## Appendix A.1 : Results of the Single

- Laboratory Validation of EPA Method A

for Enterococci and Method B for Bacteroidales in Waters by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay

# Results of the Single-Laboratory Validation of EPA Method A for Enterococci and Method B for Bacteroidales in Waters by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay

June 2010

# U.S. Environmental Protection Agency Office of Water (4303T) 1200 Pennsylvania Avenue, NW Washington, DC 20460i 

## Acknowledgments

The contributions of the following persons and organi zations to this study are gratefully acknowledged: Participant Laboratories

 New York State Department of Health Environmental Bi ology Laboratory: Ellen Braun-Howland and Stacey Chmura

 Mycometrics, LLC: King-Teh Lin and Pi-shiang Lai Volunteer Participants (Sample Collection)

• Amtest Laboratories

• Analytica International, Incorporated

• Analytical Laboratories, Incorporated

• Analytical Services Incorporated

• Anatek Laboratories

• Bay County Laboratory

• Biological Consulting Services

• BioVir

• CH Diagnostics

• City of Everett, Water Pollution Control Facility

• Energy Laboratories

• EPA Office of Research and Development, Nati onal Risk Management Research Laboratory

• Hampton Roads Sanitation District

• HML

• King County Environmental

• Lacey-Olympia-Tumwater

• Madison Metropolitan Sewerage District

• Massachusetts Water Resources Authority

• Narragansett Bay Commission

• Orange County Sanitation District

• Oregon DEQ (Bend Office) near Klamath

• Pittsburgh Water and Sewer Authority

• San Francisco Water

• State of Hawaii Department of Health , Environmental Microbiology Section

• SVL Analytical, Incorporated

• Texas A&M University – College Station

• Texas A&M University – Corpus Christi

• University of Hawaii Water Resources Research Center

• University of Rhode Is land Watershed Program

• University of South Florida

• University of Southern Mississippi

• US Department of Agriculture – Western Regional Research Center

• USEPA Region 1 Laborator y

• USEPA Region 10 Laboratory

• US Geological Survey, Ohio Water Microbiology Laboratory

• Utah Department of Health

• Wisconsin State Laboratory of Hygieneii 

## Disclaimer

Mention of company names, trade names, or commercial pr oducts does not constitute endorsement or recommendation for

use.

Questions concerning this report should be addressed to:

Robin K. Oshiro Engineering and Analysis Division (4303T) U.S. EPA Office of Water, Office of Science and Technology 1200 Pennsylvania Avenue, NW Washington, DC 20460 oshiro.robin@epa.gov or OSTCWAMethods@epa.goviii 

## TABLE OF CONTENTS

Executive Summary .............................................................................................................. ................................................vii

Section 1.0 Background ..................................................................................................................................................... 1
1.1 Summary of the Methods

............................................................................................................................... .........................1

Section 2.0 Study Objectives and Study Design.............................................................................................. .................. 2

2.1 Study Objectives

............................................................................................................................... ........................................2
2.2 Technical Approach: Identification of Laboratories ............................................................................................................2 2.2.1 Analytical Laboratories........................................................................................................ ....................... 2 2.2.2 Volunteer Participants......................................................................................................... ........................ 2
2.3 Technical Approach: Laboratory-Prepared Cell Suspensions ............................................................................................3
2.4 Technical Approach: BioBallTM Spikes

............................................................................................................................... ..3
2.5 Technical Approach: Preliminary Analyses .........................................................................................................................3
2.6 Technical Approach: Single-laboratory Validation Study Analyses .................................................................................5 2.6.1 Assessment of Method Performance............................................................................................... ............ 5 2.6.2 Assessment of Method Performance Across Multiple Fresh or Marine Waters......................................... 5 2.6.3 Development of Draft Quantitative QC Criteria fo r Initial (IPR) and Ongoing (OPR) Method/Laboratory Performance Assessments........................................................................................................................... 6 2.6.4 Development of Quantitative QC Cr iteria for Matrix Spikes (MS)............................................................ 6 2.6.5 Quality Control (QC) Analyses.................................................................................................. ................. 6

Section 3.0 Study Implementation ........................................................................................................... ........................ 11

3.1 Study Management

............................................................................................................................... ..................................11
3.2 Schedule

............................................................................................................................... ....................................................11
3.3 Participant Laboratories

............................................................................................................................... ..........................11

Section 4.0 Data Reporting a nd Validation.................................................................................................. .................... 14

4.1 Data Reporting

............................................................................................................................... .........................................14
4.2 Data Validation

............................................................................................................................... ........................................14
4.3 Censored Data

............................................................................................................................... ..........................................14

Section 5.0 Results........................................................................................................................ ................................... 15

5.1 Methods A Preliminary Results

............................................................................................................................... .............15
5.2 Methods A and 1600 PBS Sample Results .........................................................................................................................15
5.3 Methods A and 1600 Fresh Water a nd Marine Water Sample Results ...........................................................................16
5.4 Method A 25 Fresh Water and 25 Marine Water Sample Results ...................................................................................18
5.5 Method B Preliminary Results

............................................................................................................................... ...............21
5.6 Method B PBS Sample Results

............................................................................................................................... .............21
5.7 Method B Fresh Water and Marine Water Sample Results ..............................................................................................22
5.8 Method B 25 Fresh Water and 25 Marine Water Sample Results ..................................................................................23

Section 6.0 Development of Draft QC Acceptance Criteria .................................................................................... ........ 27

6.1 Initial Precision and Recovery (IPR)

............................................................................................................................... ....27iv Section 7.0 Assessment of Methods A and B Perform ance: Data Analysis and Discussion ........................................... 33

7.1 Method A

............................................................................................................................... ..................................................33 7.1.1 Reference Matrix (PBS): Method A and Associated Method 1600 Analyses .......................................... 33 7.1.2 Fresh and Marine Water Matrices Collected by Each Laboratory: Method A and Associated Method 1600 Analyses.................................................................................................................. ......................... 34 7.1.3 Twenty five Matrices (Fresh and Marine): Method A .............................................................................. 35
7.2 Method B

............................................................................................................................... ..................................................35 7.2.1 Reference Matrix (PBS): Method B............................................................................................... ........... 36 7.2.2 Fresh and Marine Water Matrices Collected by Each Laboratory: Method B.......................................... 36 7.2.3 Twenty five Matrices (Fresh and Marine): Method B .............................................................................. 36

Section 8.0 Conclusion ..................................................................................................................... ............................... 38

Section 9.0 Refere nces..................................................................................................................... ................................ 39

Section 10.0 Acronyms....................................................................................................................... ............................... 40v 

## List of Tables

Table 1. Summary of Validation Study Analyses Conducted for Method A ( Enterococcus )........................................... 6

Table 2. Method B (Bacteroidales) Validation Study Analyses to be Performed by Each Participant Laboratory .......... 9

Table 3. Schedule for the Single-laboratory Validation of Methods A and B............................................................... .. 11

Table 4. Volunteer Participants that Collected and Shi pped Samples to the Analytical Laboratories............................ 12

Table 5. Summary of E. faecalis Results from Methods A (n=4) and 1600 (n=2) for PBS Samples ............................. 16

Table 6. Summary of E. faecalis Results from Methods A (n=4) and 1600 (n=2) Fresh Water Samples ...................... 17

Table 7. Summary of E. faecalis Results from Methods A (n=4) and 1 600 (n=2) Marine Water Samples.................... 17

Table 8. Summary of E. faecalis Method A Results from Twenty Five Fresh Water Matrices (n=4)............................ 18

Table 9. Summary of E. faecalis Method A Results from Twenty Five Marine Water Matrices (n=4) ......................... 20

Table 10. Summary of B. thetaiotaomicron Results from Method B for PBS Samples (n=4) ......................................... 22

Table 11. Summary of B. thetaiotaomicron Results from Method B for Fresh Water Samples (n=4)............................. 22

Table 12. Summary of B. thetaiotaomicron Results from Method B for Marine Water Samples (n=4) .......................... 23

Table 13. Summary of B. thetaiotaomicron Results from Twenty Five Fresh Water Matrices (n=4).............................. 23

Table 14. Summary of B. thetaiotaomicron Results from Twenty Five Marine Water Matrices (n=4) ........................... 25

Table 15. Calculated Draft Initial Precision and Recovery (IPR) Acceptance Criteria for Method A (Enterococcus) .... 28

Table 16. Calculated Draft Initial Precision and Recovery (IPR) Acceptance Criteria for Method B (Bacteroidales) .... 29

Table 17. Calculated Draft Matrix Spike Precision and Recovery Acceptance Criteria for Method A (Enterococcus)... 31

Table 18. Calculated Dr aft Matrix Spike Precision and Recovery Acceptance Criteria for Method B (Bacteroidales)... 32vi 

## List of Appendices

Appendix A: Enterococci (Method A) Study-Specific Instructions

Appendix B: Enterococcus faecalis Spiking Protocol

Appendix C: Bacteroidales (Method B) Study-Specific Instructions

Appendix D: Bacteroides thetaiotaomicron Spiking Protocol

Appendix E: Laboratory Capabilities Checklistvii 

## Executive Summary

This report presents the results of the U.S. Environmental Protection Agency’s (EPA’s) single-laboratory validation study (the “Study”) of the quantitative polymerase chain reacti on (qPCR) procedures for the quantitative detection of enterococci and Bacteroidales, in recreational (fresh and marine) water matrices based on the amplification and detection of target gene fragments of these organisms. Method A [Enterococci in Water by Taqman® qPCR assay and Method B [Bacteroidales in Water by Taqman® qPCR assay] were followed during the Study. The purposes of the Study were to optimize the methods and assess method performance in the si ngle laboratory environment across multiple matrices, and develop draft quantitative quality contro l (QC) acceptance criteria.

Two analytical laboratories, one with access to fresh water ma trices and the other with access to marine water matrices, participated in the Study. During th e Study each laboratory an alyzed unspiked and spiked phosphate buffered saline (reference matrix) samples and fresh or marine water samp les according to Methods A, B and 1600 and study-specific instructions (Appendices A and C), as appropria te. Each laboratory spiked samples with Enterococcus faecalis ( E. faecalis ) or Bacteroides thetaiotaomicron ( B. thetaiotaomicron ) vendor-prepared (BioBallTM) and/or laboratory-prepared cell suspensions. Samples were spiked in accordance with method-specific spiking protocols (“Spiking Protocol,” Appendices B and D). Results from samples spiked with E. faecalis BioBalls (Method A) and laboratory-prepared B. thetaiotaomicron (Method B) cell suspensions were used to assess method performance.

To assess potential matrix issues across multip le fresh and marine water matrices from different geographic regions across the US, thirty-seven volunteer laboratories collected and shippe d recreational water samples to the analytical laboratories for analyses. The Study was conducted over a 5 month period (August 2009 through December 2009).

Results of the single-laboratory validation study indicate that Methods A and B are suitable for multi-laboratory validation in both fresh and marine waters. In addition, data generated during the Study enabled the development of draft performance criteria for use during the multi-laboratory validation studies.Methods A and B Single-Laboratory Validation Study Report

June 2010 1 

## Section 1.0 Background

Enterococci and Bacteroidales are bacteria commonly found in the feces of humans and other warm-blooded animals. Although these organisms can be persistent in the environmen t, their presence in water is an indication of fecal pollution and the possible presence of enteric pathogens. The significance of these organisms in recreational fresh or marine water samples stems from the direct epidemiological relationship ob served between the density of these organisms and the risk of gastrointestinal illness associated with swimming (References 9.1 and 9.2).

Enterococci and Bacteroidales, as measured by molecular methods, are candidate measures of recreational water quality, based on epidemiological studies which were used to establish relationships between health effects and water quality. While current EPA methods are culture-based and require a minimum of 24 hours for results, molecular methods would allow for same day results. In response to stakeholders request for rapid methods for monitoring recreational water quality, EPA has conducted a single-laboratory validation study of Methods A (enterococci [Reference 9.3]) and B (Bacteroidales [Reference 9.4]) in fresh and marine water matrices.

1.1 Summary of the Methods

Methods A and B describe quantitative polymerase chain reac tion (qPCR) procedures for the quantitative detection of enterococci and Bacteroidales bacteria in fresh and marine water matrices based on the amplification and detection of a specific region of ribosomal RNA genes (23S and 16S, respectively) in these organisms.

In Method A, water samples are filtered to collect enteroco cci on membrane filters. Following filtration, total DNA is solubilized from the filter retentate using a glass bead disruption/extraction procedure (bead b eating). Enterococci target DNA sequences present in the clarified extract are detected by the real time polymerase chain reaction (PCR) technique using TaqMan® reagents and the TaqMan® probe system. Th e TaqMan® system signals the formation of PCR products by a process involving enzymatic hydrolysis (5’exonuclease activity) of a fluorogenically-labeled oligonucleotide probe when hybridized to the target sequence undergoing amplification.

In Method B, water samples are filtered to collect Bacteroi dales on membrane filters. Following filtration, total DNA is solubilized from the filter retentate using a glass bead beati ng procedure. Bacteroidales target DNA sequences present in the clarified extract are detected by the real time P CR technique using TaqMan® reag ents and the TaqMan® probe system. The TaqMan® system signals the formation of PCR products during primer extension by a process involving polymerase-directed enzymatic hydrolysis (5’exonuclease ac tivity) of a fluorogenically-labeled oligonucleotide probe when hybridized to the target sequence.Methods A and B Single-Laboratory Validation Study Report

June 2010 2 

## Section 2.0 Study Objectives and Study Design

2.1 Study Objectives

The following objectives were established for the Study:

• Assess method performance (recovery and precision) of Methods A and B in a reference matrix (phosphate buffered saline [PBS]) spiked with vendor-prepared (B ioBallTM) and/or laboratory-prepared spikes

• Assess the performance of Methods A and B across multiple recreational fresh and marine water matrices

• Assess matrix interferences for Methods A and B across multiple fresh and marine recreational water matrices

• Analyze samples using Method 1600 (culture-based method) for Enterococcus (Reference 9.5)

• Assess performance (recovery and precision) of a culture protocol for Bacteroidales

• Establish draft quantitative quality control (QC) accepta nce criteria for initial and ongoing laboratory and method performance assessments for Methods A and B

• Establish draft quantitative matrix spike QC acceptance criteria for Methods A and B

• Assess the consistency of cell calibrators and DNA standards

• Use the results to improve the Methods and proceed to multi-laboratory validation, and pote ntial promulgation at Part 136 The following data quality objective was established for the Study:

• Data produced under this study must be generated according to the analytical and quality assurance (QA)/QC procedures in Methods A, B, and 1600 and study-specific in structions (Appendices A and C). This will ensure that data will be of known and reliable quality , and allow EPA to use the results of the study to identify the need for further revision of the methods.

2.2 Technical Approach: Identification of Laboratories

The Study required two analytical laborator ies and 37 volunteer participants to collect matrix samples. The roles of these laboratories and volunteer participants are described below.

2.2.1 Analytical Laboratories
Two analytical laboratories were chosen that were representative of the general user community, with experience analyzing ambient water samples for enterococci and/or Bacteroidales using qPCR techniques, and access to either fresh or marine recreational water matrices. A detailed Laboratory Capa bilities Checklist (Appendix E) was used to collect this information from laboratories and to screen potential participants to ensure that laboratories were qualified. Analytical laboratories pro cessed and analyzed PBS and fresh or marine recreational water samples. In addition, analytical reagents were supplied to the laborat ories for consistency.

2.2.2 Volunteer Participants
To reduce cost, 37 volunteer participants were recruited to co llect either fresh or marine or both matrix samples. Volunteers had access to the matrices of interest and the ab ility to collect and ship samples on the same day. To reduce the burden, EPA provided all sample collection and shipping supplies.Methods A and B Single-Laboratory Validation Study Report

June 2010 3 2.3 Technical Approach: Laboratory-Prepared Cell Suspensions

Results from samples spiked with laboratory-prepared cell su spensions were used to develop QC acceptance criteria. Samples were spiked with the appropriate laboratory-prepared organism Enterococcus faecalis ( E. faecalis ) or Bacteroides thetaiotaomicron ( B. thetaiotaomicron ) for Method A and B, respectively] cell suspensions to assess method performance (Method B) and develop QC acceptance criteria. Each participant laboratory prepared and enumerated cell suspensions of E. faecalis (ATCC® # 29212TM) and B. thetaiotaomicron (ATCC® # 29741TM) according to the appropriate Spiking Protocols (Appendices B and D) and determin ed optical density (OD) according to Method A or B. To determine the spike concentration participant laboratori es enumerated cell suspensions on the same day that samples were spiked.

2.4 Technical Approach: BioBallTM Spikes

Results from samples spiked with BioBalls (commercially av ailable flow cytometer counted enumerated spikes) were used to assess method performance and to develop QC acceptan ce criteria. During preliminary analyses each participant laboratory spiked samples with BioBallTM spikes of E. faecalis (ATCC® # 29212TM) and B. thetaiotaomicron (ATCC® # 29741TM) according to the appropriate Spiking Protocols (A ppendix B or D). In addition participant laboratories enumerated BioBalls on the day of analysis in triplicate, using the spread plate technique and tryptic soy agar (TSA) according to the appropriate Spiking Protocols (Appendix B or D) to confirm stability of the BioBalls. The “lot mean value” provided by the manufacturer was u sed as the “true spike concentration.”

Due to problems with B. thetaiotaomicron BioBalls when used as calibrator sampl es (e.g., non-detects or very high cycle threshold (CT) values [e.g., 38 CT]) obser ved during Method B preliminary analyses, B. thetaiotaomicron BioBallTM spikes were not used during validation study analyses.

2.5 Technical Approach: Preliminary Analyses

Preliminary analyses using sterile PBS were conducted during the first two to three weeks of the study for each organism (Enterococci followed by Bacteroidales). The following analyses were conducted during preliminary assessment:

Method A

• Labs prepared, extracted, purified, quantified, and analyzed DNA standards using E. faecalis (KwikStikTM) laboratory-prepared cell suspensions and generated DNA standard curves according to Method A and study-specific instructions.

• Labs analyzed two vendor-prepared DNA standards [a synthetic gene with native target sequence (SG-NT), and a synthetic gene with artificial sequences containing native primer sites bracketing a heterologous probe site (SG-AS)] and generated DNA standard curves according to Method A and study-specific instructions.

• Labs prepared, extracted, purified, analyzed, and quantifie d sets of initial and ongoing calibrator samples using bothlaboratory-prepared (Kwik-StikTM) and vendor-prepared [BioBallTM] suspensions according to Method A and studyspecific instructions.

• Labs evaluated two different salmon DNA sample processing controls (SPC [Sketa 2 and Sketa 22]).

• Labs evaluated the use of a DNA purification kit (GeneRite).

• Labs spiked 100-mL PBS and fresh or marine water samples with both E. faecalis laboratory-prepared cell suspensions and BioBalls (separately) at three di fferent levels: 100, 320, a nd 1000 cells per 100 mL.

• Labs analyzed crude (undiluted and diluted [1:5]) and purified (undiluted and diluted [1:5]) extracts.

• Labs processed and analyzed unspiked and spiked PBS and fresh or marine water samples according to Methods A and 1600 and study-specific instructions.Methods A and B Single-Laboratory Validation Study Report

June 2010 4 • Labs analyzed one, 100-mL unspiked st erile PBS (method blank) for enterococci and salmon DNA (Sketa 2 and Sketa 22)

.

• Additional quality control analyses were conducte d as described in Section 2.6.5, below.

Results of the preliminary an alyses were utilized as follows:

• Results from enterococci preliminary analyses were used to redu ce the number of options (e.g., Sketa 2 or 22), evaluated during preliminary analyses for Method B (Bacteroidales), as appropriate.

• Results from spiked PBS and spiked/unspiked fresh and marine water samples were used to determine the spike level(s) for Method A validation study analyses.

• Results from preliminary analyses were also used to ev aluate laboratory performance and identify any issues (e.g., interferences, faulty materials) that needed to be addressed prior to the start of the Method A validation study.

Method B

• Labs prepared, extracted, purified, quantified, and analyzed DNA standards using B. thetaiotaomicron (KwikStikTM) laboratory-prepared cell suspensions and generated DNA standard curves according to Method B and study-specific instructions.

• Labs prepared, extracted, analyzed, and quantified set s of initial and ongoing calibrator samples using both laboratory-prepared (Kwik-StikTM) and vendor-prepared [Bio BallTM] spikes according to Method B and study-specific instructions.

• Labs evaluated a single salmon DNA SPC (Sketa 22).

• Labs spiked 100-mL PBS and fresh or marine water samples with both B. thetaiotaomicron laboratory-prepared cell suspensions and BioBalls (separately) bacteria at three di fferent levels: 10,000, 32,000, and 100,000 cells per 100 mL.

• Labs analyzed crude (undiluted and diluted [1:5]) extracts.

• Labs processed and analyzed unspiked and spiked PBS and fresh or marine water samples according to Method B and study-specific instructions.

• Labs analyzed one, 100-mL unspiked sterile PBS (method blank) for Bacteroidales and salmon DNA (Sketa 22)

.

• Labs evaluated a culture protocol for Bacteroides to determine if it was appropriate for use during the study.

• Additional quality control analyses were conducte d as described in Section 2.6.5, below.

Results of the preliminary an alyses were utilized as follows:

• Results from Bacteroidales preliminary analyses were used to dete rmine the approach for validation study analyses, as appropriate.

• Results from spiked PBS and spiked/unspiked fresh and marine water samples were used to determine the spike level(s) for the validation study analyses. Results from pr eliminary analyses were also used to evaluate laboratory performance and identify any issues (e.g., inte rferences, faulty materials) that needed to be addressed prior to the start of the validation study.

• Results of the evaluation of the culture method were nega tive, thus a culture component was not included during the assessment of Method B.Methods A and B Single-Laboratory Validation Study Report

June 2010 5 2.6 Technical Approach: Single-laboratory Validation Study Analyses

Validation analyses are summarized in Tables 1 and 2 . Method A samples were also analyzed using Method 1600. However, a culture component was not used to evaluate samples analyzed according to Method B due to the poor performance of the culture method during preliminary analy ses. Validation study analyses included the following:

• For Method A, laboratories prepared, extracted, purified, analyzed, and quantified ongoing calibrator samples using E. faecalis laboratory-prepared cell suspensions and BioBalls accord ing to Method A and study-specific instructions. In addition, SPC samples (salmon DNA [Sketa 22]) were analyzed according to Method A.

• For Method B, laboratories prepared, extracted, analy zed, and quantified ongoing calibrator samples using B. thetaiotaomicron laboratory-prepared cell suspensions according to Method B and study-specific instructions. In addition, SPC samples (salmon DNA [Sketa 22]) were analyzed according to Method B.

• Laboratories spiked 100-mL PBS and fr esh or marine water samples with E. faecalis laboratory-prepared and BioBallTM spikes at three concentrations (100, 320, and 1000 cells per 100 mL). Unspiked and spiked samples were filtered, extracted, purified, and analyzed according to Method A and study-specific instructions. In addition, unspiked and spiked PBS, fresh and marine water repl icates were analyzed according to Method 1600.

• Laboratories spiked 100-mL PBS and fr esh or marine water samples with B. thetaiotaomicron laboratory-prepared cell suspensions at three concentrations (10,000, 32,000 , and 100,000 cells per 100 mL). Unspiked and spiked samples were filtered, extracted, and analyzed according to Method B and study-specific instructions.

• For Method A, purified undiluted extracts were analyzed.

• For Method B, crude diluted (1:5) extracts were analyzed.

• Laboratories quantified target sequences in unspiked and spiked samples usi ng calibrator samples and DNA standard curves according to the appropriate methods and study-specific instructions.

• Laboratories analyzed eight replicates (4 unspiked and 4 sp iked) for each of the 25 fresh or 25 marine recreational water samples from different geographic areas across the USA according to Methods A or B and study-specific instructions.

• Laboratories analyzed one 100-mL unspiked ster ile PBS (method blank) sample for enterococci

, Bacteroidales

, and salmon DNA. Additional quality control analyses were completed as described in Section 2.6.5, below.

2.6.1 Assessment of Method Performance

To assess method performance (recovery and precision), each laboratory analyzed 12, 100-mL sterile PBS samples and 12, 100-mL fresh or marine water samples spiked with spiked with E. faecalis (ATCC® # 29212TM) or B. thetaiotaomicron (ATCC® # 29741TM) BioBalls and/or laboratory-prepar ed cell suspensions, four replicates at each spike level. Samples were spiked at three different concentra tions (cells per 100 mL); 100, 320, and 1000 or 10,000, 32,000, and 100,000 for enterococci and Bacteroidales, resp ectively. Recoveries were assessed by quantifying E. faecalis or B. thetaiotaomicron in the spiked samples (minus the ambient concentrations asse ss ed from unspiked PBS and fresh or marine water samples) as determined by Method A or B an alyses. Precision was assessed based on the relative standard deviation of the four replicate recoveries.

2.6.2 Assessment of Method Performance Across Multiple Fresh or Marine Waters

Results from the 25 fresh and marine water samples from diffe rent geographical regions were used to evaluate potential matrix inhibition. Recovery and precision were calculated as described in Section 2.6.1.Methods A and B Single-Laboratory Validation Study Report

June 2010 6 2.6.3 Development of Draft Quantitative QC Criteria for Initial (IPR) and Ongoing (OPR) Method/Laboratory Performance Assessments

To collect the data necessary to develop draft quantitativ e QC (recovery and precision) criteria to assess laboratory performance during the multi-laboratory validation study, each analytical laboratory analyz ed 12, 100-mL sterile PBS samples spiked with E. faecalis (ATCC® # 29212TM) or B. thetaiotaomicron (ATCC® # 29741TM) BioBalls and/or laboratory-prepared cell suspensions, four replicates at each spike level. PBS samples were spiked at three different concentrations (cells per 100 mL) 100, 3 20, and 1000 or 10,000, 32,000, and 100, 000 for enterococci and Bacteroidales, respectively.

2.6.4 Development of Quantitative QC Criteria for Matrix Spikes (MS)

To collect the data necessary to develop draft matrix spike QC (recovery and precision) criteria to assess matrix inhibition during the multi-laboratory validation study, each analytical laboratory analyzed 12, 100-mL fresh or marine water samples spiked with E. faecalis (ATCC® # 29212TM) or B. thetaiotaomicron (ATCC® # 29741TM) BioBalls and/or laboratory-prepared cell suspensions, four replicates at each spike level. Fresh and marine samples were spiked at three different concentrations (cells per 100 mL) 100, 320, and 1000 or 10,000, 32,000, and 100,000 for enterococci and Bacteroidales, respectively.

2.6.5 Quality Control (QC) Analyses

Participating laboratories completed the following QC requirements during each week of study analyses, as appropriate: media sterility checks, filter extraction blanks, dilution wa ter sterility checks, method blanks (sterile PBS), positive controls, and negative ( no template) controls. E. faecalis (ATCC® # 29212TM) or B. thetaiotaomicron (ATCC® # 29741TM) served as the positive control organism.

Table 1. Summary of Validation Study Analyses Conducted for Method A ( Enterococcus )

Sample

Type Description Method 1600: No. Samples Method A: No. Samples

(Sample Processing

Control) Method A: No. Samples

(Target) Purpose of Analysis

Laboratory-prepared E. faecalis (Kwik-StikTM) calibrator samples analyzed by Method A using bead beating and GeneRite (purified) NA 3 (3 replicates × 1 concentration) 3 (3 replicates × 1 concentration) Ongoing Sample Processing Controls & Calibrator Samples E. faecalis (BioBallTM) calibrator samples analyzed by Method A using bead beating and GeneRite (purified) Concentration: 1000 cells NA 3 (3 replicates × 1 concentration) 3 (3 replicates × 1 concentration) QC check and DNA quantitation

E. faecalis (Kwik-StikTM) spiked samples analyzed by Method A using bead beating and GeneRite (purified) Concentrations: 100, 320, & 1000 cells 6 (2 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations) Sterile PBS (Reference

Matrix) E. faecalis (BioBallTM) spiked samples analyzed by Method A using bead beating and GeneRite (purified) Concentrations: 100, 320, & 1000 cells 6 (2 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations) Assess method performance & development of draft QC criteria for initial and ongoing performanceMethods A and B Single-Laboratory Validation Study Report

June 2010 7 Sample

Type Description Method 1600: No. Samples Method A: No. Samples

(Sample Processing

Control) Method A: No. Samples

(Target) Purpose of Analysis

Sterile PBS (Reference

Matrix) Unspiked method blank analyzed for enterococci by Method A using bead beating and GeneRite (purified) 1 2 minimum (1 method blank per run) 2 minimum (1 method blank per run) QC check

Laboratory-prepared E. faecalis (Kwik-StikTM) calibrator samples analyzed by Method A using bead beating and GeneRite (purified) NA 3 (3 replicates × 1 concentration) 3 (3 replicates × 1 concentration) Ongoing Sample Processing Controls & Calibrator Samples E. faecalis (BioBallTM) calibrator samples analyzed by Method A using bead beating and GeneRite (purified) Concentration: 1000 cells NA 3 (3 replicates × 1 concentration) 3 (3 replicates × 1 concentration) QC check and DNA quantitation

Laboratory-prepared E. faecalis (Kwik-StikTM) spiked samples analyzed by Method A using bead beating and GeneRite (purified) Concentrations: 100, 320, & 1000 cells 6 (2 replicates x 3 concentrations) 12 (4 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations)

E. faecalis (BioBallTM) spiked samples analyzed by Method A using bead beating and GeneRite (purified) Concentrations: 100, 320, & 1000 cells 6 (2 replicates x 3 concentrations) 12 (4 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations) Fresh Water

Unspiked samples analyzed for enterococci by Method A using bead beating and GeneRite (purified) 2 4 (2 samples, 2

analytes) 4 (2 samples, 2

analytes) Assess method performance & development of draft QC criteria for matrix spikes

Sterile PBS Unspiked method blank analyzed for enterococci by Method A using bead beating and GeneRite (purified) 1 1 minimum (1 method blank per

analyte, per run) 1 minimum (1 method blank per

analyte, per run) QC check

Laboratory-prepared E. faecalis (Kwik-StikTM) calibrator samples analyzed by Method A using bead beating and GeneRite (purified) NA 3 (3 replicates × 1 concentration) 3 (3 replicates × 1 concentration) Ongoing Sample Processing Controls & Calibrator Samples E. faecalis (BioBallTM) calibrator samples analyzed by Method A using bead beating and GeneRite (purified) Concentration: 1000 cells NA 3 (3 replicates × 1 concentration) 3 (3 replicates × 1 concentration) QC check and DNA quantitation

Laboratory-prepared E. faecalis (Kwik-StikTM) spiked samples analyzed by Method A using bead beating and GeneRite (purified) Concentrations: 100, 320, & 1000 cells 6 (2 replicates x 3 concentrations) 12 (4 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations)

E. faecalis (BioBallTM) spiked samples analyzed by Method A using bead beating and GeneRite (purified) Concentrations: 100, 320, & 1000 cells 6 (2 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations) Marine Water

Unspiked samples analyzed for enterococci by Method A using bead beating and GeneRite (purified) 2 2 2 Assess method performance & development of draft QC criteria for matrix spikesMethods A and B Single-Laboratory Validation Study Report

June 2010 8 Sample

Type Description Method 1600: No. Samples Method A: No. Samples

(Sample Processing

Control) Method A: No. Samples

(Target) Purpose of Analysis

Sterile PBS Unspiked method blank analyzed for enterococci by Method A using bead beating and GeneRite (purified) 1 1 minimum (1 method blank per run) 1 minimum (1 method blank per run) QC check

Multiple Fresh and Marine Matrices

Ongoing Sample Processing Controls & Calibrator Samples E. faecalis (BioBallTM) calibrator samples analyzed by Method A using bead beating and GeneRite (purified) Concentration: 1000 cells NA 3 (3 replicates × 1 concentration) 3 (3 replicates × 1 concentration) QC check and DNA quantitation

Unspiked samples analyzed for enterococci by Method A using bead beating and GeneRite (purified) NA 100 (4 replicates × 25 matrices) 100 (4 replicates × 25 matrices)

Fresh Water E. faecalis (BioBallTM) spiked samples analyzed by Method A using bead beating and GeneRite (purified) NA 100 (4 replicates × single spike concentration × 25 matrices) 100 (4 replicates × single spike concentration × 25 matrices)
Unspiked samples analyzed for background enterococci by Method A using bead beating and GeneRite (purified) NA 100 (4 replicates × 25 matrices) 100 (4 replicates × 25 matrices)

Marine Water E. faecalis (BioBallTM) spiked samples analyzed by Method A using bead beating and GeneRite (purified) NA 100 (4 replicates × single spike concentration × 25 matrices) 100 (4 replicates × single spike concentration × 25 matrices) Assess matrix inhibition

Sterile PBS Unspiked method blank analyzed for enterococci by Method A using bead beating and GeneRite (purified) NA 1 minimum (1 method blank per run) 1 minimum (1 method blank per run) QC checkMethods A and B Single-Laboratory Validation Study Report

June 2010 9 Table 2. Method B (Bacteroidales) Validation Study Anal yses to be Performed by Each Participant Laboratory

Sample

Type Description Method B: No. Samples (Sample Processing Control) Method B: No. Samples (Target) Purpose of Analysis

Ongoing Sample Processing Controls & Calibrator Samples Laboratory-prepared B. thetaiotaomicron(KwikStikTM) calibrator samples analyzed by Method B using bead beating (crude extract) 3 (3 replicates × 1 concentration) 3 (3 replicates × 1 concentration) QC check and DNA quantitation

Laboratory-prepared B. thetaiotaomicron(KwikStikTM) spiked samples analyzed by Method B using bead beating (crude extract) Concentrations: 10,000, 32,000 & 100,000 cells 12 (4 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations) Assess method performance & development of draft QC criteria for initial and ongoing performance Sterile PBS (Reference

Matrix) Unspiked method blank analyzed for Bacteroidales by Method B

, using bead beating (crude extract) 1 minimum (1 method blank per run) 1 minimum (1 method blank per run) QC check

Ongoing Sample Processing Controls & Calibrator Samples Laboratory-prepared B. thetaiotaomicron(KwikStikTM) calibrator samples analyzed by Method B using bead beating (crude extract) 3 (3 replicates × 1 concentration) 3 (3 replicates × 1 concentration) QC check and DNA quantitation

Laboratory-prepared B

. thetaiotaomicron(KwikStikTM) spiked samples analyzed by Method B using bead beating (crude extract) Concentrations: 10,000, 32,000, & 100,000 cells 12 (4 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations) Fresh Water

Unspiked samples analyzed for Bacteroidales by Method B using bead beating (crude extract) 2 2 Assess method performance & development of draft QC criteria for matrix spikes

Sterile PBS Unspiked method blank analyzed for Bacteroidales by Method B using bead beating (crude extract) 1 minimum (1 method blank per run) 1 minimum (1 method blank per run) QC check

Ongoing Sample Processing Controls & Calibrator samples Laboratory-prepared B. thetaiotaomicron(KwikStikTM) calibrator samples analyzed by Method B using bead beating (crude extract) 3 (3 replicates × 1 concentration) 3 (3 replicates × 1 concentration) QC check and DNA quantitation

Laboratory-prepared B

. thetaiotaomicron(KwikStikTM) spiked samples analyzed by Method B using bead beating (crude extract) Concentrations: 10,000, 32,000, & 100,000 cells 12 (4 replicates × 3 concentrations) 12 (4 replicates × 3 concentrations) Marine Water

Unspiked samples analyzed for Bacteroidales by Method B using bead beating (crude extract) 2 2 Assess method performance & development of draft QC criteria for matrix spikes

Sterile PBS Unspiked method blank analyzed for Bacteroidales by Method B using bead beating (crude extract) 1 minimum (1 method blank per run) 1 minimum (1 method blank per run) QC checkMethods A and B Single-Laboratory Validation Study Report

June 2010 10 Sample

Type Description Method B: No. Samples (Sample Processing Control) Method B: No. Samples (Target) Purpose of Analysis

Multiple Fresh and Marine Matrices Ongoing Sample Processing Controls & Calibrator Samples Laboratory prepared B. thetaiotaomicron (KwikStikTM) analyzed by Method B using bead beating (crude extract) 3 (3 replicates × 1 concentration) 3 (3 replicates × 1 concentration) QC check and DNA quantitation

Unspiked samples analyzed for Bacteroidales by Method B using bead beating (crude extract) 100 (4 replicates × 25 matrices) 100 (4 replicates × 25 matrices)

Fresh Water Laboratory prepared B. thetaiotaomicron (KwikStikTM) spiked samples analyzed by Method B using bead beating (crude extract) Concentration: 32,000 cells 100 (4 replicates × single spike concentration × 25 matrices) 100 (4 replicates × single spike concentration × 25 matrices)

Unspiked samples analyzed for Bacteroidales by Method B using bead beating (crude extract) 100 (4 replicates × 25 matrices ) 100 (4 replicates × 25 matrices )

Marine Water Laboratory prepared B. thetaiotaomicron (KwikStikTM) spiked samples analyzed by Method B using bead beating (crude extract) Concentration: 32,000 cells 100 (4 replicates × single spike concentration × 25 matrices) 100 (4 replicates × single spike concentration × 25 matrices) Assess matrix inhibition

Sterile PBS Unspiked method blank analyzed for Bacteroidales by Method B using bead beating (crude extract) 1 minimum (1 method blank per run) 1 minimum (1 method blank per run) QC checkMethods A and B Single-Laboratory Validation Study Report

June 2010 11 

## Section 3.0 Study Implementation

3.1 Study Management

This Study was designed under the direction of the Office of Science and Technology, Engineering and Analysis Division within the U.S. Environmental Protecti on Agency’s (EPA’s) Office of Water. The EPA technical lead was Robin K. Oshiro. Coordination of activities for the Study was performed by the CSC Microbiology and Biochemistry Studies Group.

3.2 Schedule

The Study schedule is provided in Table 3

.
Table 3. Schedule for the Single-laboratory Validation of Methods A and B Analysis Start Date

Method A
Preliminary August 3, 2009/August 10, 2009
PBS and laboratory fresh or marine samples September 7, 2009
Twenty five matrix samples September 14, 2009
Additional Marine samples March 24, 2010

Method B
Preliminary October 5, 2009
Twenty five matrix samples October 26, 2009 (Fresh)/November 2, 2009 (Marine)
PBS and laboratory fresh or marine samples November 16, 2009

3.3 Participant Laboratories

The 2 participating analytical laboratories involved in the Study are provided below.

• New York State Department of Health Environmenta l Biology Laboratory – Elle n Braun-Howland and Stacey Chmura, Wadsworth Center, Empire State Plaza, Albany, NY 12237

• Mycometrics, LLC – King-Teh Lin and Pi-shiang Lai, 11 Deerpark Drive, Monmouth Junction, NJ 08852

No endorsement of these laboratories is implied, nor should any be inferred.Methods A and B Single-Laboratory Validation Study Report

June 2010 12 Table 4. Volunteer Participants that Collected a nd Shipped Samples to the Analytical Laboratories a

Amtest Laboratories Aaron Young 13600 Northeast 126 Place, Suite C, Kirkland, WA 98034 Analytica International, Incorporated Kari Hagen 475 Hall Street, Fairbanks, AK 99701
Analytical Laboratories, Incorporated Sandy Koch 1804 N. 33 rd Street, Boise, ID 83703 Analytical Services Incorporated Paul Warden 130 Allen Brook Lane, Willison, VT 05495

Anatek Laboratories Kathy Sattler 504 East Sprague Avenue #D, Spokane, WA 99202 Bay County Laboratory Carol Monti 3420 Transmitter Road, Panama City, FL 32404

Biological Consulting Services of North Florida Troy Scott Source Molecular Corporation 4985 Southwest 74 th Court, Miami, FL 33155 BioVir Rick Danielson 685 Stone Road Unit #6, Benicia, CA 94510

CH Diagnostics Ana Luisa Child 512 5 th Street, Berthoud, CO 80513 City of Everett, Water Pollution Control Facility Chris Merwede 4027 4 th Street SE, Everett, WA 98205

Energy Laboratories Randy Ogden 2393 Salt Creek Highway, Casper, WY 82602-3258 EPA Office of Research and Development National Risk Management Research Laboratory Mark Meckes 26 West Martin Luther King Drive, Cincinnati, OH 45268
Hampton Roads Sanitation District Robert Maunz 1432 Air Rail Avenue, Virginia Beach, VA 23455 HML Dustin Smith 912 West McGalliard, Muncie, IN 47303-1702

King County Environmental Eric Thompson 322 West Ewing Street, Seattle, WA 98119-1507 Lacey-Olympia-Tumwater (LOTT) Wayne Robinson 500 Adams Street North, Olympia, WA 98501

Madison Metropolitan Sewerage District Paul Nehm 1610 Moorland Road, Madison, WI 53713 Massachusetts Water Resources Authority Steve Rhode 190 Tafts Avenue, Winthrop, MA 02152

Narragansett Bay Commission Jim Kelly 2 Earnest Street, Providence, RI 02905 Orange County Sanitation District Charlie McGee 10844 Ellis Avenue, Fountain Valley, CA 92708-7018

Oregon DEQ (Bend Office) Steve Kirk 475 Northeast Bellevue, Suite 110, Bend, OR 97701 Pittsburgh Water and Sewer Authority Stanley States 900 Freeport Road, Pittsburgh, PA 15238

San Francisco Water Mike Casteel 1000 El Camino Real, Millbrae, CA 94030 State of Hawaii Department of Health Environmental Microbiology Section Myron Honda 2725 Waimano Home Road, Pearl City, HI 96782
SVL Analytical, Incorporated Linda Johann 2195 Iron Wood Court, Suite C, Coerd’Alene, ID 83814 Texas A&M University – College Station Suresh Pillai 418D Kleberg Center 2472 Tamu s, College Station, TX 77843

Texas A&M University – Corpus Christi Joanna Mott 6300 Ocean Drive, Unit 5802, Corpus Christi, TX 78412 University of Hawaii Water Resources Research Center Gayatri Vithanage Holmes Hall 283 University of Hawaii, Honolulu, HI 96822Methods A and B Single-Laboratory Validation Study Report

June 2010 13 University of Rhode Island Watershed Program Linda Green 105 Coastal Institute 1 Greenhous e Rd, Kingston, RI 02881 University of South Florida Jody Harwood Department of In tegrative Biology 4202 East Fowler Avenue, Tampa, FL 33620 University of Southern Mississippi Rudolph D. Ellender Department of Biological Sciences 2609 West 4 th Street, Hattiesburg, MS 39406-0001 US Department of Agriculture Western Regional Research Center Robert Mandrell 800 Buchanan Street, Albany, CA 94710

USEPA Region 1 Laboratory Jack Paar 11 Technology Drive, North Chelmsford, MA 01863-2431 USEPA Region 10 Laboratory Stephanie Bailey 7411 Beach Drive East, Port Orchard, WA 98366

US Geological Survey Ohio Water Microbiology Laboratory Rebecca Bushon 6480 Doubletree Avenue, Columbus, OH 43229-1111 Utah Department of Health Larry Scanlon 46 North Medical Crive, Salt Lake City, UT 84113

Wisconsin State Laboratory of Hygiene Jeremy Olstadt 2601 Agriculture Drive, Madison, WI 53718-6780

a No endorsement of these laboratories is im plied, nor should any be inferred. Partic ipant laboratories have been randomly assi gned numbers for purposes of presenting data in this report.Methods A and B Single-Laboratory Validation Study Report

June 2010 14 

## Section 4.0 Data Reporting and Validation

4.1 Data Reporting

Laboratories submitted the following data to the CSC Microbiology and Biochemistry Studies Group for review and validation:

• Completed cover sheet with sample collection and QC information

• Completed sample-specific reporting forms

• Completed qPCR calculations spreadsheets

• Documentation of any additional information that would assist in evaluating the data

Volunteers submitted the following data to the CSC Micr obiology and Biochemistry Studies Group for review and validation:

• Completed sample collection forms

4.2 Data Validation

The CSC Microbiology and Biochemistry Studies Group used data review checklists to ensure that each data package was complete and that each sample result met the study-specific and method-specific requirements. Items reviewed for each sample included confirmation of the following: original fo rms were submitted; QC checks were conducted and exhibited the appropriate response; method-specific incubation times a nd temperatures were met; media and reagents were used within expiration dates; media sterility checks were performed and were acceptable, positive and negative controls were performed and exhibited the appropriate response; DNA extractions, PCR preparation, and PCR assays were performed according to each method; and calculations were correct.

This process was performed independently by two data reviewers, each of whom entered the results into separate spreadsheets designed for data review and validation for this St udy. The results were compared to verify consistency and identify potential data entry errors.

4.3 Censored Data

During the evaluation of validation study samples, results be low the analytical range of Methods A, B, and 1600 (lessthan results, also referred to as left-censored) were ob served (e.g., non-detects [CT values], <1 CFU/100 mL). For Methods A and B, non-detects were replaced with “0” prior to data analyses. For Method 1600, the censoring limit (the “less than” value itself [1]) was used in data analysis for these samples.Methods A and B Single-Laboratory Validation Study Report

June 2010 15 

## Section 5.0 Results

This section includes results of Method A preliminary anal yses (Section 5.1), Methods A and 1600 unspiked/spiked PBS samples (Section 5.2), Methods A and 1600 unspiked/spiked fresh and marine water samples collected by each lab (Section 5.3), Method A unspiked/spiked 25 fresh and 25 ma rine water samples (Section 5.4), Method B preliminary analyses (Section 5.5.), Method B unspiked/spiked PBS sa mples (Section 5.6), Method B unspiked/spiked fresh and marine water samples collected by each lab (Section 5.7), and Method B unspiked/spiked 25 fresh and 25 marine water samples (Section 5.8). Only valid results are included in th is section. Section 6 describes the development of draft QC acceptance criteria and Section 7 includes a discussion of method performance.

5.1 Methods A Preliminary Results

Preliminary results of the comparison of crude and purifie d extracts indicated that for Method A purified extracts may produce more consistent results, thus purified extracts were evaluated during Method A study analyses. However, EPA decided that since the majority of its results at the time of the study were of crude extracts, all subsequent Method A analyses (e.g., multi-laboratory validation) and Method B (s ingle- and multi-laboratory va lidation) would be conducted using crude extracts.

Method A analyses included the following: Environmental m aster mix, purified undiluted extracts, two spike types (laboratory-prepared and BioBalls), and three spike levels (100, 320, and 1000 cells).

5.2 Methods A and 1600 PBS Sample Results

Methods A and 1600 results from unspiked/spik ed PBS sample analyses are provided in Table 5 . Results from PBS samples spiked with E. faecalis BioBallTM spikes were used to assess me thod performance (see discussion in Section 7.1.1). Results from PBS samples spiked with either E. faecalis laboratory-prepared cell suspensions or BioBallTM spikes were used to develop draft QC acceptance criteria for use in assessing initial and ongoing method/laboratory performance for use during the multi-laborat ory validation stud y (see discussion in Section 6).Methods A and B Single-Laboratory Validation Study Report

June 2010 16 Table 5. Summary of E. faecalis Results from Methods A (n=4) and 1600 (n=2) for PBS Samples

Spike Level CFU/100 mL c Mean TSC a /100 mL Mean Percent Recovery Lab Spike Category Spike Type TSC a, b /CFU (CFU/100 mL) Method 1600 Method A 1600 (%) Method A (%) 1600 RSD (%) d Method A RSD (%) d
Unspiked NA e <1 29

100 121 88 100 1589 76.86 272.86 9.12 65.55

320 387.2 370 320 4810 88.84 261.21 10.28 55.00

1000 Lab-prep 6.06

1210 940 1020 8240 80.91 143.56 5.78 22.06 Unspiked NA e <1 33

100 107.9 84 104 315 86.19 69.00 15.21 34.44

320 315 340 300 1263 101.27 102.99 8.87 36.75 1

1000 BioBallTM 3.79

1173.4 1220 1220 6251 103.89 139.74 0.00 18.12 Unspiked NA e <1 0

100 89.33 92 84 485 97.39 54.98 6.50 35.57

320 285.87 270 360 2430 109.84 86.07 20.27 19.60

1000 Lab-prep 9.88

893.33 940 660 5879 89.44 66.63 24.78 39.31 Unspiked NA e <1 0

100 107.9 96 92 280 86.19 58.79 3.04 34.86

320 315 250 290 1400 85.40 100.60 10.51 17.79 2

1000 BioBallTM 4.42

1173.4 760 1200 5690 83.43 109.78 31.78 24.90

a Target sequence copy b Based on calibrator samples

c Since only two samples were tested, the mean was not calculated d Relative standard deviation

e Not applicable

5.3 Methods A and 1600 Fresh Water and Marine Water Sample Results

Methods A and 1600 results from unspiked/spiked fresh wa ter and marine water sample analyses are provided in Tables 6 and 7 . Results from fresh water (Table 6) and marine water (Table 7) samples spiked with E. faecalis BioBallTM spikes were used to assess method performance (see discussion in Sec tion 7.1.2). Results from fresh and marine water samples spiked with either E. faecalis laboratory-prepared cell suspensions or BioB allTM spikes were used to develop draft QC acceptance criteria for use in assessing initial and ongoing method/laboratory performance for use during the multilaboratory validation study (see discussion in Section 6).Methods A and B Single-Laboratory Validation Study Report

June 2010 17 Table 6. Summary of E. faecalis Results from Methods A (n=4) and 1600 (n=2) Fresh Water Samples

Spike Level CFU/100 mL c Mean TSC a /100 mL Mean Percent Recovery Spike Category Spike Type TSC a, b /CFU (CFU/100 mL) Method 1600 Method A 1600 (%) Method A (%) 1600 RSD (%) d Method A RSD (%) d
Unspiked NA e <1 262

100 89.3 124 76 731 110.82 53.18 34.28 23.08

320 285.9 270 260 2214 92.35 69.14 2.68 15.05

1000 Lab-prep 9.88

893.3 840 960 6029 100.63 65.37 9.44 9.69
Unspiked NA e <1 206

100 107.9 104 96 418 91.75 44.31 5.71 57.32

320 315 240 310 1264 86.98 76.03 18.06 31.92

1000 BioBallTM 4.42

1173.4 1040 1240 4570 97.07 84.18 12.42 9.99

a Target sequence copy b Based on calibrator samples

c Since only two samples were tested, the mean was not calculated d Relative standard deviation

e Not applicable

Table 7. Summary of E. faecalis Results from Methods A (n=4) and 1600 (n=2) Marine Water Samples
Spike Level CFU/100 mL c Mean TSC a /100 mL Mean Percent Recovery
Lab Spike Category Spike Type TSC a, b /CFU (CFU/100 mL) Method 1600 Method A 1600 (%) Method A (%) 1600 RSD(%) d Method A RSD(%) d Unspiked NA e 3 8102

100 121 88 108 2138 78.51 -1042.75 14.89 -13.24

320 387.2 310 340 2794 83.16 -290.03 6.59 -13.27

1000 Lab-prep 6.06

1210 1040 920 8782 80.74 11.89 8.69 164.77
Unspiked NA e 3 9233

100 107.9 112 120 17,368 104.73 4236.48 5.01 34.11

320 315 320 350 15,624 105.40 1305.11 6.39 18.82 1

1000 BioBallTM 3.79

1173.4 920 1080 39,408 84.97 884.84 11.35 32.44
Unspiked NA e 817

100 97.3 1485 169.73 2.82

320 311.5 3045 110.81 14.33

1000 Lab-prep 8.67

973.3 ND f ND f

10,494 ND e

123.74 ND e

47.86 Unspiked NA e 739

100 107.9 1190 296.43 44.92

320 315 1889 162.78 36.71 2

1000 BioBallTM 3.62

1173.4 ND f ND f

3935 ND e

91.82 ND e

9.40

a Target sequence copy b Based on calibrator samples

c Since only two samples were tested, the mean was not calculated d Relative standard deviation

e Not applicable f Not done - samples were not analyzed using Method 1600Methods A and B Single-Laboratory Validation Study Report

June 2010 18 5.4 Method A 25 Fresh Water and 25 Marine Water Sample Results

Method A results from unspiked/spiked fresh and marine water sample analyses are provided in Tables 8 and 9 . Twenty five fresh water and twenty five marine water samples we re assayed from 16 and 9 states, respectively. Results from fresh and marine water samples spiked with E. faecalis BioBallTM spikes were used to assess matrix inhibition (see discussion in Section 7.1.3).
Table 8. Summary of E. faecalis Method A Results from Twenty Five Fresh Water Matrices (n=4)
Method A Sample ID Spike Source Spike Type Mean TSC a /100 mL Mean Recovery (%) SD b (%) RSD c (%) Background Above Spike Level

Unspiked NA d 12,018 1 315 Lake BioBall TM 12,233 16.77 236.61 1411.30 X

Unspiked NA d 1279 2 315 Lake BioBall TM 2995 133.57 37.38 27.98
Unspiked NA d 80 3 315 Lake BioBall TM 3973 303.02 87.48 28.87
Unspiked NA d 986 4 315 Reservoir BioBall TM 6035 393.00 135.01 34.35
Unspiked NA d 656 5 315 Pond BioBall TM 3963 257.41 51.99 20.20
Unspiked NA d 171 6 315 Lake BioBall TM 3276 241.67 50.36 20.84
Unspiked NA d 588 7 315 River BioBall TM 2368 138.49 49.05 35.42
Unspiked NA d 107 8 315 Lake BioBall TM 1818 133.13 17.85 13.41
Unspiked NA d 13,132 9 315 Lake BioBall TM 19,634 404.16 130.06 32.18 X

Unspiked NA d 4591 10 315 River BioBall TM 5211 38.52 26.48 68.74 X

Unspiked NA d 1120 11 315 Lake BioBall TM 753,726 46,783 93,448 199.75

Unspiked NA d 2,786,751 12 315 Lake BioBall TM 2,366,986 -26,093 98,387 -377.06 X

Unspiked NA d 2464 13 315 River BioBall TM 2984 32.32 19.20 59.41 X

Unspiked NA d 451 14 315 Lake BioBall TM 1189 45.84 9.58 20.90Methods A and B Single-Laboratory Validation Study Report

June 2010 19 Table 8. Summary of E. faecalis Method A Results from Twenty Five Fresh Water Matrices (n=4) (cont.)

Method A Sample ID Spike Source Spike Type Mean TSC a /100 mL Mean Recovery (%) SD b (%) RSD c (%) Background Above Spike Level

Unspiked NA d 36 15 945 Lake BioBall TM 3511 72.00 7.35 10.20

Unspiked NA d 5959 16 945 River BioBall TM 9407 71.43 31.01 43.42

Unspiked NA d 1977 17 1173 River BioBall TM 5175 76.40 31.20 40.83

Unspiked NA d 333 18 1173 River BioBall TM 3286 70.55 17.18 24.36

Unspiked NA d 452 19 1173 Lake BioBall TM 4938 107.18 11.29 10.53

Unspiked NA d 613 20 1173 Lake BioBall TM 5163 108.70 12.74 11.72

Unspiked NA d 1730 21 1173 River BioBall TM 5466 89.26 12.56 14.07

Unspiked NA d 3939 22 1173 Creek BioBall TM 8080 98.93 17.93 18.12

Unspiked NA d 2932 23 1173 Lake BioBall TM 8669 137.06 31.06 22.66

Unspiked NA d 370 24 1173 Lake BioBall TM 5618 125.38 70.45 56.19

Unspiked NA d 1599 25 1173 Creek BioBall TM 7932 151.29 69.95 46.24

a Target sequence copy b Standard deviation

c Relative standard deviation d Not applicableMethods A and B Single-Laboratory Validation Study Report

June 2010 20 Table 9. Summary of E. faecalis Method A Results from Twenty Five Marine Water Matrices (n=4)

Method A Sample ID Spike Spike Type Mean TSC a /100 mL Mean Recovery (%) SD b (%) RSD c (%) Background Above Spike Level

Unspiked NA d 903 1 315 BioBall TM 4104 332.87 186.23 55.95
Unspiked NA d 67 2 315 BioBall TM 610 56.47 12.31 21.80
Unspiked NA d 3199 3 315 BioBall TM 5344 223.10 107.22 48.06 X

Unspiked NA d 56 4 315 BioBall TM 521 48.35 11.44 23.66
Unspiked NA d 489 5 315 BioBall TM 1193 73.20 18.99 25.94
Unspiked NA d 68 6 315 BioBall TM 995 96.40 20.68 21.46
Unspiked NA 277 7 315 BioBall TM 616 35.20 37.67 107.03
Unspiked NA d 729 8 315 BioBall TM 1653 96.06 98.88 102.94
Unspiked NA d 16 9 315 BioBall TM 1655 138.42 18.83 13.60
Unspiked NA d 122 10 315 BioBall TM 1337 102.57 11.61 11.32
Unspiked NA d 500 11 315 BioBall TM 1762 106.57 26.04 24.43
Unspiked NA d 438 12 315 BioBall TM 2328 159.56 46.55 29.17
Unspiked NA d 861 13 315 BioBall TM 2122 106.47 45.41 42.65
Unspiked NA d 814 14 315 BioBall TM 2360 130.54 36.72 28.13
Unspiked NA d 10,941 15 315 BioBall TM 12,148 101.90 364.43 357.64 X

Unspiked NA d 258 16 315 BioBall TM 1225 81.68 20.30 24.86
Unspiked NA d 208 17 315 BioBall TM 1118 63.14 2.46 3.89
Unspiked NA d 131 18 315 BioBall TM 1128 69.12 16.67 24.12Methods A and B Single-Laboratory Validation Study Report

June 2010 21 Table 9. Summary of E. faecalis Method A Results from Twenty Five Marine Water Matrices (n=4) (cont.)

Method A Sample ID Spike Spike Type Mean TSC a /100 mL Mean Recovery (%) SD b (%) RSD c (%) Background Above Spike Level

Unspiked NA d 309 19 315 BioBall TM 1483 81.46 21.57 26.48
Unspiked NA d 4636 20 315 BioBall TM 5220 40.47 43.61 107.76 X

Unspiked NA d 291 21 315 BioBall TM 2037 121.13 35.39 29.22
Unspiked NA d 227 22 315 BioBall TM 2484 156.52 180.21 115.14
Unspiked NA d 3523 23 315 BioBall TM 4895 95.13 58.05 61.03 X

Unspiked NA d 487 24 315 BioBall TM 1188 48.57 23.40 48.19
Unspiked NA d 473 25 315 BioBall TM 2056 109.80 9.43 8.59

a Target sequence copy b Standard deviation

c Relative standard deviation d Not applicable

5.5 Method B Preliminary Results

Evaluation of a culture-based membrane f iltration procedure followed by plating on Bacteroides bile esculin (BBE) agar and incubating under anaerobic conditions resulted in very low recoveries and inconsistent results between replicates. Based on these results, use of a culture method was not included during study analyses. In addition to issues with BBE, both laboratories had difficulty using TSA with 5% sheep bl ood to enumerate laboratory-prepared cell suspensions. To address this issue an alternate medium CDC anaerobe 5% blood agar was evaluated. Based on the results of this evaluation CDC anaerobe 5% blood agar was used to enumerat e laboratory-prepared cell suspensions for all subsequent analyses.

Method B analyses included the following: Universal master mix, crude diluted extracts, a single spike type (laboratoryprepared cell suspensions), and three spike le vels (10,000, 32,000, and 100,000 cells).

5.6 Method B PBS Sample Results

Method B results from spiked PBS sample analyses are provided in Table 10 . Results from PBS samples spiked with B. thetaiotaomicron laboratory-prepared cell suspensions were used to assess method performance (see discussion in Section 7.2.1) and develop draft QC acceptance criteria for use in assessing initial and ongoing method/laboratory performance for use during the multi-laboratory validati on study (see discussion in Section 6).Methods A and B Single-Laboratory Validation Study Report

June 2010 22 Table 10. Summary of B. thetaiotaomicron Results from Method B for PBS Samples (n=4) Spike Level Mean TSC a /100 mL Mean Percent Recovery Lab Spike Category Spike Type TSC a, b /CFU (CFU/100 mL) Method B Method B (%) Method B RSD c (%)
Unspiked NA d 0

10,000 84,667 197, 463 167.58 6.62

32,000 270,933 719, 133 163.57 18.48 1

100,000 Lab-prep 1.26

846,667 2,106,056 168.68 4.69

Unspiked NA d 0

10,000 46,667 27, 006 320.92 4.14

32,000 149,333 83, 710 310.86 3.61 2

100,000 Lab-prep 0.18

466,667 236,958 281.59 11.40

a Target sequence copy b Based on calibrator samples

c Relative standard deviation d Not applicable

5.7 Method B Fresh Water and Marine Water Sample Results

Method B results from unspiked/spiked fresh water and marine water sample analyses are provided in Tables 11 and 12

. Results from fresh water and mari ne water samples spiked with B. thetaiotaomicron laboratory-prepared cell suspensions were used to assess method performance (see discussion in Sec tion 7.2.2) and develop draft QC acceptance criteria (see discussion in Section 6).

Table 11. Summary of B. thetaiotaomicron Results from Method B for Fresh Water Samples (n=4)
Spike Level Mean TSC a /100 mL Mean Percent Recovery Spike Category Spike Type TSC a, b /CFU (CFU/100 mL) Method B Method B (%) Method B RSD c (%)
Unspiked NA d 644

10,000 46,667 29, 074 337.84 11.60

32,000 149,333 77, 023 283.64 16.86

100,000 Lab-prep 0.18

466,667 249,964 296.28 3.72

a Target sequence copy b Based on calibrator samples

c Relative standard deviation d Not applicableMethods A and B Single-Laboratory Validation Study Report

June 2010 23 Table 12. Summary of B. thetaiotaomicron Results from Method B for Marine Water Samples (n=4)

Spike Level Mean TSC a /100 mL Mean Percent Recovery Spike Category Spike Type TSC a, b /CFU (CFU/100 mL) Method B Method B (%) Method B RSD c (%)

Unspiked NA d 3871

10,000 84,667 182, 827 167.58 6.62

32,000 270,933 562, 829 163.57 18.48

100,000 Lab-prep 1.26

846,667 1,805,184 168.68 4.69

a Target sequence copy b Based on calibrator samples

c Relative standard deviation d Not applicable

5.8 Method B 25 Fresh Water and 25 Marine Water Sample Results

Method B results from unspiked/spiked fresh and marine water sample analyses are provided in Tables 13 and 14

. . Twenty five fresh water and twenty five marine water samp les were assayed from 16 and 9 states, respectively. Results from fresh and marine water samples spiked with B. thetaiotaomicron laboratory-prepared cell suspensions were used to assess matrix inhibition (see discussion in Section 7.2.3).

Table 13. Summary of B. thetaiotaomicron Results from Twenty Five Fresh Water Matrices (n=4)

Method B Sample ID Source Spike Spike Type Mean TSC a /100 mL Mean Recovery (%) SD b (%) RSD c (%) Background Above Spike Level

Unspiked NA d 520 1 Reservoir 31,360 Lab-prep 533 9 86.29 10.03 11.62
Unspiked NA d 19,398 2 Lake 31,360 Lab-prep 26,43 1 125.95 31.25 24.81 X

Unspiked NA d 21,758 3 River 31,360 Lab-prep 24,52 0 49.46 87.48 176.86 X

Unspiked NA d 0 4 Lake 31,360 Lab-prep 0 0.00 0.00
Unspiked NA d 44,065 5 Lake 31,360 Lab-prep 50,47 9 114.86 80.56 70.14 X

Unspiked NA d 3290 6 Lake 31,360 Lab-prep 7855 81.73 85.57 104.70

Unspiked NA d 10,338 7 River 31,360 Lab-prep 18,38 9 144.16 46.96 32.57 X

Unspiked NA d 99 8 Lake 31,360 Lab-prep 8928 158.10 11.23 7.10
Unspiked NA d 18,330 9 Lake 31,360 Lab-prep 19,36 5 18.54 27.13 146.32 XMethods A and B Single-Laboratory Validation Study Report

June 2010 24 Table 13. Summary of B. thetaiotaomicron Results from Twenty Five Fresh Water Matrices (n=4) (cont.)

Method B Sample ID Source Spike Spike Type Mean TSC a /100 mL Mean Recovery (%) SD b (%) RSD c (%) Background Above Spike Level

Unspiked NA d 761 10 Lake 31,360 Lab-prep 10,48 1 174.07 28.30 16.26
Unspiked NA d 14,424 11 River 31,360 Lab-prep 19,37 0 88.57 65.50 73.95 X

Unspiked NA d 10,166 12 Lake 31,360 Lab-prep 17,74 0 135.63 20.84 15.37 X

Unspiked NA d 22,387 13 River 31,360 Lab-prep 31,48 0 162.82 39.02 23.96 X

Unspiked NA d 31,465 14 River 31,360 Lab-prep 38,86 1 132.43 79.97 60.39 X

Unspiked NA d 107,932 15 River 31,360 Lab-prep 58,570 -883.93 46.10 -5.22 X

Unspiked NA d 477 16 Creek 31,360 Lab-prep 597 1 98.38 16.78 17.06 Unspiked NA d 2468 17 Creek 31,360 Lab-prep 14,98 1 155.09 4.91 3.16
Unspiked NA d 4157 18 Lake 31,360 Lab-prep 18,79 8 181.47 17.16 9.45
Unspiked NA d 2083 19 Lake 31,360 Lab-prep 897 5 85.42 13.27 15.53
Unspiked NA d 41,432 20 River 31,360 Lab-prep 41,93 1 6.19 35.78 577.86 X

Unspiked NA d 6462 21 Lake 31,360 Lab-prep 13,57 6 88.17 12.26 13.91
Unspiked NA d 2041 22 Lake 31,360 Lab-prep 13,43 7 141.24 12.73 9.01
Unspiked NA d 669 23 Pond 31,360 Lab-prep 560 0 61.12 10.27 16.80
Unspiked NA d 56,481 24 Lake 31,360 Lab-prep 71,538 186.61 120.88 64.78 X

Unspiked NA d 1090 25 Lake 31,360 Lab-prep 716 7 75.32 5.84 7.75

a Target sequence copy b Standard deviation

c Relative standard deviation d Not applicableMethods A and B Single-Laboratory Validation Study Report

June 2010 25 Table 14. Summary of B. thetaiotaomicron Results from Twenty Five Marine Water Matrices (n=4)

Method B Sample ID Spike Spike Type Mean TSC a /100 mL Mean Recovery (%) SD b (%) RSD c (%) Background Above Spike Level

Unspiked NA d 63 1 34,000 Lab-prep 21,16 6 42.67 5.06 11.85
Unspiked NA d 309 2 34,000 Lab-prep 21,92 6 43.71 4.96 11.35
Unspiked NA d 2241 3 34,000 Lab-prep 27,65 2 51.38 5.19 10.10
Unspiked NA d 164 4 34,000 Lab-prep 20,65 1 41.43 10.26 24.77
Unspiked NA d 440 5 34,000 Lab-prep 24,89 8 49.46 4.84 9.78
Unspiked NA d 778 6 34,000 Lab-prep 29,81 0 58.71 6.55 11.16
Unspiked NA d 17,028 7 34,000 Lab-prep 42,12 7 50.75 5.53 10.90
Unspiked NA d 3031 8 34,000 Lab-prep 28,11 6 50.73 7.82 15.42
Unspiked NA d 9765 9 34,000 Lab-prep 34,49 4 50.01 8.46 16.92
Unspiked NA d 181 10 34,000 Lab-prep 25,83 0 51.86 7.27 14.03
Unspiked NA d 6661 11 34,000 Lab-prep 24,78 1 36.64 3.72 10.16
Unspiked NA d 2422 12 34,000 Lab-prep 23,18 4 41.98 6.27 14.93
Unspiked NA d 5421 13 34,000 Lab-prep 27,98 2 45.62 4.13 9.06
Unspiked NA d 1113 14 34,000 Lab-prep 25,34 3 49.00 9.65 19.69
Unspiked NA d 981 15 34,000 Lab-prep 23,85 9 46.26 11.48 24.81
Unspiked NA d 226 16 34,000 Lab-prep 21,27 7 42.57 3.58 8.41 Unspiked NA d 8850 17 34,000 Lab-prep 33,11 3 41.99 5.35 12.74 Unspiked NA d 6198 18 34,000 Lab-prep 25,89 3 34.09 8.89 26.08Methods A and B Single-Laboratory Validation Study Report

June 2010 26 Table 14. Summary of B. thetaiotaomicron Results from Twenty Five Marine Water Matrices (n=4) (cont.)

Method B Sample ID Spike Spike Type Mean TSC a /100 mL Mean Recovery (%) SD b (%) RSD c (%) Background Above Spike Level Unspiked NA d 13,887 19 34,000 Lab-prep 35,40 5 37.24 11.19 30.05 Unspiked NA d 542 20 34,000 Lab-prep 22,53 6 38.07 12.63 33.18 Unspiked NA d 894 21 34,000 Lab-prep 14,23 4 23.09 15.11 65.44
Uns p iked NA d 26,023 22 34,000 Lab-prep 39,44 0 23.22 2.26 9.71 Unspiked NA d 1774 23 34,000 Lab-prep 649 6 8.17 1.39 17.00 Unspiked NA d 23,030 24 34,000 Lab-prep 39,18 0 27.95 2.08 7.43 Unspiked NA d 2605 25 34,000 Lab-prep 25,07 1 38.88 3.84 9.88

a Target sequence copy b Standard deviation

c Relative standard deviation d Not applicableMethods A and B Single-Laboratory Validation Study Report

June 2010 27 

## Section 6.0 Development of Dr aft QC Acceptance Criteria

This section describes the development of quantitative QC ac ceptance criteria in a reference matrix (PBS) and two different matrices of interest (fresh water and marine wate r) to support future assessments of laboratory and method performance. All data analyses described below were perform ed using the results of PBS and fresh or marine water samples spiked with either E. faecalis or B. thetaiotaomicron laboratory-prepared cell suspensions or BioBalls.

Because analyses were performed by only a single laboratory for each matrix of interest, and by only two laboratories in the reference matrix, outlier analyses were not performed for these data.

6.1 Initial Precision and Recovery (IPR)

QC acceptance criteria for initial precision and recovery (IPR) were developed based on the results from PBS (reference matrix) samples spiked with laboratory-prepared cell suspensions during the Study and based on the results from PBS samples spiked with BioBalls, as these QC analyses will be performed using PBS as the reference matrix by laboratories using the method during monitoring. Separate QC acceptance cr iteria were calculated for each spike level, spike type, and method.

The IPR criteria were calculated based on within and between laboratory variance components. These variance components were calculated with PROC MIXED from the SAS version 8 program using the maximum likelihood method of estimation on the recovery results. Details on the maximum likelihood estimation can be found in the user’s guide for this program (Reference 9.6).

Estimates of between laboratory variance and within laboratory variance were labeled s 2 L and s 2

w , respectively.

The combined standard deviation for IPR samples (s c ) is:

2 2
2 1 2 

### ) 1
4 1 ()1(

w T L
T L

i i

c 

### s n s n n

s ++ += 

# ∑

=

Where: L = number of laboratories for the given spike level and method

n i = number of PBS sample results for laboratory i for the given spike level and method

n T = total number of PBS sample results from all laboratories for the given spike level and method

Upper and lower limits for the mean recovery of four IPR samples were then calculated as:

cdf mean 

### stX * );975.0 

### ±

Where: X mean = the mean recovery of all PBS samp les, and for the given spiking procedure

df is calculated using Satterthwaite’s estimate as given below:Methods A and B Single-Laboratory Validation Study Report

June 2010 28 

### Ln s n
L s n n s df

T w T L
T L

i i c



### − ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛
+

+

− ⎥ ⎥ ⎥ ⎥

⎦ ⎤

⎢ ⎢ ⎢ ⎢

⎣ ⎡

⎟ ⎟ ⎟ ⎟

⎠ ⎞

⎜ ⎜ ⎜ ⎜

⎝ ⎛

+ = 

# ∑

= 2

2 2

2
2 1 2 4



### * 1
4 1

1 *1

The precision criterion for IPR samples was calculated as a maximum relative standard deviation (RSD) of four PBS sample results. The pooled RSD for each laboratory was calcul ated by dividing the pooled within-laboratory standard deviation calculated previously by the overall mean recove ry for that laboratory and matrix, as shown below:

mean w pool 

### X s RSD 2



### =

Where: S w and X
mean are the pooled within-lab standard deviation and mean recovery calculated above

The maximum RSD was then calculated as:

pool L 

### RSD F RSD * ),3;95.0( max 

### =

Where: L = the total number of laboratories for the given spike level and method.

The calculated IPR QC acceptance criteria are provided in Tables 15 and 16 for Methods A and B, respectively.

Table 15. Calculated Draft Initial Precision and Recovery (IPR) Acceptance Criteria for Method A (Enterococcus)

Performance test BioBallTM spike acceptance criteria Laboratory–prepared spike acceptance criteria 100 cell spike

• Mean percent recovery

• Precision (as maximum relative standard deviation of 4 samples) 34%

− 94%

68% Detect

− 690%

169%

320 cell spike

• Mean percent recovery

• Precision (as maximum relative standard deviation of 4 samples) 63%

− 140%

55% Detect

− 596%

128%

1000 cell spike

• Mean percent recovery

• Precision (as maximum relative standard deviation of 4 samples) 81%

− 169%

46% Detect

− 435%

60%Methods A and B Single-Laboratory Validation Study Report

June 2010 29 Table 16. Calculated Draft Initial Precision and Recovery (IPR) Acceptance Criteria for Method B (Bacteroidales)

Performance test Laboratory–prepared spike acceptance criteria

10,000 cell spike

• Mean percent recovery

• Precision (as maximum relative standard deviation of 4 samples) Detect – 1167%

21%

32,000 cell spike

• Mean percent recovery

• Precision (as maximum relative standard deviation of 4 samples) Detect

− 963%

13%

100,000 cell spike

• Mean percent recovery

• Precision (as maximum relative standard deviation of 4 samples) Detect

− 710%

21%

6.2 Matrix Spike (MS) Recovery and Precision

QC acceptance criteria for matrix spikes (MS) were devel oped based on laboratory-spiked sample data from the spiked fresh water and marine water matrices used in the validation study. Separate QC acceptance criteria were calculated for each spike level, spike type, and method.

Recovery criteria were based on estimates of multiple variance components (between laboratory and within laboratory). The within laboratory estimate was calculated using PR OC MIXED from SAS version 9 using the maximum likelihood method of estimation on the recovery results. Because sa mples from each matrix only were analyzed by a single laboratory, the between laboratory component could not be estimated directly using study data. Instead, the between laboratory estimate was calculated based on between laborator y/within laboratory variance differences observed in past studies. Details on the maximum likelihood estimation can be found in the user’s guide for this program (Reference 9.6). Estimates of between laboratory variance and within laboratory variance were labeled s 2 L and s 2 w, respectively.

The combined standard deviation for MS samples (s c ) is:

2 

### ) 1 1(3.2

w T c 

### s n s ⎥ ⎦ ⎤ ⎢ ⎣ ⎡
++=

Where: 2.3 = an adjustment made based on the estimate that between-laboratory variability is approximately 1.15x within-laboratory variability

n T = total number fresh or marine water sample results from the laboratory for the given spike level and methodMethods A and B Single-Laboratory Validation Study Report

June 2010 30 Upper and lower limits for the mean recovery of a single MS/matrix spike duplicate (MSD) recovery was then calculated

as:

c n mean 

### stX

T 

### * )1;975.0

− 

### ±

Where: X mean = the mean recovery of all fresh water samples, and for the given spiking procedure

The precision criterion for MS samples was calculated as a maximum relative standard deviation (RSD) of four MS results. The pooled RSD for each laboratory was calculated by dividing the within-laboratory standard deviation calculated previously by the overall mean recovery for th at spike level, method, and matrix, as shown below:

mean w pool 

### X s RSD 2



### =

Where: S w and X
mean are the within-lab standard deviati on and mean recovery calculated above

The maximum relative percent difference (RPD) was then calculated as:

pool 

### RSD F RPD **2 )3,1;95.0( max 

### =

Where: L = the total number of laboratories for the given spike level and method, and 2 = a factor used to convert an RSD to an RPD

The calculated MS QC acceptance criteria are listed in Tables 17 and 18 for Methods A and B, respectively.Methods A and B Single-Laboratory Validation Study Report

June 2010 31 Table 17. Calculated Draft Matrix Spike Precision and Recovery Acceptance Criteria for Method A (Enterococcus)

Performance test BioBallTM spike acceptance criteria Laboratory–prepared spike acceptance criteria

Fresh Water 100 cell spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) Detect

− 197%

258% Detect

− 127%

104%

320 cell spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) Detect

− 222%

144% Detect

− 132%

68%

1000 cell spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) 34

− 135%

45% 27

− 103%

44%

Marine Water a 100 cell spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) Detect – 1095%

202% 141% – 198%

13%

320 cell spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) Detect

− 521%

165% 16% – 206%

65%

1000 cell spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) 40%

− 144%

42% Detect

− 479%

215%

a Draft criteria were developed using only Laboratory 2 dataMethods A and B Single-Laboratory Validation Study Report

June 2010 32 Table 18. Calculated Draft Matrix Spike Precision and Recovery Acceptance Criteria for Method B (Bacteroidales)

Performance test Laboratory–prepared spike acceptance criteria

Fresh Water

10,000 cell spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) 103% – 573%

52%

32,000 cell spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) Detect – 571%

76%

100,000 cell spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) 230%

− 362%

17%

Marine Water

10,000 spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) 101% – 234%

30%

32,000 cell spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) Detect – 345%

83%

100,000 cell spike

• Mean percent recovery for 4 MS samples

• Precision (as maximum relative percent difference) 121%

− 216%

21%Methods A and B Single-Laboratory Validation Study Report

June 2010 33 

## Section 7.0 Assessment of Methods A and B Performance: Data Analysis and Discussion

Methods A and B performance was evaluated through the evaluation of precision and recovery in reference matrix (PBS) and fresh water and marine water samples spiked with either laboratory-prepared cell suspensions or BioBallTM spikes. Because the B.thetaiotaomicron BioBallTM spikes did not meet study requir ements, laboratory-prepared cell suspensions were used to assess method performance for Method B. Outlier analyses were not conducted for the assessment of method performance; therefore all valid data were included in the assessment of method performance.

Recoveries between matrices were compared for each organism and matrix type (fresh and marine water) using one-way ANOVAs. Because matrix was included in each model as a random effect, this test assessed whether the between-matrix variability was significantly greater than 0, rather than identifying recovery diffe rences for specific matrices. For each matrix type/organism, only those matrices for which the sp ike level exceeded background levels were included in the comparison.

7.1 Method A

Based on preliminary analyses the following variables we re included in the assessment of method performance for Method A in PBS and fresh and marine water samples: undiluted purified extracts, SPC (Sketa 22) , BioBallTM spikes, and three spike levels (100 cells , 320 cells, and 1000 cells).

7.1.1 Reference Matrix (PBS): Method A and Associated Method 1600 Analyses

PBS samples were analyzed by both of the participant laboratories.

• PBS samples spiked with E. faecalis laboratory-prepared cell suspensions

o For Laboratory 1, Method A mean recoveries for PBS samp les spiked with laboratory-prepared cell suspensions were high with recoveries ranging from 273%, 261%, and 144% for 100, 320, and 1000 cell spikes, respectively. RSDs were 66%, 55%, and 22%, for 100, 320, and 1000 cell spikes, respectively.

o For Laboratory 2, Method A mean recoveries for PBS samp les spiked with laboratory-prepared cell suspensions were 55%, 86%, and 67% for 100, 320, and 1000 cell spik es, respectively. RSDs were 36%, 20%, and 39%, for 100, 320, and 1000 cell spikes, respectively.

• PBS samples spiked with E. faecalis BioBallTM spikes

o For Laboratory 1, Method A mean recoveries for PBS sa mples spiked with BioBallTM spikes were 69%, 103%, and 140% for 100, 320, and 1000 cell spikes, respectivel y. RSDs were 34%, 37%, and 18%, for 100, 320, and 1000 cell spikes, respectively.

o For Laboratory 2, Method A mean recoveries for PBS sa mples spiked with BioBallTM spikes were 59%, 101%, and 110% for 100, 320, and 1000 cell spikes, respectivel y. RSDs were 35%, 18%, and 25%, for 100, 320, and 1000 cell spikes, respectively.

Method A mean recoveries were more consis tent between both laboratories when BioBalls were used to spike samples. Method 1600 mean recoveries were similar for all three spike levels and both spike types for the two laboratories (Table 5).Methods A and B Single-Laboratory Validation Study Report

June 2010 34 7.1.2 Fresh and Marine Water Matrices Collected by Each Laboratory: Method A and Associated Method 1600 Analyses

Fresh water samples were analyzed by one laboratory, while the other laboratory analyzed marine water samples.

• Fresh water samples spiked with E. faecalis laboratory-prepared cell suspensions

o Method A mean recoveries for fresh water samples spiked with laboratory-prepared cell suspensions were 53%, 69%, and 65% for 100, 320, and 1000 cell spikes, respec tively. RSDs were 23%, 15%, and 10%, for 100, 320, and 1000 cell spikes, respectively.

o Method 1600 mean recoveries for fresh water samples spiked with laboratory-prepared cell suspensions were 111%, 92%, and 101% for 100, 320, and 1000 cell spikes, respectively. RSDs were 34%, 3%, and 9%, for 100, 320, and 1000 cell spikes, respectively.

• Fresh water samples spiked with E. faecalis BioBallTM spikes

o Method A mean recoveries for fresh water samples spiked with BioBallTM spikes were 44%, 76%, and 84% for 100, 320, and 1000 cell spikes, respectively. RSDs were 57%, 32%, and 10%, for 100, 320, and 1000 cell spikes, respectively.

o Method 1600 mean recoveries for fresh water samples spik ed with BioBallTM spikes were 92%, 87%, and 97% for 100, 320, and 1000 cell spikes, respectively. RSDs were 6%, 18%, and 12%, for 100, 320, and 1000 cell spikes, respectively. Method A mean recovery for the 100 cell spike level was lowe r than recoveries in PBS samples (Section 7.1.1) suggesting that a spike level of 100 cells may be at the limit of det ection for fresh water samples. Method 1600 mean recoveries were similar for all three spike levels and both spike types.

• Marine water samples spiked with E. faecalis laboratory-prepared cell suspensions

o Laboratory 1: Method A mean recoveries for marine water samples spiked with laboratory-prepared cell suspensions at 100 and 320 cells were negative (-) 1043 a nd (-) 290%, because ambient enterococci levels were greater than the 100 and 320 cell spike levels based on unspiked samples analyzed using Method A. For the 1000 cell spike level the mean recovery was 12% and this low recovery could also be due to the high background levels. RSDs were (-) 13%, (-) 13%, and 165%, for 100, 320, and 1000 cell spikes, respectively.

o Laboratory 1: Method 1600 mean recoveries for marine water samples spiked with laboratory-prepared cell suspensions were 79%, 83%, and 81% for 100, 320, and 1 000 cell spikes, respectively. RSDs were 15%, 7%, and 9%, for 100, 320, and 1000 cell spikes, respectively. The unspiked marine water samples analyzed using Method 1600 did not indicate high ambient concentrations of enterococci.

o Laboratory 2: Method A mean recoveries for marine water samples spiked with laboratory-prepared cell suspensions were 170%, 111%, and 124% for 100, 320, and 1000 cell spikes, respectively. RSDs were 3%, 14%, and 48%, for 100, 320, and 1000 cell spikes, respectively.

• Marine water samples spiked with E. faecalis BioBallTM spikes

o Laboratory 1: Method A mean recoveries for marine wa ter samples spiked with BioBallTM spikes were 4237%, 1305%, and 884% for 100, 320, and 1000 cell spikes, respectively. RSDs were 34%, 19%, and 32%, for 100, 320, and 1000 cell spikes, respectively.

o Laboratory 1: Method 1600 mean recoveries for marine water samples spiked with BioBallTM spikes were 105%, 105%, and 85% for 100, 320, and 1000 cell spikes, respectiv ely. RSDs were 5%, 6%, and 11%, for 100, 320, and 1000 cell spikes, respectively.Methods A and B Single-Laboratory Validation Study Report

June 2010 35 o Laboratory 2: Method A mean recoveries for marine water samples spiked with BioBallTM spikes were 297%, 163%, and 92% for 100, 320, and 1000 cell spikes, resp ectively. RSDs were 45%, 37%, and 9%, for 100, 320, and 1000 cell spikes, respectively. For Laboratory 1, Method A mean recoveries were very differe nt between the two spike types; laboratory-prepared spikes resulted in negative or very low recoveries while recoveries for samples spiked with BioBalls were very high. However, Method 1600 mean recoveries were similar for all three spike leve ls and both spike types. Spike type had less of an effect on recovery for Laboratory 2 than Laboratory 1.

7.1.3 Twenty five Matrices (Fresh and Marine): Method A

Each laboratory analyzed a total of twenty five matrix samples over a three week period. E ach sample was split into eight replicates (4 unspiked and 4 spiked). Samples were spiked with E. faecalis BioBalls.

• Fresh water samples

o Eleven of the twenty five fresh water samples had ambient concentrations of enterococci greater than 320 cells per 100 mL. Due to the high background levels observe d the spike level was increased to approximately 1000 cells for samples 15 – 25. High ambient levels can make it difficult to accurately determine the recovery of spiked cells because the background may mask the spike.

o Method A mean recovery for sample site 11 was extremely high (46,783%) which was due to one replicate with a recovery of 186,955%, skewing the results. The other three replicates resulted in recoveries of 71%, 57%, and 50%. Method A mean recovery for sample site 11 would be 59% if the fourth replicate was not included.

o Due to an extremely high ambient enterococci concentrati on, mean recovery for site 12 was negative (-) 26,093%.

o For 14 of the 25 sample sites, mean recoveries ranged from 71%

− 151%.

o For four of the sites, mean recoveries were between 17%

− 46%, of which three of these sites had ambient enterococci concentrations greater than the spike level of 320 cells.

o For five of the 25 sample sites, mean recoveries ranged from 242%

− 404%.

• Marine water samples

o Four of the twenty five marine water samples had ambient concentrations of enterococci greater than 320 cells per 100 mL.

o For 18 of the 25 sample sites, mean recoveries ranged from 63%

− 159%.

o For five of the sites, mean recoveries were between 35%

− 57%.

o Mean recoveries were 223% and 333% for sample sites 3 and 1, respectively.

Method A mean recoveries for both fresh and marine water matr ices indicate that the method is appropriate for analyses of enterococci in both matrices.

7.2 Method B Based on preliminary analyses the following variables we re included in the assessment of method performance for Method B in PBS and fresh and marine wa ter samples: crude extracts diluted 1:5, Sketa 22 (SPC), laboratory-prepared cell suspensions, and three spike levels (10, 000 cells, 32,000 cells, and 100,000 cells).Methods A and B Single-Laboratory Validation Study Report

June 2010 36 7.2.1 Reference Matrix (PBS): Method B

PBS samples were analyzed by both of the particip ant laboratories. Due to recovery issues with B. thetaiotaomicron BioBalls only laboratory-prep ared cell suspensions were used to spike samples.

• PBS samples spiked with B. thetaiotaomicron laboratory-prepared cell suspensions

o For Laboratory 1, Method B mean recoveries for PBS samp les spiked with laboratory-prepared cell suspensions were 168%, 164%, and 169% for 10,000, 32,000, and 100,000 cell spikes, respectively. RSDs were 7%, 19%, and 5%, for 10,000, 32,000, and 10 0,000 cell spikes, respectively.

o For Laboratory 2, Method B mean recoveries for PBS samp les spiked with laboratory-prepared cell suspensions were 321%, 311%, and 282% for 10,000, 32,000, and 100,000 cell spikes, respectively. RSDs were 4%, 4%, and 11%, for 10,000, 32,000, and 100,000 cell spikes, respectively.

7.2.2 Fresh and Marine Water Matrices Collected by Each Laboratory: Method B

Fresh water samples were analyzed by one of the laboratories, while the other labor atory analyzed marine water samples.

• Fresh water samples spiked with B. thetaiotaomicron laboratory-prepared cell suspensions

o Method B mean recoveries for fresh water samples spiked with laboratory-prepared cell suspensions were 338%, 284%, and 296% for 10,000, 32,000, and 100,000 cell spikes, respectively. RSDs were 12%, 17%, and 4%, for 10,000, 32,000, and 100,000 cell spikes, respectively.

• Marine water samples spiked with labor atory-prepared cell suspensions

o Method B mean recoveries for marine water samples spik ed with laboratory-prepared cell suspensions were 168%, 164%, and 169% for 10,000, 32,000, and 100,000 cell spikes, respectively. RSDs were 7%, 18%, and 5%, for 10,000, 32,000, and 100,000 cell spikes, respectively.

Method B mean recoveries for both fresh and marine water sa mples spiked with laboratory-prepared cell suspensions were similar to mean recoveries for PBS (Sec tion 7.2.1) at each of the laboratories.

7.2.3 Twenty five Matrices (Fresh and Marine): Method B

Each laboratory analyzed a total of twenty five matrix samp les over a two week period. E ach sample was split into eight replicates (4 unspiked and 4 spiked).

• Fresh water samples

o Twelve of the twenty five fresh water samples had ambien t or background concentrations of Bacteroidales greater than 32,000 cells per 100 mL. High ambient levels can ma ke it difficult to accurately determine the recovery of spiked cells because the background may mask the spike.

o Method B mean recovery for site 4 was 0%, which was proba bly due to matrix inhibition. All four unspiked and four spiked replicates were non-detect for both the Bacteroidales and salmon DNA (SPC).

o Due to an extremely high ambient Bacteroidales concentra tion, mean recovery for site 15 was negative (-) 884%.

o For 17 of the 25 sample sites, mean recoveries ranged from 61%

− 163%.

o For three of the sample sites, mean recoveries were between 3%

− 50%, all three of these sites had ambient Bacteriodales concentrations greater than the spike le vel of 32,000 cells. High ambient levels can make it difficult to recovery spiked cells.Methods A and B Single-Laboratory Validation Study Report

June 2010 37 o For three of the 25 sample sites, mean recoveries ranged from 174%

− 187%.

• Marine water samples

o None of the twenty five marine water samples had ambi ent concentrations of Bacteroidales greater than 32,000 cells per 100 mL.

o For 24 of the 25 sample sites, mean recoveries ranged from 23%

− 59%.

o Mean recovery for sample site 23 was 8% indicating that ma trix inhibition may be an issue. However, CT values for salmon DNA (SPC) were consistent with values (approximately 20 CT) for other matrices. Method B mean recoveries for both fresh and marine water matrices indicate that Method B may be appropriate for analyses of Bacteroidales in both matrices.Methods A and B Single-Laboratory Validation Study Report

June 2010 38 

## Section 8.0 Conclusion

Results of the single-laboratory validation study indicate that Methods A and B are suitable for multi-laboratory validation in both fresh and marine waters. The data generated during the Study enabled the development of draft performance criteria for use during the multi-laboratory validation studies that were included in revised versions of Methods A and B. The revised methods were made available to the public by putting them on EPA’s Clean Water Act methods website (http://www.epa.gov/waterscience/methods / ).Methods A and B Single-Laboratory Validation Study Report

June 2010 39 

## Section 9.0 References

9.1 Cabelli, V. J., A. P. Dufour, M. A. Levin, L. J. Mc Cabe, and P. W. Haberman, 1979. Relationship of Microbial Indicators to Health Effects at Marine Bathi ng Beaches. Amer. Jour. Public Health. 69:690-696.

9.2 Dufour, A.P. 1984. Health Effects Criteria for Fresh R ecreational Waters, EPA-600/1-84-004. Office of Research and Development, USEPA.

9.3 USEPA. 2009. EPA Method A: Enterococci in Water by TaqM an® Quantitative Polymerase Chain Reaction (qPCR) Assay , Draft May 2009.

9.4 USEPA. 2009. EPA Method B: Bacteroidales in Water by T aqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay , Draft October 2009.

9.5 USEPA. 2006. EPA Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl- β -D-Glucoside Agar (mEI) , EPA-821-R-06-009, July 2006.

9.6 SAS Institute Inc. 1994. SAS/STAT User’s Guide, Volu me 2, GLM-VARCOMP. Version 6, 4th Edition, June 1994.Methods A and B Single-Laboratory Validation Study Report

June 2010 40 

## Section 10.0 Acronyms

ATCC American Type Culture Collection BBE Bacteroides bile esculin (agar) CT Cycle threshold GLM General linear model IPR Initial precision and recovery MS Matrix spike OD Optical density OPR Ongoing precision and recovery PBS Phosphate buffered saline QA Quality assurance QC Quality control qPCR Quantitative polymerase chain reaction RPD Relative percent difference RSD Relative standard deviation SAS Statistical analysis software SPC Sample processing control SD Standard deviation TSA Tryptic soy agar TSC Target sequence copy

## Appendix A

Enterococci ( Method A ) Study InstructionsA-1 

## Method A Single-Laboratory Validation Study: Assessment of Method Performance and Development of Preliminary Quantitative QC Criteria (Phase IV)
September 2, 2009

Summary of Objectives

 Characterize recovery and precision of Method A

 Establish preliminary quantitative QC accepta nce criteria for initial and ongoing method performance assessments of Method A

 Characterize matrix inhibition of Method A across multiple fresh and marine water matrices



### Summary of Analyses
Laboratories will complete the following accordi ng to Method A and study-specific instructions:

 Enumerate laboratory-prepared spiking susp ensions and determine cell concentration (OD) of the spiking suspension

 Prepare and analyze unspiked PBS and fresh or marine water samples according to Method A and Method 1600

 Prepare and analyze PBS and fresh or marine water (single source) samples spiked with BioBallTM and laboratory-prepared E. faecalis spiking suspensions at three different levels according to the Spiking Protocol, Method A, and Method 1600

 Prepare and analyze unspiked and spiked (single level) fresh or marine water (25 different sources each) samples and one method blank (PBS) according to Method A

 Prepare, extract, and analyze ongoing calibrator samples using laboratory- and vendorprepared E. faecalis cell preparations

Based on Phase II and III data received from both labs, all samples will be purified prior to analysis and only undiluted purified extracts will be evaluated for the remainder of the study. Study analyses will include the preparation of unspiked and spiked PBS (reference matrix) and water (fresh or marine) samples according to Method A and the Spiking Protocol. During week one of the study (method recovery and precision), samples will be filtered and replicate filters analyzed according to Method 1600, and following filter extraction, purified extracts (undiluted) will be analyzed for enterococci (Entero assay ) and salmon DNA (SPC-Sketa 22) according to Method A. During week two of the study (matrix inhibition), 25 fresh or marine water samples (unspiked and spiked) will be analyzed for enterococci (Entero assay) and salmon DNA (SPCSketa 22) according to Method A. During both weeks, ongoing calibrators (laboratory- prepared and BioBallTM) will be prepared and extracted as described for Phase II initial calibrator samples using a single BioBallTM cell concentration (1000 CFU BioBallTM). Following filter extraction, calibrator extracts will be purified and analyzed (undiluted) for entero cocci (Entero assay) and salmon DNA (SPC-Sketa 22) according to Method A. After PCR analyses, the ratio of target sequence quantities recovered from PBS and fresh or marine water compared to those recovered fro m ongoing calibrator samples will be used in theA-2 ΔΔ C T comparative cycle threshold calculation method to quantify DNA in PBS and fresh water samples.



### Media/Reagents/Supplies

 Undiluted laboratory-prepared E. faecalis calibrator cell suspensions (frozen 10 μ L calibrators from Phase I) – 3 calibrators

 Laboratory-prepared E. faecalis spiking suspensions

 BioBallTM E. faecalis spiking preparations (100, 320 and 1000 CFU)

 Phosphate buffered saline (PBS) – 5 L mi nimum (includes 39 x 100 mL bottles)

 Brain heart infusion broth (BHIB) – 200 mL

 Brain heart infusion agar (BHIA) plates – 20 plates

 0.45 F m polycarbonate filters – 259

 AE buffer, pH 9.0 – 10 mL minimum

 Salmon DNA/extraction buffer (0.2 μg/mL solution) – 100 mL

 GeneRite DNA purification kit – 259 Extraction tubes with glass beads and DNAsure columns, binding buffer, wash buffer, and elution buffer

 1.7 mL sterile microcentrifuge tubes

 Method A PCR Reagents:

 PCR grade water

 Bovine serum albumin (BSA, 2 mg/mL solution)

 Enterococcus primer/probe working stock solution (Entero)

 Salmon DNA primer/probe working stock solution (Sketa-22)

 TaqMan® Environmental PCR Master Mix

 Method 1600 Reagents

 Membrane Enterococcus indoxyl- β -D-glucoside agar (mEI) – 28 plates

 GN6 membrane filters - 28


### Week One – Preliminary Method Recovery and Precision Assessment
Week one analyses will focus on developing prelim inary quantitative QC criteria for initial and ongoing method performance assessment (method recovery and precision criteria).



### Procedures
Preparation and Enumeration of Spiking Suspensions

 Propagate and enumerate laboratory-prepared spiking suspensions according to study instructions (Phase I) and Method A.

Note : BioBalls will not be enumerated during Phase IV analyses.

 Record results and all associated calculations on data reporting forms provided for the study.

Spiking and Analyses of Sterile PBS Samples

 Label 39, 100-mL aliquots of sterile PBS as indicated below (see also Table 2 ):

o 3, Unspiked (1 Method 1600/2 Method A)

o 100 CFU

 6, lab-prepared (2 Method 1600/4 Method A)

 6, BioBallTM (2 Method 1600/4 Method A)A-3 o 320 CFU

 6, lab-prepared (2 Method 1600/4 Method A)

 6, BioBallTM (2 Method 1600/4 Method A)

o 1000 CFU

 6, lab-prepared (2 Method 1600/4 Method A)

 6, BioBallTM (2 Method 1600/4 Method A)

 Spike 6, 100-mL PBS replicates at each of the three levels (100, 320, and 1000 CFU) using the laboratory-prepared E. faecalis spiking suspension according to the Spiking Protocol.

 Spike 6, 100-mL PBS replicates at each of the three levels (100, 320, and 1000 CFU) using BioBallTM spikes according to the Spiking Protocol.

Water Sample Collection and Spiking

 Collect a 5 L bulk fresh or marine water sample and transport to the laboratory on ice (do not freeze). In the laboratory, split the bulk samp le into the following 100 mL aliquots and label as indicated below (see also Table 2):

o 3, Unspiked (1 Method 1600/2 Method A)

o 100 CFU

 6, lab-prepared (2 Method 1600/4 Method A)

 6, BioBallTM (2 Method 1600/4 Method A)

o 320 CFU

 6, lab-prepared (2 Method 1600/4 Method A)

 6, BioBallTM (2 Method 1600/4 Method A)

o 1000 CFU

 6, lab-prepared (2 Method 1600/4 Method A)

 6, BioBallTM (2 Method 1600/4 Method A)

 Spike 6, 100-mL sample replicates at each of the three levels (100, 320, and 1000 CFU) using the laboratory-prepared E. faecalis spiking suspension according to the Spiking Protocol.

 Spike 6, 100-mL sample replicates at each of the three levels (100, 320, and 1000 CFU) using BioBallTM spikes according to the Spiking Protocol.
Membrane Filtration and Method 1600 Analyses

 Mix samples (Method 1600 samples; 26 total) by shaking 25 times.

 For each matrix (PBS and either fresh or marine water), spike type (laboratory- prepared and BioBallTM) and spike level, filter sample volumes indicated below:

 Unspiked – 100 mL

 100 CFU Spike – 25 mL

 320 CFU Spike – 10 mL

 1000 CFU Spike – 5 mLA-4  Filter unspiked and spiked samples and rinse the si des of the funnel at least twice with sterile PBS (Method A, Section 11.4).

Note: Use a sterile funnel for each matrix and always filter the lowest E. faecalis spike concentration (e.g., 100 CFU spike, then 320 CFU spike, etc.) first. The unspiked PBS (method blank) should be filtered l ast using a sterile funnel.

 Use sterile forceps to aseptically remove filters and place onto mEI plates. Incubate at 41°C ± 0.5°C for 24 ± 2 hours according to Method 1600.

 Count all colonies ≥ 0.5 mm in diameter with a blue halo (regardless of colony color) as enterococci. Also record the number of atypical colonies (e.g., pink to red colonies without halos, off white colonies without halos).

 Fax results to Rashmi Ghei at (703) 461-8056.
Membrane Filtration and Filter Extraction/Extract Processing – Method A

 Mix samples (Method A samples; 52 total) by shaking 25 times.

 Filter unspiked and spiked samples (entire 100 mL volumes) and rinse the sides of the funnel at least twice with sterile PBS (Method A, Section 11.4).

Note: Use a sterile funnel for each matrix and always filter the lowest E. faecalis spike concentration (e.g., 100 CFU spike, then 320 CFU spike, etc.) first. The unspiked PBS sample (method blank) should be f iltered last using a sterile funnel.

 Place individual filters into appropriately labeled extraction tubes (52 total) and extract according to Method A (Section 11.5).

 Recover as much of each sample extract and clarified supernatant as possible (e.g., 400 μ L).

 Prepare two unspiked sample extracts and 4 replicate extracts (Method A samples) of each matrix, spike level and format (e.g., PBS - BioBallTM 100 CFU) for DNA purification (GeneRite) according to instructions (bel ow) for 2-step DNAsure column loading of calibrator extracts (e.g., 400 μ L sample plus 800 μ L binding buffer).

 Load samples onto DNAsure columns, wash, and elute samples in 100 μ L elution buffer.

 Refrigerate purified undiluted samples until analyzed.

Preparation, Extraction and Processing of Ongoing Calibrator Samples 1. Laboratory-prepared calibrators

 Thaw (on ice) a total of 3 calibrator sa mples (prepared during Phase 1) and add 990 μ L of AE buffer to each calibrator. Thoroughly mix by vortexing.

 Prepare a total of 3 extraction tubes containing glass beads by placing one polycarbonate filter into each extracti on tube and adding 590 μ L of salmon DNA/extraction buffer.

 Mix diluted calibrator cells thoroughly and spot 10 μ L of each diluted suspension onto the polycarbonate filter in each extraction tube.

 Perform extraction of calibrator samples acco rding to Method A bead-beating procedure (Sections 11.3.9 – 11.3.12).

 Recover as much of the clarified extracts as possible (e.g., 400 μ L) and purify (GeneRite) 300 μ L of each extract as described in Method A (Sections 11.2.9 – 11.2.14).

2. BioBallTM calibrators

 Prepare a total of 3 extraction tubes containing glass beads by placing one polycarbonate filter into each extracti on tube and adding 550 μ L of salmon DNA/extraction buffer.A-5  Aseptically add a BioBallTM (1000 CFU; 3 rep licates) to each of 3 appropriately labeled 1.7 mL microcentrifuge tubes and suspend each BioBallTM in 50 μ L AE buffer.

 Using a clean micropipette tip for each ce ll suspension, thoroughly mix by gentle pipetting and spot the entire 50 μ L volume onto the polycarbonate filter in the appropriately labeled extraction tube

 Extract samples according to Method A bead-beating procedure (Sections 11.3.9- 11.3.12).

 Recover the entire volumes (e.g., 400 μ L) of crude (Section 11.3.11) and clarified (Section 11.3.12) extracts (supernatants) from each sample and transfer to a sterile 1.7 mL microcentrifuge tube. Record total sample (clarified extracts) volumes.

Purification of Calibrator Extracts 1. Laboratory-prepared calibrators

 Process the calibrator (3 total) extracts (i.e., 300 μ L each) using the DNA purification procedure for preparation of genomic DNA standards (Method A, Sections 11.2.9 - 11.2.14).

 Refrigerate purified undiluted samples until analyzed.

2. BioBallTM calibrators

Due to the low level of cells (1000 CFU) used to prepare the BioBallTM calibrators a modified DNA purification protocol will be used to ensure maximum recovery of target DNA. Calibrator extracts (3 replicates) will be further purified using the DNA purification kit (GeneRite) prior to PCR analysis.

 Add 2 volumes of Binding Buffer (GeneRite kit) to each sample extract (e.g., add 800 μ L binding buffer to 400 μ L sample) and vortex to mix.

Note: Each sample mixture must be loaded onto a DNAsure column in two steps since the maximum holding capacity of a column is 900 μ L.

 Add approximately half of each sample (e.g., 600 μ L) to individual labeled DNAsure columns inserted into collection tubes and cen trifuge at top speed for 1 minute. Discard the flow through from collection tubes.

 Add the remaining portion of each sample to the appropriate DNAsure column, centrifuge and discard flow through from collection tubes.

 Transfer each DNAsure column to a clean collection tube.

 Add 500 μ L EZ-Wash Buffer to each column and centrifuge at top speed for 1 minute. Discard flow through from each column. Repeat wash using 500 μ L EZ-Wash Buffer.

 Transfer each column to a sterile 1.7 mL microcentrifuge tube and add 50 μ L DNA Elution Buffer to each column. Centrifuge for 30 seconds at top speed. Repeat DNA elution step using a second 50 μ L of DNA Elution Buffer to obtain a final volume of 90- 100 μ L/sample.

 Refrigerate purified undiluted samples until analyzed.A-6 PCR Preparation and Analysis

Note : To minimize potential environmental DNA contam ination, routinely treat all work surfaces with a 10% bleach solution, allowing bleach to contact the work surface for a minimum of 15 minutes prior to rinsing with sterile water. Further decontaminate work area surfaces using UV light for 15 minutes prior to assay set up if possible. Set up PCR reactions (Entero and SPC-Sketa-22 assays) as follows:

 Prepare Entero and SPC-Sketa-22 PCR Assay Mixes on the day of use according to Method A (Table 2) and Table 1 below:

Table 1. PCR Assay Mix Composition

Rea g ent V olume/Reaction 1

Sterile H 2 O 1.5 μ L

BSA 2.5 μ L

Ta q Man® master mix 12.5 μ L Primer/probe workin g stock solution 2 3.5 μ L
1 Multiply by # samples (plus 1) to be analyzed per day 2 See Phase II Table 1 for assay-specific primer/probe working stocks

 Set up individual PCR reactions to contain 20 μ L of the appropriate assay mix and 5 μ L of each sample (undiluted replicates) as specified in Table 2 (below) and Method A (Section 11.8).
Note: 5 μ L of each sample will be used for each assay format (10 μ L total/sample).

 Include at least 1 no template control (NTC) containing 5 μ L PCR grade water instead of template (for each PCR assay) with each set of samples analyzed.

 Analyze PCR reactions according to Method A (Section 11.8 and Appendix B).

 When PCR run(s) is/are complete, save and export the Results Table and Analysis Settings according to Method A, Appendix B.

 Input appropriate data (e.g., CT values) into the study-specific Excel data reporting form.

 Email results to Robin K Oshiro at oshiro.robin@epa.gov and Yildiz Chambers at ychambers@csc.com

.

 Fax hard copy data reporting forms to Yildiz Chambers at 703.461.8056.A-7 Table 2. Summary of Week One Validation Study Analyses Sample Description Method 1600 Method A PCR Reactions a

PBS (spiked) Lab-prepared (100, 320, and 1000 CFU) 6 , 2 reps x 3 concentrations 24 , 4 reps × 3 concentrations x 2 assays

BioBallTM (100, 320, 1000 CFU) 6 , 2 reps x 3 concentrations 24 , 4 reps × 3 concentrations x 2 assays

Water Matrix (spiked) Lab-prepared (100, 320, and 1000 CFU) 6 , 2 reps x 3 concentrations 24 , 4 reps × 3 concentrations x 2 assays

BioBallTM (100, 320, 1000 CFU) 6 , 2 reps x 3 concentrations 24 , 4 reps × 3 concentrations x 2 assays

Water Matrix (unspiked) Unspiked 1 4 , 2 reps × 2 assays

PBS (unspiked) Method Blank 1 4 , 2 reps × 2 assays

Ongoing Calibrators NA 12, 3 reps x 2 formats x 2 asays

No template Controls NA 4 -6 , 1 rep × 2 assays × per run

Total Analyses 26 120-122

a PCR Assays include Entero and Sketa-22



### Week Two – Matrix Inhibition Analyses

Week two will focus on assessing matrix inhibition through analyses of multiple fresh and marine water samples collected from recreational sites across the nation. These analyses will include sample spiking, sample processing, and assay form ats as determined previously. Samples will be collected and shipped to each laboratory during the weeks of September 14 th , 21 st , and 28 th

.


### Procedures

Sample Preparation and Spiking Participating laboratories will be provided with e ither fresh or marine water samples (1 L per sample, 25 total samples) collected from various sites across the nation. Samples should be processed on the day of receipt as follows:

 Thoroughly mix each sample by vigorously shaking the bottles a minimum of 25 times.

 Prepare 8 - 100 mL subsamples from each orig inal grab sample and label replicate subsamples with the sample ID and designate each as either unspiked (4 replicates) or spiked (4 replicates).

 Spike 4 replicate subsamples with a BioBallTM (320 CFU per replicate) according to the Spiking Protocol and mix thoroughly.A-8  Prepare a 100 mL PBS method blank (unspiked) each week that matrix samples are processed.

 Process unspiked and spiked replicates a nd method blank(s) as described below.

Membrane Filtration and Filter Extraction/Extract Processing – Method A

 Mix samples by shaking 25 times.

 Filter unspiked and spiked samples (entire 100 mL volumes) and rinse the sides of the funnel at least twice with sterile PBS (Method A, Section 11.4).

Note: Use a sterile funnel for each matrix and always filter the lowest E. faecalis spike concentration (e.g., 100 CFU spike, then 320 CFU spike, etc.) first. The unspiked PBS sample (method blank) should be f iltered last using a sterile funnel.

 Place individual filters into appropriately labe led extraction tubes and extract according to Method A (Section 11.5).

 Recover as much of each sample extract and clarified supernatant as possible (e.g., 400 μ L).

 Prepare clarified sample extracts unspiked and spiked) for DNA purification (GeneRite) according to previous instructions for 2-step DNAsure column loading of calibrator extracts (e.g., 400 μ L sample plus 800 μ L binding buffer).

 Load samples onto DNAsure columns, wash, and elute samples in 100 μ L elution buffer.

 Refrigerate purified undiluted samples until analyzed.

Preparation, Extraction and Processing of Ongoing Calibrator Samples

 Prepare a total of 3 extraction tubes containing glass beads by placing one polycarbonate filter into each extracti on tube and adding 550 μ L of salmon DNA/extraction buffer.

 Aseptically add a BioBallTM (1000 CFU; 3 rep licates) to each of 3 appropriately labeled 1.7 mL microcentrifuge tubes and suspend each BioBallTM in 50 μ L AE buffer.

 Using a clean micropipette tip for each ce ll suspension, thoroughly mix by gentle pipetting and spot the entire 50 μ L volume onto the polycarbonate filter in the appropriately labeled extraction tube

 Extract samples according to Method A bead-beating procedure (Sections 11.3.9- 11.3.12).

 Recover the entire volumes (e.g., 400 μ L) of crude (Section 11.3.11) and clarified (Section 11.3.12) extracts (supernatants) from each sample and transfer to a sterile 1.7 mL microcentrifuge tube. Record total sample (clarified extracts) volumes.

 Add 2 volumes of Binding Buffer (GeneRite kit) to each sample extract (e.g., add 800 μ L binding buffer to 400 μ L sample) and vortex to mix.

Note: Each sample mixture must be loaded onto a DNAsure column in two steps since the maximum holding capacity of a column is 900 μ L.

 Add approximately half of each sample (e.g., 600 μ L) to individual labeled DNAsure columns inserted into collection tubes and cen trifuge at top speed for 1 minute. Discard the flow through from collection tubes.A-9  Add the remaining portion of each sample to the appropriate DNAsure column, centrifuge and discard flow through from collection tubes.

 Transfer each DNAsure column to a clean collection tube.

 Add 500 μ L EZ-Wash Buffer to each column and centrifuge at top speed for 1 minute. Discard flow through from each column. Repeat wash using 500 μ L EZ-Wash Buffer.

 Transfer each column to a sterile 1.7 mL microcentrifuge tube and add 50 μ L DNA Elution Buffer to each column. Centrifuge for 30 seconds at top speed. Repeat DNA elution step using a second 50 μ L of DNA Elution Buffer to obtain a final volume of 90- 100 μ L/sample.

 Refrigerate purified undiluted samples until analyzed.

PCR Preparation and Analysis Set up PCR reactions (Entero and SPC-Sketa-22 assays) as follows:

 Prepare Entero and SPC-Sketa-22 PCR Assay Mixes on the day of use according to Method A (Table 2) and Table 1 (above):

 Set up individual PCR reactions to contain 20 μ L of the appropriate assay mix and 5 μ L of each sample (undiluted) as specified in Table 3 (below) and Method A (Section 11.8).

Note: 5 μ L of each sample will be used for each assay format (10 μ L total/sample).

 Include at least 1 no template control (NTC) containing 5 μ L PCR grade water instead of template (for each PCR assay) with each set of samples analyzed.

 Analyze PCR reactions according to Method A (Section 11.8 and Appendix B).

 When PCR run(s) is/are complete, save and export the Results Table and Analysis Settings according to Method A, Appendix B.

 Input appropriate data (e.g., CT values) into the study-specific Excel data reporting form.

 Email results to Robin K Oshiro at oshiro.robin@epa.gov and Yildiz Chambers at ychambers@csc.com

.

 Fax hard copy data reporting forms to Yildiz Chambers at 703.461.8056.

Table 3. Summary of Week Two Validation Study Analyses Sample Description Method A PCR Reactions a

PBS (unspiked) Method Blank 3 , 1 rep x 2 assays

Water Matrix (unspiked) Background 200 , 25 samples x 4 reps × 2 assays

Water Matrix (spiked) BioBallTM (320 CFU) 200 , 25 samples x 4 reps × 2 assays

Ongoing Calibrators (BioBallTM 1000 CFU) 6, 3 reps x 2 assays
No template Controls 10-12 , 1 rep × 2 assays × per run Total Analyses 420-422

a PCR Assays include Entero and Sketa-22

## Appendix B

**Enterococcus faecalis** Spiking ProtocolB-1 

## Enterococcus faecalis Spiking Protocol for Single-Laboratory Validation of Method A

July 21, 2009

The purpose of this protocol is to provide laboratories with Enterococcus faecalis spiking procedures for the single-laboratory validation study of Method A in fresh water and marine water samples. During this study, laboratories will use laboratory-prepared and BioBall TM spikes. The following sections are included in this protocol:

Laboratory-Prepared Spiking Suspensions Section 1: Laboratory-Prepared Sp iking Suspensions and Enumeration Section 2: Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration Section 3: Calculation of Spiked E. faecalis Percent Recovery

BioBallTM Spikes Section 4: BioBallTM Sample Spiking and Enumeration Section 5: Calculation of Bi oBallTM Spike Percent Recovery

1.0 Laboratory-Prepared Spiking Suspensions and Enumeration

1.1 Preparation of Laboratory-Prepared Undiluted Spiking Suspension

1.2 Stock Culture ( Enterococcus faecalis ) . Prepare a stock culture by inoculating a brain heart infusion agar (BHIA) slant with Enterococcus faecalis ATCC #29212 and incubating at 35°C ± 0.5°C for 20 ± 4 hours. This stock culture may be stored in the dark at room temperature for up to 30 days.

1.3 Phosphate-Buffered Saline (PBS)

. Prepare PBS according to Study Instructions.

1.4 Brain Heart Infusion Broth (BHIB) . Prepare 100 mL of BHIB according to manufacturer’s instructions.

1.5 Brain Heart Infusion Agar (BHIA) . Prepare BHIA according to study instructions.

1.6 Enterococcus faecalis Spiking Suspension (Undiluted) . From the stock culture of Enterococcus faecalis ATCC #29212 (Section 1.2), transfer a small loopful of growth to the BHIB (Section 1.4) and vigorously shake a minimum of 25 times. In cubate at 35°C ± 0.5°C for 21 ± 3 hours. The resulting spiking suspension contains approximately 1.0 × 10 9 enterococci colony forming units (CFU) per mL. This is referred to as the “undiluted spiking suspension.”

1.7 Proceed to Section 2.0 for sample spiking and e numeration of spiking suspension (Section 1.6).B-2 2.0 Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration

Since the objective of spiking the sample is to establis h percent recovery, it is necessary to determine the concentration of E. faecalis in the laboratory-prepared undiluted spiking suspension (Section 1.6). This section provides instructions for sample spiking (2 .1) and spiking suspension enumeration (2.2).

2.1 Sample spiking Please be sure to vigorously shake the spiking susp ensions in the steps belo w, to ensure accurate sample spiking and spiking suspension enumeration.

2.1.1 Dilute spiking suspension Perform steps 2.1.1.1 through 2.2.6 using the laboratory-prepared undiluted spiking suspension (Section 1.6).

2.1.1.1 Mix the undiluted spiking suspension by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipette to transfer 1.0 mL of the undiluted spiking suspension to 99 mL of sterile phosphate-buffered saline; cap and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “A.” A 1.0-mL volume of dilution “A” is equivalent to a concentration of 1 × 10 7 CFU per mL (10 -2 mL of the original undiluted spiking suspension).

2.1.1.2 Mix spiking suspension “A” (Section 2.1.1.1) by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipette to transfer 1.0 mL to 99 mL of sterile phosphate-buffered saline; cap a nd mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “B.” A 1.0-mL volume of dilution “B” is equivalent to a concentration of 1 × 10 5 CFU per mL (10 -4 mL of the original undiluted spiking suspension).

2.1.1.3 Mix spiking suspension “B” (Section 2.1.1.2) by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipette to transfer 11.0 mL to 99 mL of sterile phosphate-buffered saline; cap a nd mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “C.” A 1.0-mL volume of dilution “C” is equivalent to a concentration of 1 × 10 4 CFU per mL (10 -5 mL of the original undiluted spiking suspension).

2.1.1.4 Mix spiking suspension “C” (Section 2.1.1.3) by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipette to transfer 11.0 mL to 99 mL of sterile phosphate-buffered saline; cap a nd mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “D.” A 1.0-mL volume of dilution “D” is equivalent to 1 × 10 3 CFU per mL (10 -6 mL of the original undiluted spiking suspension).

2.1.1.5 Mix spiking suspension “D” (Section 2.1.1.4) by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipette to transfer 11.0 mL to 99 mL of sterile phosphate-buffered saline; cap a nd mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “E.” A 1.0-mL volume of dilution “E” is equivalent to 1 × 10 2 CFU per mL (10 -7 mL of the original undiluted spiking suspension).B-3 2.1.2 Spike samples

2.1.2.1 100 CFU Spike To spike the sample, add 1.0 mL of sp iking suspension dilution “E” (Section 2.1.1.5) to 100 mL of unspiked sample and mix by vigorously shaking the bottle a minimum of 25 times. This is the “spiked” sample with a concentration of 100 CFU per 100 mL. The volume (mL) of undiluted spiking suspension added is 1 × 10 -7 mL/100 mL. This is referred to as V spiked per 100 mL sample in Section 3.2 below. Analyze the spiked sample according to Study Instructions.

2.1.2.2 320 CFU Spike To spike the sample, add 3.2 mL of sp iking suspension dilution “E” (Section 2.1.1.5) to 100 mL of sample and mix by vigorously shaking the bottle a minimum of 25 times. This is the “spiked” sample with a concentration of 320 CFU per 100 mL. The volume (mL) of spiking suspension added is 1 × 10 -7 mL/100 mL. This is referred to as V spiked per 100 mL sample in Section 3.2 below. Analyze the spiked sample according to Study Instructions.

2.1.2.3 1000 CFU Spike To spike the sample, add 1.0 mL of sp iking suspension dilution “D” (Section 2.1.1.4) to 100 mL of sample and mix by vigorously shaking the bottle a minimum of 25 times. This is the “spiked” sample with a concentration of 1000 CFU per 100 mL. The volume (mL) of spiking suspension added is 1 × 10 -6 mL/100 mL. This is referred to as V spiked per 100 mL sample in Section 3.2. Analyze the spiked sample according to Study Instructions.

2.2 Enumeration of Spiking Suspensions (Prepared in Section 2.1.1)

2.2.1 Prepare brain heart infusion agar (BHIA) according to manufacturer’s directions; add 12 - 15 mL of BHIA per 100 × 15 mm Petri dish a nd solidify. Ensure the agar surfaces are dry. Note : Agar plates must be dry and free from co ndensation prior to use. To ensure the agar surfaces are dry, plates should be ma de several days in advance and stored inverted at room temperature or dried using a laminar-flow hood.

2.2.2 Each of the following will be conducted in triplicate, resulting in the evaluation of nine spread plates:

 Pipet 0.1 mL of dilution “C” (Section 2.1.1. 3) onto surface of pre-dried BHIA plate (10 -6 mL of the original spiking suspension).

 Pipet 0.1 mL of dilution “D” (Section 2.1. 1.4) onto surface of pre-dried BHIA plate (10 -7 mL of the original spiking suspension).

 Pipet 0.1 mL of dilution “E” (Section 2.1.1. 5) onto surface of pre-dried BHIA plate (10 -8 mL of the original spiking suspension).

2.2.3 For each spread plate use a sterile bent glass rod or spreader to distribute inoculum over the surface of the medium by rotating the dish by hand or on a turntable. Note : Please ensure that inoculum is evenly distributed over entire surface of the plate.

2.2.4 Allow inoculum to absorb into the medium completely.

2.2.5 Invert plates and incubate at 35°C ± 0.5°C for 20 ± 4 hours.B-4 2.2.6 Count and record number of colonies per plate. Refer to Section 3.0 for calculation of spiking suspension concentration.

3.0 Calculation of Spiked E. faecalis Percent Recovery (Laboratory-Prepared Undiluted Spiking Suspension) Spiked E. faecalis percent recovery will be calculated in three steps as indicated in Sections 3.1 through 3.3 below. Note : The calculated numbers in the table examples provided below have been rounded at the end of each step. If your laboratory recalculates the examples using a spreadsheet and rounds only after the final cal culation (Step 3), the percent recoveries may be slightly different.

3.1 Step 1: Calculate Concentration of E. faecalis (CFU/mL) in Undiluted Spiking Suspensions

3.1.1 The number of E. faecalis (CFU/mL) in the undiluted spiking suspensions will be calculated using all BHIA plates from Section 2.2.2 yielding counts within the ideal range of 30 to 300 CFU per plate.

3.1.2 If the number of colonies exceeds the upper range (i.e., >300) or if the colonies are not discrete, results should be recorded as “too numerous to count” (TNTC).

3.1.3 Calculate the concentration of E. faecalis (CFU/mL) in the undiluted spiking suspensions according to the following equation. Example calculations are provided in Table 1

.

E. faecalis undiluted spike = (CFU 1 + CFU 2 + ... + CFU n ) / (V 1 + V 2 + ... + V n )

Where,

E. faecalis undiluted spike = E. faecalis (CFU/mL) in undiluted spiking suspension CFU = Number of colony forming units from BHIA plates yielding counts within the ideal range of 30 to 300 CFU per plate V = Volume of undiluted sample on each BHIA plate yielding counts within the ideal range of 30 to 300 CFU per plate n = Number of plates with counts within the ideal range

Table 1. Example Calculations of E. faecalis Spiking Suspension Concentration

Examples CFU / plate (triplicate analyses) from BHIA plates in Section 2.2.2 E. faecalis CFU / mL in undiluted spiking suspension ( E. faecalis undiluted spike ) * 10 -6 mL plates 10 -7 mL plates 10 -8 mL plates

Example 1 TNTC, TNTC, TNTC 94, 106, 89 9, 11, 28 (94+106+89) / (10 -7 +10 -7 +10 -7 ) =
289 / (3.0 x 10 -7 ) =

9.6 x 10 8 CFU / mL

Example 2 TNTC, TNTC, TNTC 32, 55, 72 8, 5, 3 (32+55+72) / (10 -7 +10 -7 +10 -7 ) =
159 / (3.0 x 10 -7 ) =

5.3 x 10 8 CFU / mL

* E. faecalis undiluted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per plateB-5 3.2 Step 2: Calculate “True” Spiked E. faecalis (CFU/mL) Calculate the true concentration of spiked E. faecalis (CFU/100 mL) according to the following equation. Example calculations are provided in Table 2

.

T Spiked E. faecalis = ( E. faecalis undiluted spike ) × (V spiked per 100 mL sample )

Where, T Spiked E. faecalis = Number of spiked E. faecalis (CFU/100 mL) E. faecalis undiluted spike = E. faecalis (CFU/mL) in undiluted spiking suspension (calculated in Section 3.1.3) V spiked per mL sample = mL of undiluted spiking suspension per 100 mL sample (e.g., Section 2.1.2.2)

Table 2. Example Calculations of Spiked E. faecalis

E. faecalis undiluted spike (Table 1 above) V spiked per mL sample (Section 2.1.2.2 above) T Spiked E. faecalis

9.6 × 10 8 CFU / mL 1.0 × 10 -7 mL per 100 mL of sample (9.6 × 10 8 CFU / mL) × (1.0 × 10 -7 mL / 100 mL) = 96 CFU / 100 mL

5.3 × 10 8 CFU / mL 1.0 × 10 -7 mL per 100 mL of sample (5.3 × 10 8 CFU / mL) × (1.0 × 10 -7 mL / 100 mL) = 53 CFU / 100 mL

3.3 Step 3: Calculate Percent Recovery

3.3.1 Calculate percent recovery (R) using the following equation.

R = 100 H (N s - N u )

T Where, R = Percent recovery N s = E. faecalis (CFU/100 mL) in the spiked sample N u = E. faecalis (CFU/100 mL) in the unspiked sample T Spiked E. faecalis = True spiked E. faecalis (CFU/100 mL) in spiked sample (Section 3.2)

3.3.2 Example percent recovery calculations are provided in Table 3

.
Table 3. Example Percent Recovery Calculations

N s (CFU / 100 mL) N u (CFU / 100 mL) T Spiked E. faecalis (CFU / 100 mL) Percent recovery (R)

947 3 a 960 100 × (947 – 3) / 960 = 98%

288 <1 301 100 × (288 - 1) / 301 = 95%

85 <1 96 100 × (85 - 1) / 96 = 88%

49 <1 53 100 × (49 - 1) / 53 = 91%

a The result of 3 CFU is acce ptable if the sample was a re creational water – if the samp le is PBS, it would not be acceptable.B-6 4.0 BioBallTM Sample Spiking and Enumeration During the validation study, each laboratory will enumerate E. faecalis in the BioBalls so that percent recovery can be evaluated. This section provides instructions for sample spiking (Section 4.1) and spiking suspension enumeration (Section 4.2).

Upon receipt, BioBalls should be stored at -20°C. Preparation of BioBalls prior to spiking is not necessary, as they can be spiked directly into the sample once the vial is opened.

4.1 Sample Spiking
4.1.1 Open BioBallTM vial by removing the cap. Aseptically add 1 BioBallTM to 100 mL of sample for each concentration (100, 320, and 1000 CFU). Mix by vigorously shaking the bottles a minimum of 25 times.
4.1.2 Spike and analyze samples according to Study Instructions.

4.2 Enumeration of BioBallTM

4.2.1 Prepare BHIA according to manufacturer’s di rections. Add 12 – 15 mL of BHIA per 100 × 15 mm Petri dish and let solidify

. Note : Agar plates must be dry and free from condensation prior to use. To ensure agar surfaces are dry, plates should be made several days in advance and stored inverted at room temperature or dried using a laminar-flow hood.

4.2.2 The following will be conducted in triplicate, resulting in the evaluation of three spread plates per concentration (100, 320, 1000 CFU) for a total of nine plates.

 Open BioBallTM vial by removing the cap and aseptically add one BioBallTM to 1.0 mL of sterile PBS. Allow BioBallTM to dissolve completely.

 Mix suspension to homogenize. Pipet 0.1 mL of the BioBallTM suspension onto a 100 × 15 mm BHIA plate.

4.2.3 For each spread plate, use a sterile bent glass rod or spreader to distribute BioBallTM inoculum over the medium surface by rotating the dish by hand or on a turntable. Note: Please ensure that inoculum is evenly di stributed over entire surface of plate.

4.2.4 Allow inoculum to absorb into medium completely.

4.2.5 Invert plates and incubate at 35°C ± 0.5°C for 20 ± 4 hours.

4.2.6 For each dilution, count and r ecord the sum of the number of colonies (colony forming units [CFU]) on the triplicate plates and multiply the sum by a factor of 10 to account for the volume of the BioBallTM suspension plated. For example, the 320 CFU BioBallTM results in plate counts of 30, 24 and 29. The sum is 83; 83 × 10 = 830. The average of the plates is 277 CFU and represents the “true” spiked E. faecalis . Refer to Section 5.0 for calculation of E. faecalis concentration in BioBallTM.

5.0 Calculation of BioBallTM Spike Percent Recovery
Spiked BioBall J percent recovery will be calculated following the steps indicated below. Note : The calculated numbers in the examples provided in the tables below have been rounded at the end of each step. If your laboratory recalculates th e examples using a spreadsheet and rounds only after the final calculation (Step 2), the percent recoveries may be slightly different.B-7 5.1 Step One: Calculate Mean E. faecalis per BioBallTM and “True” Spiked E. faecalis (CFU/100 mL)

The mean concentration of E. faecalis (CFU) in the BioBalls will be calculated using all three BHIA plates for each concentration (100, 320, and 1000 CFU) from Section 4.2.2. Since one BioBallTM is spiked per 100 mL sample, use the mean number of E. faecalis per BioBallTM as the

A true @ spiked E. faecalis. For example, the triplicate plate counts for the 320 CFU BioBallTM concentration are as follows:

T Spiked E. faecalis (CFU / 100 mL) = [(30 + 24 + 29) ×10] / 3 = 277 per BioBallTM

Where, T Spiked E. faecalis = True spiked E. faecalis (CFU/100 mL) in spiked sample

5.2 Step Two: Calculate Percent Recovery

Calculate percent recovery (R) using the following equation:

R = 100 × (N s - N u ) T

Where, R = Percent recovery N s = E. faecalis (CFU/100 mL) in the spiked sample (or CFU per BioBallTM) N u = E. faecalis (CFU/100 mL) in the unspiked sample T Spiked E. faecalis = True spiked E. faecalis or CFU/100 mL in spiked sample (Section 5.1).

Percent recovery example calculations are provided in Table 4

.
Table 4. Percent Recovery Example Calculations

Concentration N s (CFU / 100 mL) N u (CFU / 100 mL) T (CFU / 100 mL) Percent recovery (R)

100 98 <1 92 100 H (98 - 1) / 92 = 105%

320 305 10 300 100 H (305 - 10) / 300 = 98%

1000 890 <1 975 100 × (890 – 1) / 975 = 91%

## Appendix C

Bacteroidales (Method B) Study InstructionsC-1 

## Method B Single-Laboratory Validation Study: Assessment of Method Performance – Matrix Inhibition Analyses

Revised October 28, 2009

Summary of Objectives

The objective of this study is to characterize Me thod B matrix inhibition across multiple fresh and marine water matrices



### Summary of Analyses
Laboratories will complete the following accordi ng to Method B and study-specific instructions:

 Prepare and analyze unspiked and spiked (single level) fresh or marine water (25 different sources) samples and one me thod blank (PBS) according to Method B

 Prepare, extract, and analyze ongoing calibrator samples using laboratory-prepared B. thetaiotaomicron cell preparations During this study phase Method B matrix inhibition will be assessed through analysis of multiple fresh and marine water samples collected from recreational sites across the nation. Study analyses will include the preparation of unsp iked PBS and both unspiked and spiked fresh or marine water samples according to Method B and the Spiking Protocol. During the study samples will be filtered and following filter extraction, crude extracts will be diluted and analyzed for Bacteroidales (Bact assay) and salmon DNA (SPC-Sketa 22) according to Method B. Ongoing calibrators will be prepared and extr acted as described for Phase II initial calibrator samples using a laboratory-prepared suspension. Following filter extraction, diluted calibrator extracts will be analyzed for Bacteroidales (Bact assay) and salmon DNA (SPC-Sketa 22) according to Method B. After PCR analyses, the ratio of target sequence quantities recovered from PBS and fresh or marine water samples compared to those recove red from ongoing calibrator samples will be used in the ΔΔ C T comparative cycle threshold calculation method to quantify DNA in PBS and fresh water samples.



### Media/Reagents/Supplies

 Phosphate buffered saline (PBS) – 5 L minimum

 0.45 μm polycarbonate filters – 204

 AE buffer, pH 9.0 – 10 mL minimum

 Extraction tubes with glass beads – 204

 Salmon DNA/extraction buffer (0.2 μg/mL solution) – 100 mL

 1.7 mL sterile microcentrifuge tubes

 Method B PCR Reagents:

 PCR grade water

 Bovine serum albumin (BSA, 2 mg/mL solution)

 B. thetaiotaomicron primer/probe working stock solution (Bact)

 Salmon DNA primer/probe working stock solution (Sketa-22)

 TaqMan® Universal PCR Master MixC-2 

### Matrix Inhibition Analyses

Sample Preparation and Spiking Participating laboratories will be provided with e ither fresh or marine water samples (1 L per sample, 25 total samples) collected from various sites across the nation. Samples should be processed on the day of receipt as follows:

 Thoroughly mix each sample by vigorously shaking the bottles a minimum of 25 times.

 Prepare 8 - 100 mL subsamples from each orig inal grab sample and label replicate subsamples with the sample ID and designate each as either unspiked (4 replicates) or spiked (4 replicates).

 Spike 4 replicate subsamples with 32,000 CFU using a laboratory-prepared suspension and mix thoroughly.

 Prepare a 100 mL PBS method blank (unspiked) each week that matrix samples are processed.

 Process unspiked and spiked replicates a nd method blank(s) as described below.
Membrane Filtration and Filter Extraction/Extract Processing – Method B

 Mix samples by shaking 25 times.

 Filter unspiked and spiked samples (entire 100 mL volumes) and rinse the sides of the funnel at least twice with sterile PBS (Method B, Section 11.4).

Note: Use a sterile funnel for each matrix and always filter the unspiked samples first. The unspiked PBS sample (method blank) should be filtered last using a sterile funnel.

 Place individual filters into appropriately labe led extraction tubes and extract according to Method B (Section 11.5).

 Recover as much of each crude extract as possible (e.g., 400 μ L).

 Prepare a 5-fold dilution of each crude extract (e.g., 50 μ L crude extract plus 200 μ L AE buffer).

 Refrigerate diluted and undiluted samples until analyzed. Note : Only the diluted extracts will be assayed.

Preparation, Extraction and Processing of Ongoing Calibrator Samples

 Thaw (on ice) a total of 3 calibrator sa mples (prepared during Phase 1) and add 990 μ L AE buffer. Thoroughly mix by vortexing.

 Prepare a total of 3 extraction tubes containing glass beads by placing one polycarbonate filter into each extracti on tube and adding 590 μ L of salmon DNA/extraction buffer to each extraction tube.

 Mix diluted calibrator cells thoroughly and spot 10 μ L of each diluted suspension onto the polycarbonate filter in each extraction tube (6 total).

 Spot 10 μ L of AE buffer onto the polycarbonate f ilter of the SPC blank extraction tube.

 Perform extraction of calibrator samples acco rding to Method B bead-beating procedure (Sections 11.3.9-11.3.12).C-3  Prepare a 5-fold dilution for each extract (e.g., 50 μ L extract plus 200 μ L AE buffer).

 Refrigerate crude (undiluted and diluted) sample extracts until analyzed. Note : Only the diluted extracts will be assayed.

PCR Preparation and Analysis

Note : To minimize potential environmental DNA contam ination, routinely treat all work surfaces with a 10% bleach solution, allowing bleach to contact the work surface for a minimum of 15 minutes prior to rinsing with sterile water. Further decontaminate work area surfaces using UV light for 15 minutes prior to assay set up if possible. Set up PCR reactions (Bact and SPC-Sketa-22 assays) as follows:

 Prepare Bact and SPC-Sketa-22 PCR Assay Mixes on the day of use according to Method B (Table 2) and Table 1 below:

Table 1. PCR Assay Mix Composition

Rea g ent V olume/Reaction 1

Sterile H 2 O 1.5 μ L

BSA 2.5 μ L

Ta q Man® master mix 12.5 μ L Primer/probe workin g stock solution 2 3.5 μ L
1 Multiply by # samples (plus 1) to be analyzed per day
2 See Phase II Table 1 for assay-specific primer/probe working stocks

 Set up individual PCR reactions to contain 20 μ L of the appropriate assay mix and 5 μ L of each sample (diluted replicates) as specified in Table 2 (below) and Method B (Section 11.8).
Note: 5 μ L of each sample will be used for each assay format (10 μ L total/sample).

 Include at least 1 no template control (NTC) containing 5 μ L PCR grade water instead of template (for each PCR assay) with each set of samples analyzed.

 Analyze PCR reactions according to Method B (Section 11.8 and Appendix A).

Table 2. Summary of Matrix Inhibition Study Analyses Sample Description Method B PCR Reactions a

PBS (unspiked) Method Blank 2 , 1 rep x 2 assays

Water Matrix (unspiked) Background 200 , 25 samples x 4 reps × 2 assays

Water Matrix (spiked) Laboratory-prepared (32,000 CFU) 200 , 25 samples x 4 reps × 2 assays

Ongoing Calibrators (Laboratory-prepared ) 6, 3 reps x 2 assays
No template Controls 10-12 , 1 rep × 2 assays × per run Total Analyses 412-414

a PCR Assays include Bact and Sketa-22C-4  When PCR run(s) is/are complete, save and export the Results Table and Analysis Settings according to Method B, Appendix A.

 Input appropriate data (e.g., CT values) into the study-specific Excel data reporting form.

 Email results to Robin K Oshiro at oshiro.robin@epa.gov and Yildiz Chambers at ychambers@csc.com

.

 Fax hard copy data reporting forms to Yildiz Chambers at 703.461.8056.C-5 

## Method B (qPCR for Bacteroidales) Single-Laboratory Validation Study Assessment of Method Performance and Development of Preliminary Quantitative QC Criteria

November 9, 2009

Summary of Objectives

 Evaluation of ongoing calibrator/SPC samples from laboratory-prepared B. thetaiotaomicron cell preparations

 Evaluation of PBS and water samples spiked with laboratory-prepared B. thetaiotaomicron cells at three different levels

 Characterize recovery and precision of Method B

 Establish preliminary quantitative QC accepta nce criteria for initial and ongoing method performance assessments of Method B



### Summary of Preliminary Analyses
Laboratories will complete the following accordi ng to Method B and study-specific instructions:

 Enumerate laboratory-prepared spiking suspen sions and determine cell concentration of the spiking suspension

 Prepare and analyze unspiked PBS and fresh or marine water samples to determine ambient Bacteroidales concentrations accord ing to Method B and culture instructions

 Prepare and analyze PBS and fresh or marine (single source) water samples spiked with laboratory-prepared B. thetaiotaomicron spiking suspensions at three different levels according to the Spiking Protocol, Me thod B, and culture instructions

 Prepare, extract, and analyze ongoing calibrator samples using laboratory- prepared B. thetaiotaomicron cell preparations

 Determine B. thetaiotaomicron target sequences/calibrator from PCR analysis and cell concentration of calibrator samples from laboratory- prepared B. thetaiotaomicron cell preparations Study analyses will include the preparation of unspiked and spiked PBS (reference matrix) and water (fresh or marine) samples according to Meth od B and the Spiking Protocol. Samples will be filtered and analyzed according to culture inst ructions, or following filter extraction, samples (undiluted and diluted extracts) will be analyzed for Bacteroidales (Bact assay) and salmon DNA (SPC-Sketa 22). Ongoing calibrators will be prepared using laboratory-prepared cell suspensions and extracted as described for Phase II initial calib rator samples. Following extraction, replicate samples (undiluted and diluted extracts) will be analyzed according to Method B.

After PCR analyses, the ratio of target sequence quantities recovered from PBS and fresh or marine water compared to those recovered fro m ongoing calibrator samples will be used in the ΔΔ C T comparative cycle threshold calculation method to quantify target DNA in PBS and fresh water samples.C-6 

### Media/Reagents/Supplies

 Undiluted laboratory-prepared B. thetaiotaomicron calibrator cell suspensions (frozen 10
μ L calibrators from Phase 1) – 6 calibrators

 Phosphate buffered saline (PBS) – 10 L minimum

 Chopped meat carbohydrate broth – 6 tubes (5 mL each)

 CDC anaerobic blood agar – 20 plates

 BBE agar – 20 plates

 0.45 μm polycarbonate filters – 45

 AE buffer, pH 9.0 – 10 mL minimum

 Salmon DNA/extraction buffer (0.2 μg/mL solution) – 100 mL

 Extraction tubes with glass beads – 45 tubes

 Method A PCR Reagents:

 PCR grade water

 Bovine serum albumin (BSA, 2 mg/mL solution)

 Bacteroidales primer/probe working stock solution (Bact)

 Salmon DNA primer/probe working stock solution (Sketa-22)

 TaqMan® Universal PCR Master Mix



### Preliminary Analyses (Week One)
Week one preliminary analyses will assess spiki ng protocols and calibrators using laboratoryprepared cell suspensions.
Preparation and enumeration of spiking suspensions

 Propagate and enumerate laboratory-prepared spiking suspensions according to study instructions (Phase I) and Spiking Protocol using CDC anaerobe blood agar. Record results and all associated calculations on data reporting forms provided for the study.

Spiking and analyses of sterile PBS samples

 Label 10, 100-mL aliquots of sterile PBS as indicated below:

o 2, Unspiked (1 Method B/1 Culture)

o 1,000 CFU

 2, lab-prepared (Culture)

o 10,000 CFU

 2, lab-prepared

o 32,000 CFU

 2, lab-prepared

o 100,000 CFU

 2, lab-prepared

 Method B: Spike 2, 100-mL PBS replicates at each of the three levels (10,000, 32,000, and 100,000 CFU) using the laboratory-prepared B. thetaiotaomicron spiking suspension according to the Spiking Protocol.

 Culture: Spike 2, 100-mL PBS replicates by adding 1.0 mL of B. thetaiotaomicron spiking suspension dilution (1 × 10 3 ) “D” (Spiking Protocol, Section 2.1.1.4), to 100 mL of sample and mix by vigorously shaking the bottle a minimum of 25 times.C-7 Note : If previous counts indicate that the undiluted spiking suspension is 1.0 × 10 8 B. thetaiotaomicron CFU per mL, please add 1.0 mL of the appropriate dilution to spike samples at 1000 CFU/100 mL.

Water sample collection and spiking

 Collect a 2 L bulk fresh or marine water sample and transport to the laboratory on ice (do not freeze). In the laboratory, split the bulk sample into 100-mL aliquots (10 total) and label as indicated below:

o 2, Unspiked (1 Method B/1 Culture)

o 1,000 CFU

 2, lab-prepared (Culture)

o 10,000 CFU

 2, lab-prepared

o 32,000 CFU

 2, lab-prepared

o 100,000 CFU

 2, lab-prepared

 Method B: Spike 2, 100-mL sample replicates at each of the three levels (10,000, 32,000, and 100,000 CFU) using the laboratory-prepared B. thetaiotaomicron spiking suspension according to the Spiking Protocol.

 Culture: Spike 2, 100-mL sample replicates at 1000 CFU using the laboratory-prepared B. thetaiotaomicron spiking suspension
Membrane filtration and culture analyses

 Mix samples (6 total) by shaking 25 times.

 For each matrix (PBS and either fresh or marine water), filter sample volumes indicated below:

 Unspiked – 100 mL

 1000 CFU Spike – 5 mL

 Filter unspiked and spiked samples and rinse the si des of the funnel at least twice with sterile PBS.

Note: Use a sterile funnel for each individual sample and filter the unspiked PBS (method blank) last.

 Filter sample aliquots and rinse the funnel at least twice with 20 - 30 mL of sterile PBS. Use sterile forceps to aseptically remove filters and place onto BBE plates. Incubate at 35.0°C ± 0.5°C for 48 ± 3 hours under anaerobic conditions.
Method B membrane filtration and filter extraction/extract processing

 Mix samples by shaking 25 times.

 Filter unspiked and spiked PBS and water samples and rinse the sides of the funnel at least twice with sterile PBS (Method B, Section 11.4).C-8 Note: Use a sterile funnel for each matrix and always filter the lowest B. thetaiotaomicron spike concentration (e.g., 10,000 CFU spike, then 32, 000 CFU spike, etc.) first. The unspiked PBS (method blank) should be filtered last using a sterile funnel.

 Place individual filters into appropriately la beled extraction tubes (7 total) and extract according to Method B (Section 11.5).

 Recover as much of each sample extract and clarified supernatant as possible (e.g., 400 μ L).

 After extraction, dilute (5-fold; e.g., 50 μ L extract plus 200 μ L AE buffer) one unspiked sample extract and 2 replicate extracts (clarified supernatants) of each matrix and spike level (e.g., fresh water - 32,000 CFU) and place undilu ted and diluted extracts in refrigerator until analyzed.

Preparation, extraction and processi ng of ongoing calibrator samples 1. Laboratory-prepared calibrators

 Thaw (on ice) a total of 3 calibrator sa mples (prepared during Phase 1) and add 990 μ L of AE buffer to each calibrator. Thoroughly mix by vortexing.

 Prepare a total of 3 extraction tubes containing glass beads by placing one polycarbonate filter into each extracti on tube and adding 590 μ L of salmon DNA/extraction buffer to each extraction tube.

 Mix diluted calibrator cells thoroughly and spot 10 μ L of each diluted suspension onto the polycarbonate filter in each extraction tube.

 Perform extraction of calibrator samples acco rding to Method B bead-beating procedure (Sections 11.3.9-11.3.12).
Note: Recover as much of the clarified extracts as possible (e.g., 400 μ L).

 Prepare a 5-fold dilution for each calibrator extract (e.g., 50 μ L extract plus 200 μ L AE buffer).

 Refrigerate undiluted and diluted calibrator extracts until analyzed.

PCR preparation and analysis

Note : To minimize potential environmental DNA contam ination, routinely treat all work surfaces with a 10% bleach solution, allowing bleach to contact the work surface for a minimum of 15 minutes prior to rinsing with sterile water. Further decontaminate work area surfaces using UV light for 15 minutes prior to assay set up if possible. Set up PCR reactions (Bact and SPC assays) as follows:

 Prepare Bact and SPC (Sketa-22) PCR Assay Mixes on the day of use according to Method B (Table 2) and Table 1

. Table 1. PCR Assay Mix Composition
Reagent Volume/Reaction 1

Sterile H 2 O 1.5 μ L

BSA 2.5 μ L
TaqMan® master mix 12.5 μ L
Primer/probe working stock solution 2 3.5 μ L

1 Multiply by # samples (plus 1) to be analyzed per day
2 See Phase II Table 1 for assay-specific primer/probe working stocksC-9  Set up individual PCR reactions to contain 20 μ L of the appropriate assay mix and 5 μ L of each sample (diluted and undiluted replicates) as specified in Table 2 (below) and Method B (Section 11.8).
Note: 5 μ L of each sample will be used for each assay format (10 μ L total/sample).

 Include at least 1 no template control (NTC) containing 5 μ L PCR grade water instead of template (for each PCR assay) with each set of samples analyzed.

Table 2. Calibrator and PBS/Water Samples – Method B PCR Analyses Sample Description Undiluted/Diluted Reactions a

Calibrators Lab-prepared Undiluted 6, 3 reps × 2 assays Diluted (5-Fold) 6, 3 reps × 2 assays

PBS Lab-prepared (10,000, 32,000, and 100,000 CFU) Undiluted 12, 2 reps × 2 assays × 3 concentrations Diluted (5-Fold) 12, 2 reps × 2 assays × 3 concentrations

Water Matrix Lab-prepared (10,000, 32,000, and 100,000 CFU) Undiluted 12, 2 reps × 2 assays × 3 concentrations
Diluted (5-Fold) 12, 2 reps × 2 assays × 3 concentrations

Water Matrix Unspiked Undiluted 2, 1 rep × 2 assays Diluted (5-Fold) 2, 1 rep × 2 assays

PBS Method Blank Undiluted 2, 1 rep × 2 assays Diluted (5-Fold) 2, 1 rep × 2 assays
No template Control NA 2-4, 1 rep × 2 assays × per run

Total PCR Reactions = 70 - 72

a Assays include Bact and Sketa-22

 Analyze PCR reactions according to Method B (Section 11.8 and Appendix A).

 When PCR run(s) is/are complete, save and export the Results Table and Analysis Settings according to Method B, Appendix A.

 Input appropriate data (e.g., CT values) into the study-specific Excel data reporting form

 Email results to Robin K. Oshiro at oshiro.robin@epa.gov and Yildiz Chambers at ychambers@csc.com

.

 Fax hard copy data reporting forms to Yildiz Chambers at 703.461.8056.C-10 

### Validation Study Analyses (Week Two)
Week two analyses will focus on developing prelim inary quantitative QC criteria for initial and ongoing method performance assessment (method recovery and precision criteria).
Preparation and enumeration of spiking suspensions

 Propagate and enumerate laboratory-prepared spiking suspensions according to study instructions (Phase I) and the Spiking Protocol.

 Record results and all associated calculations on data reporting forms provided for the study.

Spiking and analyses of sterile PBS samples

 Label 19, 100-mL aliquots of sterile PBS as indicated below (see also Table 2 ):

o 3, Unspiked (2 Method B/1 culture)

o 1,000 CFU

 4, lab-prepared (Culture)

o 10,000 CFU

 4, lab-prepared

o 32,000 CFU

 4, lab-prepared

o 100,000 CFU

 4, lab-prepared

 Method B: Spike 4, 100-mL PBS replicates at each of the three levels using the laboratory-prepared B. thetaiotaomicron spiking suspension according to the Spiking Protocol.

 Culture: Culture: Spike 4, 100-mL PBS replicates by adding 1.0 mL of B. thetaiotaomicron spiking suspension dilution (1 × 10 3 ) “D” (Spiking Protocol, Section 2.1.1.4), to 100 mL of sample and mix by vigorously shaking the bottle a minimum of 25 times. L

Note : If previous counts indicate that the undiluted spiking suspension is 1.0 × 10 8 B. thetaiotaomicron CFU per mL, please add 1.0 mL of the appropriate dilution to spike samples at 1000 CFU/100 mL.

Water sample collection and spiking

 Collect a 3-L bulk fresh or marine water sample and transport to the laboratory on ice (do not freeze). In the laboratory, split the bulk sample into the following 100–mL aliquots (19 total) and label as indicated below (see also Table 3):

o 3, Unspiked (2 Method B/1 Culture)

o 1,000 CFU

 4, lab-prepared (Culture)

o 10,000 CFU

 4, lab-preparedC-11 o 32,000 CFU

 4, lab-prepared

o 100,000 CFU

 4, lab-prepared

 Method B: Spike 4, 100-mL sample replicates at each of the three levels using the laboratory-prepared B. thetaiotaomicron spiking suspension according to the Spiking Protocol.

 Culture: Spike 4, 100-mL sample replicat es at 1000 CFU using laboratory-prepared B. thetaiotaomicron spiking suspension.
Membrane filtration and culture analyses

 Mix samples (10 total) by shaking 25 times.

 For each matrix (PBS and either fresh or marine water), filter sample volumes indicated below:

 Unspiked – 100 mL

 1000 CFU Spike – 5 mL

 Filter unspiked and spiked samples and rinse the si des of the funnel at least twice with sterile PBS.

Note: Use a sterile funnel for each individual sample and filter the unspiked PBS (method blank) last.

 Filter sample aliquots and rinse the funnel at least twice with 20 - 30 mL of sterile PBS. Use sterile forceps to aseptically remove filters and place onto BBE plates. Incubate at 35.0°C ± 0.5°C for 48 ± 3 hours under anaerobic conditions.
Method B membrane filtration and filter extraction/extract processing

 Mix samples (28 total) by shaking 25 times.

 Filter unspiked and spiked samples (entire 100 mL volumes) and rinse the sides of the funnel at least twice with sterile PBS (Method B, Section 11.4).

Note: Use a sterile funnel for each matrix and always filter the lowest B. thetaiotaomicron spike concentration (e.g., 10,000 CFU spike, then 32,000 CFU spike, etc.) first. The unspiked PBS sample (method blank) should be filtered last using a sterile funnel.

 Place individual filters into appropriately labeled extraction tubes (28 total) and extract according to Method B (Section 11.5).

 Recover as much of each sample extract and clarified supernatant as possible (e.g., 400 μ L).

 After extraction, dilute (5-fold; e.g., 50 μ L extract plus 200 μ L AE buffer) both unspiked sample extracts and 4 replicate extracts (clarifi ed supernatants) of each matrix type and spike level (e.g., fresh water- lab-prepared - 32,000 CFU) and place undiluted and diluted extracts in refrigerator until analyzed.

Note : Only diluted (5-fold) samples will be analyzed during this study phase.C-12 Preparation, extraction and processi ng of ongoing calibrator samples 1. Laboratory-prepared calibrators

 Prepare a total of 3 calibrator extracts as descr ibed previously and in Method B (Sections 11.3.9 – 11.3.12).

 Prepare a 5-fold dilution for each calibrator extract (e.g., 50 μ L extract plus 200 μ L AE buffer).

 Refrigerate undiluted and diluted calibrator extracts until analyzed.

Note : Only diluted (5-fold) calibrator samples will be analyzed during this study phase.

PCR preparation and analysis Set up PCR reactions (Bact and SPC-Sketa-22 assays) as follows:

 Prepare Bact and SPC-Sketa-22 PCR Assay Mixes on the day of use according to Method B (Table 2) and Table 1 (see above).

 Set up individual PCR reactions to contain 20 μ L of the appropriate assay mix and 5 μ L of each sample (undiluted replicates) as specified in Table 3 (below):
Note: 5 μ L of each sample will be used for each assay format (10 μ L total/sample).

 Include at least 1 no template control (NTC) containing 5 μ L PCR grade water instead of template (for each PCR assay) with each set of samples analyzed.

Table 3. Summary of Week Two Validation Study Analyses Sample Description Method B PCR Reactions a

PBS Lab-prepared (10,000, 32,000, 100,000 CFU) 24 , 4 reps × 3 concentrations x 2 assays

Water Matrix Lab-prepared (10,000, 32,000, 100,000 CFU) 24 , 4 reps × 3 concentrations x 2 assays

Water Matrix Unspiked 4 , 2 reps × 2 assays

PBS Method Blank 4 , 2 reps × 2 assays

Ongoing Calibrators 6, 3 reps x 2 assays

No template Controls 4-6 , 1 rep × 2 assays × per run

Total Analyses 66-68

a PCR Assays include Bact and Sketa-22

 Analyze PCR reactions according to Method B (Section 11.8 and Appendix A).

 When PCR run(s) is/are complete, save and export the Results Table and Analysis Settings according to Method B, Appendix A.

 Input appropriate data (e.g., CT values) into the study-specific Excel data reporting form

 Email results to Robin K Oshiro at oshiro.robin@epa.gov and Yildiz Chambers at ychambers@csc.com

.



###  Fax hard copy data reporting forms to Yildiz Chambers at 703.461.8056.

## Appendix D

**Bacteroides thetaiotaomicron** Spiking ProtocolD-1 

## Bacteroides thetaiotaomicron Spiking Protocol for Single-Laboratory Validation of Method B

October 5, 2009

The purpose of this protocol is to provide laboratories with Bacteroides thetaiotaomicron spiking procedures for the single-laboratory validation stud y of Method B in fresh water and marine water samples. During this study, laboratories will use laboratory-prepared and vendor-prepared (BioBall TM ) spikes.

Note : The sample spiking procedures described in this pr otocol are intended only for samples that will be analyzed according to Method B (qPCR).

The following sections are included in this protocol:

Laboratory-Prepared Spiking Suspensions Section 1: Preparation of Laborator y-Prepared Spiking Suspensions Section 2: Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration Section 3: Calculation of Spiked B. thetaiotaomicron Percent Recovery

BioBallTM Spikes Section 4: BioBallTM Sample Spiking and Enumeration Section 5: Calculation of Bi oBallTM Spike Percent Recovery

1.0 Laboratory-Prepared Spiking Suspensions and Enumeration

1.1 Preparation of Spiking Suspension

1.1.1 Stock Culture . Rehydrate lyophilized B. thetaiotaomicron (ATCC #20741) in 0.5 mL of sterile phosphate buffered saline (PBS) and mix well to dissolve lyophilized culture. Using a sterile syringe and needle aspirate suspension and inoculate 10 mL of CMCB. Incubate in an anaerobe chamber at 35.0°C ± 0.5°C for 24 ± 2 hours. This stock culture should be stored refrigerated (do not fr eeze) and under anaerobic conditions (e.g., Gas Pak bags or anaerobe chamber).

1.1.2 B. thetaiotaomicron Spiking Suspension (Undiluted) . From the stock culture of B. thetaiotaomicron ATCC #29741 (Section 1.1.1), transfer 0.1 mL of growth broth to a fresh tube of CMCB (Section 1.2) and vigorously shake a minimum of 25 times. Incubate in an anaerobic chamber at 35°C ± 0.5°C for 24 ± 2 hours. The resulting spiking suspension contains approximately 1.0 × 10 8 B. thetaiotaomicron colony forming units (CFU) per mL. This is refe rred to as the “undiluted spiking suspension.”

1.2 Reagents/Medium

 Phosphate Buffered Saline (PBS). Prepare PBS according to Method B

 Chopped Meat Carbohydrate Broth (CMCB)

 Tryptic Soy Agar with 5% Sheep Blood (BAP).D-2 2.0 Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration

Since the objective of spiking the sample is to establis h percent recovery, it is necessary to determine the concentration of B. thetaiotaomicron in the laboratory-prepared undilu ted spiking suspension (Section 1.6). This section provides instructions for sample spiking (2.1) and spiking suspension enumeration (2.2).

2.1 Sample spiking Please be sure to vigorously shake the spiking susp ensions in the steps belo w, to ensure accurate sample spiking and spiking suspension enumeration.

2.1.1 Dilute spiking suspension Perform the following steps using the laboratory-prepared undiluted spiking suspension (Section 1.1.2).

2.1.1.1 Mix the undiluted spiking suspension by vigorously shaking the tube a minimum of 25 times. Use a sterile pipette to transfer 1.0 mL of the undiluted spiking suspension to 99 mL of sterile PBS; cap and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “ A .” A 1.0-mL volume of dilution “ A ” is equivalent to a concentration of 1 × 10 6 CFU per mL (10 -2 mL of the original undiluted spiking suspension).

2.1.1.2 Mix spiking suspension “ A ” (Section 2.1.1.1) by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipette to transfer 11.0 mL to 99 mL of sterile PBS; cap and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “ B .” A 1.0-mL volume of dilution

“ B ” is equivalent to a concentration of 1 × 10 5 CFU per mL (10 -3 mL of the original undiluted spiking suspension).

2.1.1.3 Mix spiking suspension “ B ” (Section 2.1.1.2) by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipette to transfer 11.0 mL to 99 mL of sterile PBS; cap and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “ C .” A 1.0-mL volume of dilution

“ C ” is equivalent to a concentration of 1 × 10 4 CFU per mL (10 -4 mL of the original undiluted spiking suspension).

2.1.1.4 Mix spiking suspension “ C ” (Section 2.1.1.3) by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipette to transfer 11.0 mL to 99 mL of sterile PBS; cap and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “ D .” A 1.0-mL volume of dilution

“ D ” is equivalent to 1 × 10 3 CFU per mL (10 -5 mL of the original undiluted spiking suspension).

2.1.1.5 Mix spiking suspension “ D ” (Section 2.1.1.4) by vigorously shaking the bottle a minimum of 25 times. Use a sterile pipette to transfer 11.0 mL to 99 mL of sterile PBS; cap and mix by vigorously shaking the bottle a minimum of 25 times. This is spiking suspension dilution “ E .” A 1.0-mL volume of dilution

“ E ” is equivalent to 1 × 10 2 CFU per mL (10 -6 mL of the original undiluted spiking suspension).D-3 2.1.2 Spike samples

2.1.2.1 10,000 CFU Spike To spike the sample, add 1.0 mL of spiking suspension dilution “ C ” (Section 2.1.1.3) to 100 mL of unspiked sample and mix by vigorously shaking the bottle a minimum of 25 times. This is the “spiked” sample with a concentration of 10,000 CFU per 100 mL. The volume (mL) of undiluted spiking suspension added is 1 × 10 -4 mL/100 mL. This is referred to as V spiked per 100 mL sample in Section 3.2 below. Analyze the spiked sample according to Study Instructions.

2.1.2.2 32,000 CFU Spike To spike the sample, add 3.2 mL of sp iking suspension dilution “C” (Section 2.1.1.3) to 100 mL of sample and mix by vigorously shaking the bottle a minimum of 25 times. This is the “spiked” sample with a concentration of 32,000 CFU per 100 mL. The volume (mL) of spiking suspension added is 3.2 × 10 -4 mL/100 mL. This is referred to as V spiked per 100 mL sample in Section 3.2 below. Analyze the spiked samp le according to Study Instructions.

2.1.2.3 100,000 CFU Spike To spike the sample, add 1.0 mL of spiking suspension dilution “ B ” (Section 2.1.1.2) to 100 mL of sample and mix by vigorously shaking the bottle a minimum of 25 times. This is the “spiked” sample with a concentration of 100,000 CFU per 100 mL. The volume (mL) of spiking suspension added is 1 × 10 -3 mL/100 mL. This is referred to as V spiked per 100 mL sample in Section 3.2. Analyze the spiked sample according to Study Instructions.

2.2 Enumeration of Spiking Suspensions (Prepared in Section 2.1.1)

2.2.1 Ensure tryptic soy agar with 5% sheep blood plates (BAP) are dry and free from condensation prior to use. To ensure the ag ar surfaces are dry, plates should be stored inverted at room temperature or dried using a laminar-flow hood.

2.2.2 Each of the following will be conducted in triplicate, resulting in the evaluation of nine spread plates:

 Pipet 0.1 mL of dilution “C” (Section 2.1. 1.3) onto surface of pre-dried BAP plate (10 -5 mL of the original spiking suspension).

 Pipet 0.1 mL of dilution “D” (Section 2.1. 1.4) onto surface of pre-dried BAP plate (10 -6 mL of the original spiking suspension).

 Pipet 0.1 mL of dilution “E” (Section 2.1. 1.5) onto surface of pre-dried BAP plate (10 -7 mL of the original spiking suspension).

2.2.3 For each spread plate use a sterile bent glass rod or spreader to distribute inoculum over the surface of the medium by rotating the dish by hand or on a turntable. Note : Please ensure that inoculum is evenly distributed over entire surface of the plate.

2.2.4 Allow inoculum to absorb into the medium completely.

2.2.5 Invert plates and incubate in an anaerobe chamber at 35°C ± 0.5°C for 24 ± 2 hours.D-4 2.2.6 Count and record number of colonies per plate. Refer to Section 3.0 for calculation of spiking suspension concentration.

3.0 Calculation of Spiked B. thetaiotaomicron Percent Recovery (Laboratory-Prepared Undiluted Spiking Suspension) Spiked B. thetaiotaomicron percent recovery will be calculate d in three steps as indicated in Sections 3.1 through 3.3, below. Note : The calculated numbers in the table examples provided below have been rounded at the end of each step . If your laboratory recalculates the examples using a spreadsheet and rounds only after the final calculation (Step 3), the percent recoveries may be slightly different.

3.1 Step 1: Calculate Concentration of B. thetaiotaomicron (CFU/mL) in Undiluted Spiking Suspensions

3.1.1 The concentration of B. thetaiotaomicron (CFU/mL) in the undilu ted spiking suspensions will be calculated using all BAP plates from Section 2.2.2 yielding counts within the ideal range of 30 to 300 CFU per plate.

3.1.2 If the number of colonies exceeds the upper range (i.e., >300) or if the colonies are not discrete, results should be recorded as “too numerous to count” (TNTC).

3.1.3 Calculate the concentration of B. thetaiotaomicron (CFU/mL) in the undiluted spiking suspensions according to the following equati on. Example calculations are provided in Table 1

.

B. thetaiotaomicron undiluted spike = (CFU 1 + CFU 2 + ... + CFU n ) / (V 1 + V 2 + ... + V n )
Where,

B. thetaiotaomicron undiluted spike = B. thetaiotaomicron (CFU/mL) in undiluted spiking suspension CFU = Number of colony forming units from BAP plates yielding counts within the ideal range of 30 to 300 CFU per plate V = Volume of undiluted sample on each BAP plate yielding counts within the ideal range of 30 to 300 CFU per plate n = Number of plates with counts within the ideal range

Table 1. Example Calculations of B. thetaiotaomicron Spiking Suspension Concentration

Examples CFU / plate (triplicate analyses) from BAP plates in Section 2.2.2 B. thetaiotaomicron CFU / mL in undiluted spiking suspension ( B. thetaiotaomicron undiluted spike )* 10 -6 mL plates 10 -7 mL plates 10 -8 mL plates

Example 1 TNTC, TNTC, TNTC 94, 106, 89 9, 11, 28 (94+106+89) / (10 -7 +10 -7 +10 -7 ) =
289 / (3.0 x 10 -7 ) =

9.6 x 10 8 CFU / mL

Example 2 TNTC, TNTC, TNTC 32, 55, 72 8, 5, 3 (32+55+72) / (10 -7 +10 -7 +10 -7 ) =
159 / (3.0 x 10 -7 ) =

5.3 x 10 8 CFU / mL

* B. thetaiotaomicron undiluted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per plateD-5 3.2 Step 2: Calculate “True” Spiked B. thetaiotaomicron (CFU/mL) Calculate the true concentration of spiked B. thetaiotaomicron (CFU/100 mL) according to the following equation. Example calculations are provided in Table 2

.

T Spiked B. thetaiotaomicron = ( B. thetaiotaomicron undiluted spike ) × (V spiked per 100 mL sample )

Where, T Spiked B. thetaiotaomicron = Number of spiked B. thetaiotaomicron (CFU/100 mL) B. thetaiotaomicron undiluted spike = B. thetaiotaomicron (CFU/mL) in undiluted spiking suspension (calculated in Section 3.1.3) V spiked per mL sample = mL of undiluted spiking suspension per 100 mL sample (e.g., Section 2.1.2.2)

Table 2. Example Calculations of Spiked B. thetaiotaomicron

B. thetaiotaomicron

undiluted spike (Table 1 above) V spiked per mL sample (Section 2.1.2.2 above) T Spiked B. thetaiotaomicron

9.6 × 10 8 CFU / mL 1.0 × 10 -4 mL per 100 mL of sample (9.6 × 10 8 CFU / mL) × (1.0 × 10 -4 mL /100 mL) = 96,000 CFU / 100 mL

5.3 × 10 8 CFU / mL 3.2 × 10 -4 mL per 100 mL of sample (5.3 × 10 8 CFU / mL) × (3.2 × 10 -4 mL /100 mL) = 169,600 CFU / 100 mL

3.3 Step 3: Calculate Percent Recovery

3.3.1 Calculate percent recovery (R) using the following equation.

R = 100 × (N s - N u )

T Where, R = Percent recovery N s = B. thetaiotaomicron (CFU/100 mL) in the spiked sample N u = B. thetaiotaomicron (CFU/100 mL) in the unspiked sample T Spiked B. thetaiotaomicron = True spiked B. thetaiotaomicron (CFU/100 mL) in spiked sample (Section 3.2)

3.3.2 Example percent recovery calculations are provided in Table 3

.
Table 3. Example Percent Recovery Calculations

N s (CFU / 100 mL) N u (CFU / 100 mL) T Spiked B. thetaiotaomicron (CFU / 100 mL) Percent recovery (R)

947,000 3 a 960,000 100 × (947,000 – 3) / 960,000 = 98%

28,800 <1 30,100 100 × (28,800 - 1) / 30,100 = 95%

85,000 <1 96,000 100 × (85,000 - 1) / 96,000 = 88%

a The result of 3 CFU is acce ptable if the sample was a re creational water – if the samp le is PBS, it would not be acceptable.D-6 4.0 BioBallTM Sample Spiking and Enumeration During the validation study, each laboratory will enumerate B. thetaiotaomicron in the BioBalls so that percent recovery can be evaluated. This section pr ovides instructions for sample spiking (Section 4.1) and spiking suspension enumeration (Section 4.2).

Upon receipt, BioBalls should be stored at -20°C. Preparation of BioBalls prior to spiking is not necessary, as they can be spiked directly into the sample once the vial is opened.

4.1 Sample Spiking for Method B
4.1.1 Open BioBallTM vial by removing the cap. Aseptically add 1 BioBallTM to 100 mL of sterile PBS sample for each concentration (10,000, 32,000, and 100, 000 CFU). Allow to dissolve completely. Mix by vigorously shaki ng the bottles a minimum of 25 times.
4.1.2 Spike and analyze samples according to Study Instructions.

4.2 Enumeration of BioBallTM

4.2.1 Ensure BAP are dry and free from condensation prior to use. To ensure the agar surfacesare dry, plates should be stored inverted at room temperature or dried using a laminarflow hood.

4.2.2 The following will be conducted in triplicate, resulting in the evaluation of three spread plates per concentration (10,000, 32,000 CFU) for a total of six plates.

 Open BioBallTM vial by removing the cap and aseptically add one BioBallTM to 10 mL of sterile PBS. Allow BioBallTM to dissolve completely.

 Mix suspension to homogenize. Pipet 0.1 mL of the BioBallTM suspension onto a 100 × 15 mm BAP plate.

4.2.3 For each spread plate, use a sterile bent glass rod or spreader to distribute BioBallTM inoculum over the medium surface by rotating the dish by hand or on a turntable. Note: Please ensure that inoculum is evenly di stributed over entire surface of plate.

4.2.4 Allow inoculum to absorb into medium completely.

4.2.5 Invert plates and incubate in an anaerobe chamber at 35°C ± 0.5°C for 24 ± 2 hours.

4.2.6 For each dilution, count and r ecord the sum of the number of colonies (colony forming units [CFU]) on the triplicate plates and multiply the sum by a factor of 100 to account for the volume of the BioBallTM suspensi on plated. For example, the 32,000 CFU BioBallTM results in plate counts of 300, 240 and 290. The sum is 830; 830 × 100 = 83,000. The average of the plates is 27,700 CFU and represents the “true” spiked B. thetaiotaomicron . Refer to Section 5.0 for calculation of B. thetaiotaomicron concentration in BioBallTM.

5.0 Calculation of BioBallTM Spike Percent Recovery
Spiked BioBall J percent recovery will be calculated following the steps indicated below. Note : The calculated numbers in the examples provided in the tables below have been rounded at the end of each step. If your laboratory recalculates th e examples using a spreadsheet and rounds only after the final calculation (Step 2), the percent recoveries may be slightly different.D-7 5.1 Step One: Calculate Mean B. thetaiotaomicron per BioBallTM and “True” Spiked B. thetaiotaomicron (CFU/100 mL)

The mean concentration of B. thetaiotaomicron (CFU) in the BioBalls will be calculated using all three plates (BAP) for each concentration (10,000, 32,000 CFU) from Section 4.2.2. Since one BioBallTM is spiked per 100 mL sample, use the mean number of B. thetaiotaomicron per BioBallTM as the A true @ spiked B. thetaiotaomicron. For example, the triplicate plate counts for the 32,000 CFU BioBallTM are as follows:

T Spiked B. thetaiotaomicron (CFU / 100 mL) = [(300 + 240 + 290) ×100] / 3 = 27,700 per BioBallTM

Where, T Spiked B. thetaiotaomicron = True spiked B. thetaiotaomicron (CFU/100 mL) in spiked sample

5.2 Step Two: Calculate Percent Recovery

Calculate percent recovery (R) using the following equation:

R = 100 × (N s - N u ) T

Where, R = Percent recovery N s = B. thetaiotaomicron (CFU/100 mL) in the spiked sample (or CFU per BioBallTM) N u = B. thetaiotaomicron (CFU/100 mL) in the unspiked sample T Spiked B. thetaiotaomicron = True spiked B. thetaiotaomicron or CFU/100 mL in spiked sample (Section 5.1).

Percent recovery example calculations are provided in Table 4

.
Table 4. Percent Recovery Example Calculations

Concentration N s (CFU / 100 mL) N u (CFU / 100 mL) T (CFU / 100 mL) Percent recovery (R)

10,000 9800 <1 9200 100 H (9800 - 1) / 9200 = 105%

32,000 30,500 10 30,000 100 H (30,500 - 10) / 30,000

= 98%

## Appendix E

Laboratory Capabilities ChecklistE-1 

## Laboratory Capabilities Checklist qPCR Single-Laboratory Validation Study

(May 18, 2009)

EPA plans to invite utility, research, and commercial laborator ies to bid for participation in a study to evaluate the performance of Methods A and B for analysis of recrea tional fresh water samples through a single laboratory validation study. EPA will provide the media, reagents, and standards necessary for this study.

If your laboratory is interested in participating in this study, please provide the requested information below and fax the signed, completed checklist along with your bi d package to Yildiz Chambers at 703.461.8056 by June 1, 2009

. If you have any questions pertaining to the informa tion requested below or the validation study, please do not hesitate to contact Yildiz Chambers at 703.461.2165, ychambers@csc.com

.

Section 1. Laboratory Capabilities and Experience

A. Please complete the requested laboratory cap abilities and experience information below.

Table 1. Analyst Experience

Analyst Years of experience or estimated number of samples analyzed

Real-time PCR TaqMan® probes

B. Name of primary analyst participati ng in study: _________________________________

C. Primary analyst’s years of experience performing molecular analyses: _______________

D. What certifications does your laboratory have for microbial analyses?

E. Additional comments:E-2 Section 2. Laboratory Equipment

Please be specific and list the following equipment used in your laboratory.

A. Thermocycler(s) please provide make and model:________________________________

________

____ _________________________________________________________________________________

B. Number of workstations with a dedicated laminar flow hood, UV light source, and supplies:

____ _________________________________________________________________________________

C. Number of dedicated micropipettor sets and capabilities (e.g., 200 μL, 1000 μL) of micropipettors in each
set:

_____________________________________________________________________________________
D. Brand of micropipettors

____________________________________________________________________________________

E. Bead beater (single or 8-place; please provide make and model): Yes No (circle one)

____ _________________________________________________________________________________

F. Autoclave (make and model):

_____________________________________________________________________________________

G. Spectophotometer (make and model):

_____________________________________________________________________________________

H. Hemacytometer available Yes No (circle one)

Section 3. Background Information

A. Does your laboratory have access to recreationa l fresh or marine water samples? Yes No If yes, please indicate in Table 2, below

.

B. Is your laboratory located within two hours driving distance of Yes No fresh or marine water sample collection site?

C. Has your laboratory ever participat ed in a validation study? Yes No

D. If your laboratory has experience anal yzing water samples for enterococci or Bacteroidales spp. please place a “  ” next to the sample type which you have analyzed and indicate both: the method(s) used for analysis, and typical concentrations ( Table 2, below ).E-3 Table 2. Enterococci and Bacteroidales spp. Analysis

Access? Sample Type Monitoring Frequency Enterococci/ Bacteroidales spp.

Method Typical Range

Example  Recreational marine water 2 times per month 1600 25/100 mL

Recreational ma rine water

Other:

________________________

Other:

_______________________

E. CSC recognizes that your laboratory may have access to multiple recreational marine water sites. For each collection site, please describe the location (e.g., pub lic beach, fishing pier). Please be as descriptive as possible. If there is a known source of fecal contamination (e.g. WWTP, birds), please identif y that

source.

Collection site 1:

Collection site 2:

Collection site 3:

F. Will your laboratory be prepared to collect a nd analyze water samples within 8 hours from time of collection?

Yes No

I certify that the information provided above is accurate and complete:

Primary Analyst: ___________________________________

Phone Number: ____________________________________

Email Address: _____________________________________

Lab Manager: ___________________________________________

Phone Number: __________________________________________E-4 Email Address: ___________________________________________

Laboratory Name: _____________________________________________________________________

Laboratory FedEx Shipping Address: ______

_______________ __________________ ______________

_____________________________________________________

_____________________________________________________

_____________________________________________________

Signature (lab manager): _________________________________

Date: ___________________

Additional Contact Information (if applicable)