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 20460
i

Acknowledgments

The contributions of the following persons and organizations to this study are gratefully acknowledged: 

Participant Laboratories 

 New York State Department of Health Environmental Biology 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, National 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 Island Watershed Program 

• University of South Florida 

• University of Southern Mississippi 

• US Department of Agriculture – Western Regional Research Center 

• USEPA Region 1 Laboratory 

• USEPA Region 10 Laboratory 

• US Geological Survey, Ohio Water Microbiology Laboratory 

• Utah Department of Health 

• Wisconsin State Laboratory of Hygiene 
ii

Disclaimer

Mention of company names, trade names, or commercial products 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.gov 
iii

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 for Initial (IPR) and Ongoing (OPR) Method/Laboratory 

Performance Assessments........................................................................................................................... 6

2.6.4 Development of Quantitative QC Criteria 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 and 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 and 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)...................................................................................................................................27
iv

Section 7.0 Assessment of Methods A and B Performance: 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 References..................................................................................................................................................... 39

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

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 Shipped 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 1600 (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 Draft Matrix Spike Precision and Recovery Acceptance Criteria for Method B (Bacteroidales)... 32
vi

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 Checklist 

 
vii

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 reaction (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 single laboratory environment across multiple matrices, and 

develop draft quantitative quality control (QC) acceptance criteria. 

Two analytical laboratories, one with access to fresh water matrices and the other with access to marine water matrices, 

participated in the Study. During the Study each laboratory analyzed unspiked and spiked phosphate buffered saline 

(reference matrix) samples and fresh or marine water samples according to Methods A, B and 1600 and study-specific 

instructions (Appendices A and C), as appropriate. 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 multiple fresh and marine water matrices from different geographic regions across 

the US, thirty-seven volunteer laboratories collected and shipped 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 

1 June 2010 

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 environment, 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 observed 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 reaction (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 enterococci on membrane filters. Following filtration, total DNA is 

solubilized from the filter retentate using a glass bead disruption/extraction procedure (bead beating). 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. The 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 Bacteroidales on membrane filters. Following filtration, total DNA is 

solubilized from the filter retentate using a glass bead beating procedure. Bacteroidales target DNA sequences present in 

the clarified extract are detected by the real time PCR technique using TaqMan® reagents 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 activity) of a fluorogenically-labeled oligonucleotide probe 

when hybridized to the target sequence. 
Methods A and B Single-Laboratory Validation Study Report 

2 June 2010 

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 (BioBallTM) 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) acceptance 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 potential 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 instructions (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 laboratories 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 Capabilities Checklist (Appendix E) 

was used to collect this information from laboratories and to screen potential participants to ensure that 

laboratories were qualified. Analytical laboratories processed and analyzed PBS and fresh or marine recreational 

water samples. In addition, analytical reagents were supplied to the laboratories for consistency. 

2.2.2 Volunteer Participants 

To reduce cost, 37 volunteer participants were recruited to collect either fresh or marine or both matrix samples. 

Volunteers had access to the matrices of interest and the ability 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 

3 June 2010 

2.3 Technical Approach: Laboratory-Prepared Cell Suspensions 

Results from samples spiked with laboratory-prepared cell suspensions 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 determined optical density (OD) according to Method A or B. 

To determine the spike concentration participant laboratories enumerated cell suspensions on the same day that samples 

were spiked. 

2.4 Technical Approach: BioBallTM Spikes 

Results from samples spiked with BioBalls (commercially available flow cytometer counted enumerated spikes) were 

used to assess method performance and to develop QC acceptance 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 (Appendix 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 used as the “true spike concentration.” 

Due to problems with B. thetaiotaomicron BioBalls when used as calibrator samples (e.g., non-detects or very high cycle 

threshold (CT) values [e.g., 38 CT]) observed 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 quantified sets of initial and ongoing calibrator samples using both 

laboratory-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 different levels: 100, 320, and 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 

4 June 2010 

• Labs analyzed one, 100-mL unspiked sterile PBS (method blank) for enterococci and salmon DNA (Sketa 2 and Sketa 

22).

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

Results of the preliminary analyses were utilized as follows: 

• Results from enterococci preliminary analyses were used to reduce 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 evaluate 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 sets of initial and ongoing calibrator samples using both 

laboratory-prepared (Kwik-StikTM) and vendor-prepared [BioBallTM] 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 different 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 conducted as described in Section 2.6.5, below. 

Results of the preliminary analyses were utilized as follows: 

• Results from Bacteroidales preliminary analyses were used to determine 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 preliminary analyses were also used to evaluate laboratory 

performance and identify any issues (e.g., interferences, faulty materials) that needed to be addressed prior to the start 

of the validation study. 

• Results of the evaluation of the culture method were negative, thus a culture component was not included during the 

assessment of Method B. 
Methods A and B Single-Laboratory Validation Study Report 

5 June 2010 

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 analyses. 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 according 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, analyzed, 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 fresh 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 replicates were analyzed according to Method 1600. 

• Laboratories spiked 100-mL PBS and fresh 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 using calibrator samples and DNA standard 

curves according to the appropriate methods and study-specific instructions. 

• Laboratories analyzed eight replicates (4 unspiked and 4 spiked) 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 sterile 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-prepared cell suspensions, four replicates at each spike 

level. 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. Recoveries were assessed by quantifying E. faecalis or B. 

thetaiotaomicron in the spiked samples (minus the ambient concentrations assessed from unspiked PBS and fresh or 

marine water samples) as determined by Method A or B analyses. 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 different 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 

6 June 2010 

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 quantitative QC (recovery and precision) criteria to assess laboratory 

performance during the multi-laboratory validation study, each analytical laboratory analyzed 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, 320, 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 water 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 

performance 
Methods A and B Single-Laboratory Validation Study Report 

7 June 2010 

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 

spikes 
Methods A and B Single-Laboratory Validation Study Report 

8 June 2010 

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 check 
Methods A and B Single-Laboratory Validation Study Report 

9 June 2010 

Table 2. Method B (Bacteroidales) Validation Study Analyses 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 check 
Methods A and B Single-Laboratory Validation Study Report 

10 June 2010 

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 check 
Methods A and B Single-Laboratory Validation Study Report 

11 June 2010 

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 Protection 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 Environmental Biology Laboratory – Ellen 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 

12 June 2010 

Table 4. Volunteer Participants that Collected and 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. 33rd 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 74th Court, Miami, FL 33155 

BioVir 

Rick Danielson 

685 Stone Road Unit #6, Benicia, CA 94510 

CH Diagnostics 

Ana Luisa Child 

512 5th Street, Berthoud, CO 80513 

City of Everett, Water Pollution Control Facility 

Chris Merwede 

4027 4th 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 Tamus, 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 96822 
Methods A and B Single-Laboratory Validation Study Report 

13 June 2010 

University of Rhode Island Watershed Program 

Linda Green 

105 Coastal Institute 1 Greenhouse Rd, Kingston, RI 02881 

University of South Florida 

Jody Harwood 

Department of Integrative Biology 

4202 East Fowler Avenue, Tampa, FL 33620 

University of Southern Mississippi 

Rudolph D. Ellender 

Department of Biological Sciences 

2609 West 4th 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 implied, nor should any be inferred. Participant laboratories have been randomly assigned 

numbers for purposes of presenting data in this report. 
Methods A and B Single-Laboratory Validation Study Report 

14 June 2010 

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 Microbiology 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 forms were submitted; QC checks were conducted and exhibited 

the appropriate response; method-specific incubation times and 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 Study. 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 below the analytical range of Methods A, B, and 1600 (lessthan results, also referred to as left-censored) were observed (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 

15 June 2010 

Section 5.0 Results 

This section includes results of Method A preliminary analyses (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 marine water samples (Section 5.4), Method B preliminary 

analyses (Section 5.5.), Method B unspiked/spiked PBS samples (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 this 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 purified 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 (single- and multi-laboratory validation) would be conducted 

using crude extracts. 

Method A analyses included the following: Environmental master 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/spiked PBS sample analyses are provided in Table 5. Results from PBS 

samples spiked with E. faecalis BioBallTM spikes were used to assess method 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-laboratory validation study (see discussion in Section 6). 
Methods A and B Single-Laboratory Validation Study Report 

16 June 2010 

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 water 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 Section 7.1.2). Results from fresh and marine water 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 multilaboratory validation study (see discussion in Section 6). 
Methods A and B Single-Laboratory Validation Study Report 

17 June 2010 

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 1600
Methods A and B Single-Laboratory Validation Study Report 

18 June 2010 

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 were 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 

BioBallTM 12,233 16.77 236.61 1411.30 

X 

Unspiked NA d 1279 2 

315 

Lake 

BioBallTM 2995 133.57 37.38 27.98 

Unspiked NA d 80 3 

315 

Lake 

BioBallTM 3973 303.02 87.48 28.87 

Unspiked NA d 986 4 

315 

Reservoir 

BioBallTM 6035 393.00 135.01 34.35 

Unspiked NA d 656 5 

315 

Pond 

BioBallTM 3963 257.41 51.99 20.20 

Unspiked NA d 171 6 

315 

Lake 

BioBallTM 3276 241.67 50.36 20.84 

Unspiked NA d 588 7 

315 

River 

BioBallTM 2368 138.49 49.05 35.42 

Unspiked NA d 107 8 

315 

Lake 

BioBallTM 1818 133.13 17.85 13.41 

Unspiked NA d 13,132 9 

315 

Lake 

BioBallTM 19,634 404.16 130.06 32.18 

X 

Unspiked NA d 4591 10 

315 

River 

BioBallTM 5211 38.52 26.48 68.74 

X 

Unspiked NA d 1120 

11 

315 

Lake 

BioBallTM 753,726 46,783 93,448 199.75 

Unspiked NA d 2,786,751 12 

315 

Lake 

BioBallTM 2,366,986 -26,093 98,387 -377.06 

X 

Unspiked NA d 2464 

13 

315 

River 

BioBallTM 2984 32.32 19.20 59.41 

X 

Unspiked NA d 451 14 

315 

Lake 

BioBallTM 1189 45.84 9.58 20.90 
Methods A and B Single-Laboratory Validation Study Report 

19 June 2010 

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 

BioBallTM 3511 72.00 7.35 10.20 

Unspiked NA d 5959 

16 

945 

River 

BioBallTM 9407 71.43 31.01 43.42 

Unspiked NA d 1977 

17 

1173 

River 

BioBallTM 5175 76.40 31.20 40.83 

Unspiked NA d 333 

18 

1173 

River 

BioBallTM 3286 70.55 17.18 24.36 

Unspiked NA d 452 

19 

1173 

Lake 

BioBallTM 4938 107.18 11.29 10.53 

Unspiked NA d 613 

20 

1173 

Lake 

BioBallTM 5163 108.70 12.74 11.72 

Unspiked NA d 1730 

21 

1173 

River 

BioBallTM 5466 89.26 12.56 14.07 

Unspiked NA d 3939 

22 

1173 

Creek 

BioBallTM 8080 98.93 17.93 18.12 

Unspiked NA d 2932 

23 

1173 

Lake 

BioBallTM 8669 137.06 31.06 22.66 

Unspiked NA d 370 

24 

1173 

Lake 

BioBallTM 5618 125.38 70.45 56.19 

Unspiked NA d 1599 

25 

1173 

Creek 

BioBallTM 7932 151.29 69.95 46.24 

a Target sequence copy b Standard deviation c Relative standard deviation d

 Not applicable
Methods A and B Single-Laboratory Validation Study Report 

20 June 2010 

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 BioBallTM 4104 332.87 186.23 55.95 

Unspiked NA d 67 2 

315 BioBallTM 610 56.47 12.31 21.80 

Unspiked NA d 3199 3 

315 BioBallTM 5344 223.10 107.22 48.06 

X 

Unspiked NA d 56 4 

315 BioBallTM 521 48.35 11.44 23.66 

Unspiked NA d 489 5 

315 BioBallTM 1193 73.20 18.99 25.94 

Unspiked NA d 68 6 

315 BioBallTM 995 96.40 20.68 21.46 

Unspiked NA 277 7 

315 BioBallTM 616 35.20 37.67 107.03 

Unspiked NA d 729 8 

315 BioBallTM 1653 96.06 98.88 102.94 

Unspiked NA d 16 9 

315 BioBallTM 1655 138.42 18.83 13.60 

Unspiked NA d 122 10 

315 BioBallTM 1337 102.57 11.61 11.32 

Unspiked NA d 500 11 

315 BioBallTM 1762 106.57 26.04 24.43 

Unspiked NA d 438 12 

315 BioBallTM 2328 159.56 46.55 29.17 

Unspiked NA d 861 13 

315 BioBallTM 2122 106.47 45.41 42.65 

Unspiked NA d 814 14 

315 BioBallTM 2360 130.54 36.72 28.13 

Unspiked NA d 10,941 15 

315 BioBallTM 12,148 101.90 364.43 357.64 

X 

Unspiked NA d 258 16 

315 BioBallTM 1225 81.68 20.30 24.86 

Unspiked NA d 208 17 

315 BioBallTM 1118 63.14 2.46 3.89 

Unspiked NA d 131 

18 

315 BioBallTM 1128 69.12 16.67 24.12 
Methods A and B Single-Laboratory Validation Study Report 

21 June 2010 

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 BioBallTM 1483 81.46 21.57 26.48 

Unspiked NA d 4636 20 

315 BioBallTM 5220 40.47 43.61 107.76 

X 

Unspiked NA d 291 21 

315 BioBallTM 2037 121.13 35.39 29.22 

Unspiked NA d 227 22 

315 BioBallTM 2484 156.52 180.21 115.14 

Unspiked NA d 3523 23 

315 BioBallTM 4895 95.13 58.05 61.03 

X 

Unspiked NA d 487 24 

315 BioBallTM 1188 48.57 23.40 48.19 

Unspiked NA d 473 25 

315 BioBallTM 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 filtration 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 blood 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 enumerate 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 levels (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 validation study (see discussion in Section 6). 
Methods A and B Single-Laboratory Validation Study Report 

22 June 2010 

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

were used to assess method performance (see discussion in Section 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 applicable
Methods A and B Single-Laboratory Validation Study Report 

23 June 2010 

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 samples 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 5339 86.29 10.03 11.62 

 

Unspiked NA d 19,398 2 Lake 

31,360 Lab-prep 26,431 125.95 31.25 24.81 

X 

Unspiked NA d 21,758 3 River 

31,360 Lab-prep 24,520 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 50,479 114.86 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 18,389 144.16 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 19,365 18.54 27.13 146.32 

X 
Methods A and B Single-Laboratory Validation Study Report 

24 June 2010 

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 10,481 174.07 28.30 16.26 

Unspiked NA d 14,424 11 River 

31,360 Lab-prep 19,370 88.57 65.50 73.95 

X 

Unspiked NA d 10,166 12 Lake 

31,360 Lab-prep 17,740 135.63 20.84 15.37 

X 

Unspiked NA d 22,387 13 River 

31,360 Lab-prep 31,480 162.82 39.02 23.96 

X 

Unspiked NA d 31,465 14 River 

31,360 Lab-prep 38,861 132.43 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 5971 98.38 16.78 17.06 

Unspiked NA d 2468 17 Creek 

31,360 Lab-prep 14,981 155.09 4.91 3.16 

Unspiked NA d 4157 

18 Lake 

31,360 Lab-prep 18,798 181.47 17.16 9.45 

Unspiked NA d 2083 19 Lake 

31,360 Lab-prep 8975 85.42 13.27 15.53 

Unspiked NA d 41,432 20 River 

31,360 Lab-prep 41,931 6.19 35.78 577.86 

X 

Unspiked NA d 6462 21 Lake 

31,360 Lab-prep 13,576 88.17 12.26 13.91 

Unspiked NA d 2041 22 Lake 

31,360 Lab-prep 13,437 141.24 12.73 9.01 

Unspiked NA d 669 23 Pond 

31,360 Lab-prep 5600 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 7167 75.32 5.84 7.75 

a Target sequence copy b Standard deviation c Relative standard deviation

d

 Not applicable
Methods A and B Single-Laboratory Validation Study Report 

25 June 2010 

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 21,166 42.67 5.06 11.85 

Unspiked NA d 309 2 

34,000 Lab-prep 21,926 43.71 4.96 11.35 

Unspiked NA d 2241 3 

34,000 Lab-prep 27,652 51.38 5.19 10.10 

Unspiked NA d 164 4 

34,000 Lab-prep 20,651 41.43 10.26 24.77 

Unspiked NA d 440 5 

34,000 Lab-prep 24,898 49.46 4.84 9.78 

Unspiked NA d 778 6 

34,000 Lab-prep 29,810 58.71 6.55 11.16 

Unspiked NA d 17,028 7 

34,000 Lab-prep 42,127 50.75 5.53 10.90 

Unspiked NA d 3031 8 

34,000 Lab-prep 28,116 50.73 7.82 15.42 

Unspiked NA d 9765 9 

34,000 Lab-prep 34,494 50.01 8.46 16.92 

Unspiked NA d 181 10 

34,000 Lab-prep 25,830 51.86 7.27 14.03 

Unspiked NA d 6661 11 

34,000 Lab-prep 24,781 36.64 3.72 10.16 

Unspiked NA d 2422 12 

34,000 Lab-prep 23,184 41.98 6.27 14.93 

Unspiked NA d 5421 13 

34,000 Lab-prep 27,982 45.62 4.13 9.06 

Unspiked NA d 1113 14 

34,000 Lab-prep 25,343 49.00 9.65 19.69 

Unspiked NA d 981 15 

34,000 Lab-prep 23,859 46.26 11.48 24.81 

Unspiked NA d 226 16 

34,000 Lab-prep 21,277 42.57 3.58 8.41 

Unspiked NA d 8850 17 

34,000 Lab-prep 33,113 41.99 5.35 12.74 

Unspiked NA d 6198 18 

34,000 Lab-prep 25,893 34.09 8.89 26.08 
Methods A and B Single-Laboratory Validation Study Report 

26 June 2010 

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

Sample Method B

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 35,405 37.24 11.19 30.05 

Unspiked NA d 542 20 

34,000 Lab-prep 22,536 38.07 12.63 33.18 

Unspiked NA d 894 21 34,000 Lab-prep 14,234 23.09 15.11 65.44 

Unspiked NA d 26,023 22 34,000 Lab-prep 39,440 23.22 2.26 9.71 

Unspiked NA d 1774 23 34,000 Lab-prep 6496 8.17 1.39 17.00 

Unspiked NA d 23,030 24 34,000 Lab-prep 39,180 27.95 2.08 7.43 

Unspiked NA d 2605 25 

34,000 Lab-prep 25,071 38.88 3.84 9.88 

a Target sequence copy b Standard deviation c Relative standard deviation d

 Not applicable 
Methods A and B Single-Laboratory Validation Study Report 

27 June 2010 

Section 6.0 Development of Draft QC Acceptance Criteria 

This section describes the development of quantitative QC acceptance criteria in a reference matrix (PBS) and two 

different matrices of interest (fresh water and marine water) to support future assessments of laboratory and method 

performance. All data analyses described below were performed 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 criteria 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 s2

L and s2w, respectively. 

The combined standard deviation for IPR samples (sc) 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 

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

 nT = 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: 

 mean cdf ± );975.0

* stX 

Where: 

 Xmean = the mean recovery of all PBS samples, 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 

28 June 2010 

 

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 calculated by dividing the pooled within-laboratory standard 

deviation calculated previously by the overall mean recovery for that laboratory and matrix, as shown below: 

 

mean

w

pool X

s RSD

2

=

Where: 

 Sw and Xmean are the pooled within-lab standard deviation and mean recovery calculated above 

The maximum RSD was then calculated as: 

 RSDmax = F L),3;95.0(

* RSDpool

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 

29 June 2010 

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 developed 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 PROC MIXED from SAS version 9 using the maximum likelihood 

method of estimation on the recovery results. Because samples 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 laboratory/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 s2

L and s2w, respectively. 

The combined standard deviation for MS samples (sc) 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 

 nT = total number fresh or marine water sample results from the laboratory for the given spike level and 

method 
Methods A and B Single-Laboratory Validation Study Report 

30 June 2010 

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

as: 

 mean n c stX T ± − )1;975.0

* 

Where: 

 Xmean = 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 that spike level, method, and matrix, as shown below: 

 

mean

w

pool X

s RSD

2

=

Where: 

 Sw and Xmean are the within-lab standard deviation and mean recovery calculated above 

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

 RPDmax = F )3,1;95.0( **2 RSDpool

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 

31 June 2010 

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 data 
Methods A and B Single-Laboratory Validation Study Report 

32 June 2010 

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 

33 June 2010 

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 requirements, 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 differences for specific matrices. For each 

matrix type/organism, only those matrices for which the spike level exceeded background levels were included in the 

comparison. 

7.1 Method A 

Based on preliminary analyses the following variables were 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 samples 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 samples spiked with laboratory-prepared cell suspensions 

were 55%, 86%, and 67% for 100, 320, and 1000 cell spikes, 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 samples spiked with BioBallTM spikes were 69%, 103%, 

and 140% for 100, 320, and 1000 cell spikes, respectively. RSDs were 34%, 37%, and 18%, for 100, 320, and 

1000 cell spikes, respectively. 

o For Laboratory 2, Method A mean recoveries for PBS samples spiked with BioBallTM spikes were 59%, 101%, 

and 110% for 100, 320, and 1000 cell spikes, respectively. RSDs were 35%, 18%, and 25%, for 100, 320, and 

1000 cell spikes, respectively. 

Method A mean recoveries were more consistent 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 

34 June 2010 

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, respectively. 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 spiked 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 lower than recoveries in PBS samples (Section 7.1.1) suggesting 

that a spike level of 100 cells may be at the limit of detection 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 and (-) 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 1000 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 water 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, respectively. RSDs were 5%, 6%, and 11%, for 100, 320, and 

1000 cell spikes, respectively. 
Methods A and B Single-Laboratory Validation Study Report 

35 June 2010 

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, respectively. RSDs were 45%, 37%, and 9%, for 100, 320, 

and 1000 cell spikes, respectively. 

For Laboratory 1, Method A mean recoveries were very different 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 levels 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. Each 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 observed 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 concentration, 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 matrices indicate that the method is appropriate for analyses 

of enterococci in both matrices. 

7.2 Method B 

Based on preliminary analyses the following variables were included in the assessment of method performance for 

Method B in PBS and fresh and marine water 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 

36 June 2010 

7.2.1 Reference Matrix (PBS): Method B 

PBS samples were analyzed by both of the participant laboratories. Due to recovery issues with B. thetaiotaomicron

BioBalls only laboratory-prepared 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 samples 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 100,000 cell spikes, respectively. 

o For Laboratory 2, Method B mean recoveries for PBS samples 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 laboratory 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 laboratory-prepared cell suspensions 

o Method B mean recoveries for marine water samples 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%, 18%, and 5%, 

for 10,000, 32,000, and 100,000 cell spikes, respectively. 

Method B mean recoveries for both fresh and marine water samples spiked with laboratory-prepared cell suspensions 

were similar to mean recoveries for PBS (Section 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 samples over a two week period. Each sample was split into eight 

replicates (4 unspiked and 4 spiked). 

• Fresh water samples 

o Twelve of the twenty five fresh water samples had ambient or background concentrations of Bacteroidales greater 

than 32,000 cells per 100 mL. High ambient levels can make 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 probably 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 concentration, 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 level of 32,000 cells. High ambient levels can make it 

difficult to recovery spiked cells. 
Methods A and B Single-Laboratory Validation Study Report 

37 June 2010 

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 ambient 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 matrix 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 

38 June 2010 

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 

39 June 2010 

Section 9.0 References 

9.1 Cabelli, V. J., A. P. Dufour, M. A. Levin, L. J. McCabe, and P. W. Haberman, 1979. Relationship of Microbial 

Indicators to Health Effects at Marine Bathing Beaches. Amer. Jour. Public Health. 69:690-696. 

9.2 Dufour, A.P. 1984. Health Effects Criteria for Fresh Recreational Waters, EPA-600/1-84-004. Office of Research 

and Development, USEPA. 

9.3 USEPA. 2009. EPA Method A: Enterococci in Water by TaqMan® Quantitative Polymerase Chain Reaction 

(qPCR) Assay, Draft May 2009. 

9.4 USEPA. 2009. EPA Method B: Bacteroidales in Water by TaqMan® 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, Volume 2, GLM-VARCOMP. Version 6, 4th Edition, June 

1994. 
Methods A and B Single-Laboratory Validation Study Report 

40 June 2010 

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 Instructions
A-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 acceptance 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 according to Method A and study-specific instructions: 

 Enumerate laboratory-prepared spiking suspensions 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 enterococci (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 from ongoing calibrator samples will be used in the 
A-2 

ΔΔCT 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 μL 

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 minimum (includes 39 x 100 mL bottles) 

 Brain heart infusion broth (BHIB) – 200 mL 

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

 0.45 Fm 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 preliminary 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 sample 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 mL
A-4 

 Filter unspiked and spiked samples 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 (method 

blank) should be filtered last 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 filtered 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 μL). 

 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 (below) for 2-step DNAsure column loading of 

calibrator extracts (e.g., 400 μL sample plus 800 μL binding buffer). 

 Load samples onto DNAsure columns, wash, and elute samples in 100 μL 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 samples (prepared during Phase 1) and add 990 μL 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 extraction tube and adding 590 μL of salmon DNA/extraction buffer. 

 Mix diluted calibrator cells thoroughly and spot 10 μL of each diluted suspension onto 

the polycarbonate filter in each extraction tube. 

 Perform extraction of calibrator samples according 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 μL) and purify (GeneRite) 

300 μL 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 extraction tube and adding 550 μL of salmon DNA/extraction buffer. 
A-5 

 Aseptically add a BioBallTM (1000 CFU; 3 replicates) to each of 3 appropriately labeled 

1.7 mL microcentrifuge tubes and suspend each BioBallTM in 50 μL AE buffer. 

 Using a clean micropipette tip for each cell suspension, thoroughly mix by gentle 

pipetting and spot the entire 50 μL 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 μL) 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 μL 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 μL 

binding buffer to 400 μL 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 μL. 

 Add approximately half of each sample (e.g., 600 μL) to individual labeled DNAsure 

columns inserted into collection tubes and centrifuge 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 μL EZ-Wash Buffer to each column and centrifuge at top speed for 1 minute. 

Discard flow through from each column. Repeat wash using 500 μL EZ-Wash Buffer. 

 Transfer each column to a sterile 1.7 mL microcentrifuge tube and add 50 μL DNA 

Elution Buffer to each column. Centrifuge for 30 seconds at top speed. Repeat DNA 

elution step using a second 50 μL of DNA Elution Buffer to obtain a final volume of 90-

100 μL/sample. 

 Refrigerate purified undiluted samples until analyzed. 
A-6 

PCR Preparation and Analysis

Note: To minimize potential environmental DNA contamination, 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 

Reagent Volume/Reaction1

Sterile H2O 1.5 μL

BSA 2.5 μL

TaqMan® master mix 12.5 μL

Primer/probe working stock solution2 3.5 μL 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 μL of the appropriate assay mix and 5 μL 

of each sample (undiluted replicates) as specified in Table 2 (below) and Method A 

(Section 11.8). 

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

 Include at least 1 no template control (NTC) containing 5 μL 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 formats as determined previously. Samples will be 

collected and shipped to each laboratory during the weeks of September 14th, 21st, and 28th. 

Procedures 

Sample Preparation and Spiking

Participating laboratories will be provided with either 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 original 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 and 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 filtered last using a sterile funnel. 

 Place individual filters into appropriately labeled 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 μL). 

 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 μL sample plus 800 μL binding buffer). 

 Load samples onto DNAsure columns, wash, and elute samples in 100 μL 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 extraction tube and adding 550 μL of salmon DNA/extraction buffer. 

 Aseptically add a BioBallTM (1000 CFU; 3 replicates) to each of 3 appropriately labeled 

1.7 mL microcentrifuge tubes and suspend each BioBallTM in 50 μL AE buffer. 

 Using a clean micropipette tip for each cell suspension, thoroughly mix by gentle 

pipetting and spot the entire 50 μL 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 μL) 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 μL 

binding buffer to 400 μL 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 μL. 

 Add approximately half of each sample (e.g., 600 μL) to individual labeled DNAsure 

columns inserted into collection tubes and centrifuge 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 μL EZ-Wash Buffer to each column and centrifuge at top speed for 1 minute. 

Discard flow through from each column. Repeat wash using 500 μL EZ-Wash Buffer. 

 Transfer each column to a sterile 1.7 mL microcentrifuge tube and add 50 μL DNA 

Elution Buffer to each column. Centrifuge for 30 seconds at top speed. Repeat DNA 

elution step using a second 50 μL of DNA Elution Buffer to obtain a final volume of 90-

100 μL/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 μL of the appropriate assay mix and 5 μL 

of each sample (undiluted) as specified in Table 3 (below) and Method A (Section 11.8). 

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

 Include at least 1 no template control (NTC) containing 5 μL 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 Protocol 
B-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 BioBallTMspikes. The following sections are included 

in this protocol: 

Laboratory-Prepared Spiking Suspensions 

Section 1: Laboratory-Prepared Spiking 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 BioBallTM 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. Incubate at 35°C ± 0.5°C for 21 ± 3 hours. The 

resulting spiking suspension contains approximately 1.0 × 109

 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 enumeration 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 establish 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 suspensions in the steps below, 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 × 107 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 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 × 105

 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 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 × 104

 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 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 × 103 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 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 × 102

 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 spiking 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 Vspiked 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 spiking 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 Vspiked 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 spiking 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 Vspiked 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 and solidify. Ensure the agar surfaces are 

dry. Note: Agar plates must be dry and free from condensation prior to use. To ensure 

the 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. 

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 calculation (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 = (CFU1 + CFU2 + ... + CFUn) / (V1 + V2 + ... + Vn) 

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 108 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 108

 CFU / mL

*E. faecalis undiluted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per plate
B-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. 

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

 Where, 

 TSpiked 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) TSpiked E. faecalis 

 9.6 × 108 CFU / mL 1.0 × 10-7 mL per 100 mL of 

sample

(9.6 × 108 CFU / mL) × (1.0 × 10-7 mL / 100 mL) = 

96 CFU / 100 mL

5.3 × 108 CFU / mL 1.0 × 10-7 mL per 100 mL of 

sample 

 (5.3 × 108

 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

(Ns - Nu) 

T 

 Where, 

R = Percent recovery 

Ns = E. faecalis (CFU/100 mL) in the spiked sample 

Nu = E. faecalis (CFU/100 mL) in the unspiked sample 

TSpiked 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 

Ns (CFU / 100 mL) Nu (CFU / 100 mL)

T Spiked E. faecalis 

(CFU / 100 mL) Percent recovery (R)

947 3a 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 acceptable if the sample was a recreational water – if the sample 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 directions. 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 distributed 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 record 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 BioBallJ 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 the 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 

Atrue@ spiked E. faecalis. For example, the triplicate plate counts for the 320 CFU BioBallTM 

concentration are as follows:

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

 

Where, 

 TSpiked 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 × (Ns - Nu ) 

 T 

 Where, 

 R = Percent recovery 

 Ns = E. faecalis (CFU/100 mL) in the spiked sample (or CFU per 

BioBallTM) 

 Nu = E. faecalis (CFU/100 mL) in the unspiked sample 

TSpiked 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 Ns (CFU / 100 mL) Nu (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 Instructions
C-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 Method B matrix inhibition across multiple fresh and 

marine water matrices

Summary of Analyses 

Laboratories will complete the following according 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 method 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 unspiked 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 extracted 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 recovered from ongoing calibrator samples will be used 

in the ΔΔCT 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 Mix
C-2 

Matrix Inhibition Analyses

Sample Preparation and Spiking

Participating laboratories will be provided with either 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 original 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 and 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 labeled extraction tubes and extract according to 

Method B (Section 11.5). 

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

 Prepare a 5-fold dilution of each crude extract (e.g., 50 μL crude extract plus 200 μL 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 samples (prepared during Phase 1) and add 990 μL AE 

buffer. Thoroughly mix by vortexing. 

 Prepare a total of 3 extraction tubes containing glass beads by placing one polycarbonate 

filter into each extraction tube and adding 590 μL of salmon DNA/extraction buffer to each 

extraction tube. 

 Mix diluted calibrator cells thoroughly and spot 10 μL of each diluted suspension onto the 

polycarbonate filter in each extraction tube (6 total). 

 Spot 10 μL of AE buffer onto the polycarbonate filter of the SPC blank extraction tube. 

 Perform extraction of calibrator samples according 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 μL extract plus 200 μL 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 contamination, 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 

Reagent Volume/Reaction1

Sterile H2O 1.5 μL

BSA 2.5 μL

TaqMan® master mix 12.5 μL

Primer/probe working stock solution2 3.5 μL 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 μL of the appropriate assay mix and 5 μL of 

each sample (diluted replicates) as specified in Table 2 (below) and Method B (Section 11.8). 

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

 Include at least 1 no template control (NTC) containing 5 μL 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-22 
C-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 acceptance criteria for initial and ongoing method 

performance assessments of Method B 

Summary of Preliminary Analyses 

Laboratories will complete the following according to Method B and study-specific instructions: 

 Enumerate laboratory-prepared spiking suspensions and determine cell concentration of 

the spiking suspension 

 Prepare and analyze unspiked PBS and fresh or marine water samples to determine 

ambient Bacteroidales concentrations according 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, Method 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 Method B and the Spiking Protocol. Samples will 

be filtered and analyzed according to culture instructions, 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 calibrator 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 from ongoing calibrator samples will be used in the 

ΔΔCT 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 

μL 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 spiking 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 × 103

) “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 × 108 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 sides 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 labeled 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 μL). 

 After extraction, dilute (5-fold; e.g., 50 μL extract plus 200 μL 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 undiluted and diluted extracts in refrigerator until 

analyzed. 

Preparation, extraction and processing of ongoing calibrator samples

1. Laboratory-prepared calibrators 

 Thaw (on ice) a total of 3 calibrator samples (prepared during Phase 1) and add 990 μL 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 extraction tube and adding 590 μL of salmon DNA/extraction buffer to 

each extraction tube. 

 Mix diluted calibrator cells thoroughly and spot 10 μL of each diluted suspension onto 

the polycarbonate filter in each extraction tube. 

 Perform extraction of calibrator samples according 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 μL). 

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

buffer). 

 Refrigerate undiluted and diluted calibrator extracts until analyzed. 

PCR preparation and analysis

Note: To minimize potential environmental DNA contamination, 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/Reaction1

Sterile H2O 1.5 μL 

BSA 2.5 μL

TaqMan® master mix 12.5 μL

Primer/probe working stock solution2 3.5 μL 1 Multiply by # samples (plus 1) to be analyzed per day 2 See Phase II Table 1 for assay-specific primer/probe working stocks 
C-9 

 Set up individual PCR reactions to contain 20 μL of the appropriate assay mix and 5 μL 

of each sample (diluted and undiluted replicates) as specified in Table 2 (below) and 

Method B (Section 11.8). 

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

 Include at least 1 no template control (NTC) containing 5 μL 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 preliminary 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 × 103

) “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 × 108 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-prepared 
C-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 replicates 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 sides 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 μL). 

 After extraction, dilute (5-fold; e.g., 50 μL extract plus 200 μL AE buffer) both unspiked 

sample extracts and 4 replicate extracts (clarified 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 processing of ongoing calibrator samples

1. Laboratory-prepared calibrators 

 Prepare a total of 3 calibrator extracts as described previously and in Method B (Sections 

11.3.9 – 11.3.12). 

 Prepare a 5-fold dilution for each calibrator extract (e.g., 50 μL extract plus 200 μL 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 μL of the appropriate assay mix and 5 μL 

of each sample (undiluted replicates) as specified in Table 3 (below): 

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

 Include at least 1 no template control (NTC) containing 5 μL 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 Protocol 
D-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 study of Method B in fresh water and marine water 

samples. During this study, laboratories will use laboratory-prepared and vendor-prepared (BioBallTM) 

spikes. 

Note: The sample spiking procedures described in this protocol 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 Laboratory-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 BioBallTM 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 freeze) 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 × 108 B. thetaiotaomicron colony 

forming units (CFU) per mL. This is referred 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 establish percent recovery, it is necessary to determine the 

concentration of B. thetaiotaomicron 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 suspensions in the steps below, 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 × 106 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 × 105

 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 × 104

 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 × 103

 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 × 102

 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 Vspiked 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 spiking 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 Vspiked per 100 mL sample in Section 

3.2 below. Analyze the spiked sample 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 Vspiked 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 agar 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 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 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 undiluted 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 equation. Example calculations are provided in 

Table 1. 

B. thetaiotaomicron undiluted spike = (CFU1 + CFU2 + ... + CFUn) / (V1 + V2 + ... + Vn) 

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 108

 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 108

 CFU / mL

* B. thetaiotaomicron undiluted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU 

per plate
D-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. 

 TSpiked B. thetaiotaomicron = (B. thetaiotaomicron undiluted spike) × (V spiked per 100 mL sample) 

 Where, 

 TSpiked 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) TSpiked B. thetaiotaomicron

 9.6 × 108 CFU / mL 1.0 × 10-4 mL per 100 mL of 

sample

(9.6 × 108 CFU / mL) × (1.0 × 10-4 mL /100 mL) = 

96,000 CFU / 100 mL

5.3 × 108 CFU / mL 3.2 × 10-4 mL per 100 mL of 

sample 

 (5.3 × 108

 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 × 

(Ns - Nu) 

T 

 Where, 

R = Percent recovery 

Ns = B. thetaiotaomicron (CFU/100 mL) in the spiked sample 

Nu = B. thetaiotaomicron (CFU/100 mL) in the unspiked sample 

TSpiked 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 

Ns (CFU / 100 mL) Nu (CFU / 100 mL)

T Spiked B. thetaiotaomicron

(CFU / 100 mL) Percent recovery (R)

947,000 3a 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 acceptable if the sample was a recreational water – if the sample 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 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 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 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 Ensure BAP are dry and free from condensation prior to use. To ensure the agar surfaces 

are 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 distributed 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 record 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 suspension 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 BioBallJ 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 the 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 Atrue@ spiked B. thetaiotaomicron. For example, the triplicate plate counts for 

the 32,000 CFU BioBallTM are as follows:

TSpiked B. thetaiotaomicron (CFU / 100 mL) = [(300 + 240 + 290) ×100] / 3 = 27,700 per BioBallTM 

 

Where, 

 TSpiked 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 × (Ns - Nu ) 

 T 

 Where, 

 R = Percent recovery 

 Ns = B. thetaiotaomicron (CFU/100 mL) in the spiked sample (or 

CFU per BioBallTM) 

 Nu = B. thetaiotaomicron (CFU/100 mL) in the unspiked sample 

TSpiked 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 Ns (CFU / 100 mL) Nu (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 Checklist 
E-1 

Laboratory Capabilities Checklist 

qPCR Single-Laboratory Validation Study 

(May 18, 2009) 

EPA plans to invite utility, research, and commercial laboratories to bid for participation in a study to evaluate the 

performance of Methods A and B for analysis of recreational 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 bid package to Yildiz Chambers at 703.461.8056 by June 1, 

2009. If you have any questions pertaining to the information 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 capabilities 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 participating 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 recreational 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 participated in a validation study? Yes No 

 

D. If your laboratory has experience analyzing 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 marine 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., public beach, fishing pier). Please be as descriptive 

as possible. If there is a known source of fecal contamination (e.g. WWTP, birds), please identify that 

source. 

 

Collection site 1: 

Collection site 2: 

Collection site 3: 

 

F. Will your laboratory be prepared to collect and 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)