNFG are not appropriate for L T2 microbial data analysis,
and cannot be entered into the L T2 Data Collection System. These results should not be
reported.
D. Using the E. coli counts from the appropriate dilution, E. coli CPU/100 mL is calculated based on the
following equation:
100
£ coli CPU x = £ coli CFU/100 mL
mL sample filtered
Example 1:
Filter 1 volume = 100 mL CPU = TNTC
Filter 2 volume = 10 mL CPU = 40
Filter 3 volume = 1.0 mL CPU = 9
Filter 4 volume = 0.1 mL CPU = 0
Using the guidance on countable colonies in Step A, the counts from the 10-mL plate will be used
to calculate the E. coli concentration for the sample:
100
40 £ coli CPU x = 400 £ coli CFU/100 mL
10 mL
E. If no E. coli colonies are present, the detection limit is calculated as
< largest volume filtered per 100 mL.
Example 2:
Filter 1 volume (mL) = 100 mL CPU = 0
Filter 2 volume (mL) = 10 mL CPU = 0
Filter 3 volume (mL) = 1.0 mL CPU = 0
100mL
Detection limit = = £ coli CFU/100 mL
Largest volume filtered
100mL
= <1 £ co//7100 mL
100 mL
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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£. coli CPU /100mL
10ml
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 E. coli CFU/100 mL
(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 E. coli CFU/100 mL
(100+ 10+ 1)
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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. co// CFU/100 mL
10
4.5.3 Multiple-Tube Data
In addition to the general sample data listed in Table 4-6, 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 4-10 for 15-tube most probable number methods and
Table 4-11 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
4.5.3.2.
4.5.3.1 Data Elements for Multiple-Tube Analyses
Table 4-10. Minimum Data Elements to Record for 15-Tube MPN Methods
Primary Measurements
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/read date
Incubation start/read time
Incubation start/read temperature
Additional incubation start/read date
Additional incubation start/read time
Additional incubation start/read temperature
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Section 4: E. coli Laboratory Guidance
Table 4-11. Minimum Data Elements to Record for 15-Tube Fermentation Methods
Primary Measurements
Number of positive tubes 10.0 ml_
Number of positive tubes 1.0 ml_
Number of positive tubes 0.1 ml_
Number of 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
LIB incubation start time
LIB incubation start temperature
LIB 24-hour incubation read date
LIB 24-hour incubation read time
LIB 24-hour incubation read temperature
LIB 48-hour incubation read date
LIB 48-hour incubation read time
LIB 48-hour incubation read temperature
EC-MUG incubation 24-hour read date (from 24-hour LIB)
EC-MUG incubation 24-hour read time (from 24-hour LIB)
EC-MUG incubation 24-hour read temperature (from 24-hour LIB)
EC-MUG incubation 24-hour read date (from 48-hour LIB)
EC-MUG incubation 24-hour read time (from 48-hour LIB)
EC-MUG incubation 24-hour read temperature (from 48-hour LIB)
4.5.3.2 Determination of E. coli Concentrations Using Multiple-Tube Methods2:
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 2001 Supplement to the 20th Edition of
Standard Methods, approved by the Standard Methods Committee in 1999.
Note: The analytical result can be automatically calculated using the LT2 Data Collection System.
See Section 4.6 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
Adapted from 2001 Supplement to the 20th Edition of Standard Methods 9221 C: Explanation of Bacterial
Density. This supplement is available for download at http://www.techstreet.com/cgi-bin/detail7product id=923645
(Accessed April 7, 2003).
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Section 4: E. coli Laboratory Guidance
significant dilutions and are selected according to the following criteria. Examples of significant
dilution selections are provided in Table 4-12, 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
4-12, Example A).
• If the lowest dilution (least dilute) tested has less than five tubes with positive results, select it and
the two next succeeding higher dilutions (Table 4-12, Examples B and 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 4-12,
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 4-12, Example E).
If there were not enough higher dilutions tested to select three dilutions, then select the next lower
dilution (Table 4-12, Example F).
C. MPN values need to be adjusted based on the significant dilutions series selected above. Because the
MPN/100 mL values in the table are based on the 10 mL, 1 mL, and 0.1 mL dilution series, per
method requirements, the MPN value must be adjusted if these are not the significant dilution series
selected. Use the following calculation to adjust the MPN when the 10 mL, 1 mL, and 0.1 mL
dilution series are not the significant dilution series selected:
MPN value
Analytical result =
# of mL in middle dilution
= £. co//MPN/100 mL
Table 4-12. Examples of Different Combinations of Positive Tubes (Significant Dilution Results Are
in Bo/dand Underlined)
Example
A
B
C
D
E
F
Least dilute | S 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
£. co/;7100 mL
(after adjustment)
330
48
1.8
400
400
54,000
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Section 4: E. coli Laboratory Guidance
Example A: The significant dilution series for the 5-1-0 combination of positives includes the 1
mL, 0.1 mL, and 0.01 mL dilutions. Since the 10 mL, 1 mL, and 0.1 mL dilutions
were not selected, an adjustment is necessary to account for the dilutions selected:
33
Analytical result = = 330 E. coli 1100 mL
0.1
Example B: Since the 10 mL, 1 mL, and 0.1 mL dilutions are the significant dilutions, no
adjustment is necessary and the result is 48 E. coli/100 mL.
Example C: Since the 10 mL, 1 mL, and 0.1 mL dilutions are the significant dilutions, no
adjustment is necessary and the result is 1.8 E. coli/WO mL.
Examples D and E: The significant dilution series for the 4-4-1 combination of positives includes the
1 mL, 0.1 mL, and 0.01 mL dilutions. Since the 10 mL, 1 mL, and 0.1 mL
dilutions were not selected, an adjustment is necessary to account for the
dilutions selected:
40
Analytical result = = 400 E. coli 1100 mL
0.1
Example F: The significant dilution series for the 5-5-2 combination of positives includes the 0.1 mL,
0.01 mL and .001 mL dilutions. Since the 10 mL, 1 mL, and 0.1 mL dilutions were not
selected, an adjustment is necessary to account for the dilutions selected:
540
Analytical result = = 54,000 E. coli 1100 mL
0.01
4.6 Electronic Data Reporting
During the LT2 rule, laboratories will report E. coli data electronically through EPA's LT2 Data
Collection System to the PWS staff responsible for approving and submitting monitoring results to EPA.
The LT2 Data Collection System 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 LT2 Data Collection System 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 LT2 Data Collection System for
both Cryptosporidium and E. coli samples is provided in Table 3-6, in Section 3.8, above.
The data reporting process is summarized below, in Sections 4.6.1 through 4.6.3, and discussed in detail
in the Users' Manual for the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule) Data
Collection System. The LT2 data system users' guide also provides detailed information on the laboratory
registration process. Information on the LT2 Data Collection System, as well as a downloadable users'
manual, is available at http://www.epa.gov/safewater/lt2/index.html.
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Section 4: E. coli Laboratory Guidance
4.6.1 Data Entry/Upload
The analyst or another laboratory staff member enters a subset of the data recorded at the bench (Section
4.5) into the LT2 Data Collection System either by entering the data using web forms or by uploading
data in XML format [40 CFRpart 141.707 (2)]. This information includes the following:
• Sample ID (optional)
• PWS ID
• Facility ID
• Sample collection point ID
• Sample collection date
Analytical method number
Method type
• Source water type (provided by PWS on sample collection form)
• E. CO///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 LT2 Data Collection System 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.
The laboratory should verify that all holding times and other QC specifications were met.
The laboratory's official contact is responsible for verifying the quality and accuracy of all sample results
in the laboratory, and is required to 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.
4.6.2 PWS Data Review
After the laboratory has released E. coli data electronically to the PWS using the LT2 Data Collection
System, 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 LT2 Data Collection System for the return of the data to the laboratory, you also
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."
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Section 4: E. coli Laboratory Guidance
4.6.3 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 LT2 Data Collection System. EPA and State users cannot edit the data.
4.7 Vendor Contact List for E. coli Methods
Contact information for the vendors for materials used in the methods described in Section 4 is provided
below. Footnotes indicate which vendors are associated with each method.
American Type Culture Collection1
www.atcc.org
P.O. Box 1549
Manassas, VA 20108
Sales:
Phone:(800) 638-6597
Fax: (703) 365-2750
Technical Support:
Phone:(800) 638-6597
E-mail: tech@atcc.org
Becton, Dickinson and Company2
www.bd.com
1 Becton Drive
Franklin Lakes, NJ 07417
Sales:
Phone: (800) 675-0908
Fax:(410)316-4723
Technical Support:
Phone:(800) 638-8663
Fax: (410) 527-0244
E-mail: rep .technical services@ibd.com
bioMerieux6
www.biomerieux.com
595 Anglum Drive
Hazelwood, MO 63042
Sales:
Phone: (800)638-4835
Fax: (800) 657-3053
Technical Support:
Phone: (800)638-4835
Fax: (800) 657-3053
Cole-Parmer1
www. coleparmer. com
625 East Bunker Court
Vernon Hills, IL 60061
Sales:
Phone: (800)323-4340
Fax: (847) 247-2929
E-mail: sales(g),coleparmer.com
Technical Support:
Phone: (800) 323-4340 ext.122
Fax: (847) 327-2987
E-mail: techinfo(gicoleparmer.com
Fisher Scientific1
www.fishersci.com
9999 Veterans Memorial Drive
Houston, TX 77038
Sales:
Phone: (800) 766-7000
Fax:(800)926-1166
Technical Support:
Phone: (800) 766-7000
Fax:(800)926-1166
Hach5
www.hach.com
P.O. Box 389
Loveland, CO 80539
Sales:
Phone: (800) 227-4224
Fax: (970) 669-2932
Technical Support:
Phone: (800) 227-4224
Fax: (970) 669-2932
E-mail: techhelp@hach.com
Biosynth International Inc.2
www.biosynth.com
1665 West Quincy Avenue
Naperville, IL 60540
Sales:
Phone: (800) 270-2436
Fax: (800) 276-2436
Technical Support:
Phone: (800) 270-2436
Fax: (800) 276-2436
E-mail: craig@,biosvnth.com
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Section 4: E. coli Laboratory Guidance
Hardy Diagnostics 2
www.hardvdiagnostics.com
1430 West McCoy Lane
Santa Maria, CA 93455
Sales:
Phone: (800) 266-2222
Fax: (805)346-2760
E-mail: sales@hardvdiagnostics.com
Technical Support:
Phone: (800) 266-2222 ext 5658
Fax: (805)346-2760
IDEXX 4
www.idexx.com
Sales:
Phone: (800) 321-0207 ext. 1
Fax: (207) 856-0630
Technical Support:
Phone: (800) 321-0207 ext.2
Fax: (207) 856-0630
E-mail: water@idexx.com
Pall Gelman Sciences 3
www .pall. com/gelman
600 South Wagner Road
Ann Arbor, MI 48103
Sales:
Phone: (800) 521-1520 ext.2
Fax: (734) 913-6495
Technical Support:
Phone: (800) 521-1520 ext.3
Fax: (734) 913-6495
Oxoid Inc.2
www. oxoid. com
Suite 100, 1926 Merivale Road
Nepean, Ontario K2G1E8, Canada
Sales:
Phone:(613)226-1318
Fax:(613)226-3728
E-mail: oxoid(g),oxoid.com
Technical Support:
Phone: (800) 521-1520 ext.3
Fax: (734) 913-6495
E-mail: oxoid@,oxoid.com
1 Possible vendors for all E. coli methods
2 Vendors for methods: 9221B/9221F, 9222B/9222G, 9222D/9222G, 9213D, EPA Method 1603, EPA Method 1604
3 Vendors for methods: 9222B/9222G, 9222D/9222G, 9213D, EPA Method 1603, EPA Method 1604
4 Vendor for methods: 9221F, 9223B
5 Vendor for method: m-ColiBlue24 broth
6 Vendor for API 20E for EPA Method 1604
Millipore3
www .millipore. com
80 Ashby Road
Bedford, MA 01730
Sales:
Phone: (800) 645-5476
Fax:(781)533-3110
Technical Support:
Phone: (800) 645-5476
Fax:(781)533-3110
Sigma2
www. sigma-aldrich. com
P.O. Box 14508
St. Louis, MO 63178
Sales:
Phone: (800) 325-3010
Fax: (800)325-8070
E-mail: custserv(g),sial.com
Technical Support:
Phone: (800) 325-5832
Fax:(314)286-7828
E-mail: techserv@sial.com
VWR1
www.vwr.com
405 Heron Drive
Bridgeport, NJ 08014
Sales:
Phone: (800) 932-5000
Fax: (856) 467-3336
Technical Support:
Phone: (800) 932-5000
Fax: (856) 467-3336
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SECTION 5: REFERENCES
5.1 Cornell, Kevin, et al. 2000. ICRSS - Building a Better Protozoa Data Set, J. AWWA. 91(10): 30
-43.
5.2 USEPA. 1997. Manual for the Certification of Laboratories Analyzing Drinking Water; Criteria
and Procedures; Quality Assurance: Fourth Edition. EPA 815-B-97-001.
5.3 APHA. 1998. Standard Methods for the Examination of Water and Wastewater; 20th Edition.
American Public Health Association, American Waterworks Association, Washington, D.C.
5.4 Pope, M., et al. 2002. Assessment of the effects of holding time and temperature on E. coll
concentrations in surface water samples. Appl. Environ. Micro, (submitted).
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SECTION 6: ACRONYMS
CPU
CNFG
DAPI
DIG
EPA
FA
FITC
ICR
IDC
IFA
IMS
IPR
IPX
L
LT2 rule
MPC
MPN
MS
MS/MSD
NA-MUG
NELAC
NPDWR
OPR
OPT
PBMS
PT
PWS
QA
QAP
QC
Colony-forming unit
Confluent growth
4, 6-diamidino-2-phenylindole
Differential interference contrast microscopy
U.S. Environmental Protection Agency
Immunofluorescense assay
Fluorescien isothiocyanate
Information Collection Rule
Initial demonstration of capability
Immunofluorescence assay
Immunomagnetic separation
Initial precision and recovery
Initial proficiency test
Liter
Long Term 2 Enhanced Surface Water Treatment Rule
Magnetic particle concentrator
Most probable number
Matrix spike
Matrix spike/matrix spike duplicate
Nutrient Agar (NA) with 4-methylumbelliferyl-beta-D-glucuronide (MUG)
National Environmental Laboratory Accreditation Conference
National Primary Drinking Water Regulations
Ongoing precision and recovery
Ongoing proficiency testing
Performance-based measurement system
Proficiency testing
Public water system
Quality assurance
Quality Assurance Plan
Quality control
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Section 4: E. coli Laboratory Guidance
RSD Relative standard deviation
SDWA Safe Drinking Water Act
SOP Standard operating procedure
TNTC Too numerous to count
UV Ultraviolet
Draft 68 June 2003
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