# Assessment of the Effects of Holding Time on Enterococci Concentrations in Fresh and Marine Recreational Waters and Escherichia coli Concentrations in Fresh Recreational Waters

December 2006

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

EPA-821-R-06-019i 

## Acknowledgments

This report was prepared by the CSC Microbiology and Biochemistry Studies Group under the direction of Robin K. Oshiro, of the Office of Science and Te chnology’s Engineering and Analysis Division within the U.S. Environmental Protection Agency (EPA’s) Office of Water.

The contributions of the following volunteer participan ts and organizations to this study are gratefully acknowledged:

• City of Los Angeles, Bureau of Sanitation, Environmental Monitoring Division: Gerald McGowen, Stan Asato, Ioannice Lee, Hung Pham , Marieta Ravelo, Nicanor Arzadon, and Amber Kuhn

• County Sanitation Districts of Los Angeles County, Joint Water Pollution Control Plant: Kathy Walker, Michele Padilla, and Albert Soof

• East Bay Municipal Utility District Laborator y: Daniel Mills, Wanda Poerwantoro, and Microbiology Staff

• Hampton Roads Sanitation District: Robi n Parnell, Paula Hogg, and Bob Maunz

• King County Environmental Laboratory: Kate Leone , Colin Elliott, Despina Strong, Joe Clark, Debbie Turner, Tami Alley, Karl Bruun, Bobbie Anderson, Eyob Mazengi a, Robin Revelle, and Jodeen Wieser

• Massachusetts Water Resources Authority: Steve Rhode, Mariya Gofshteyn, and Laura Ducott

• Narragansett Bay Commission: Nora Lo ugh, Cynthia Walters, and Walter Palm

• Orange County Sanitation District, Environmental Sciences Laboratory: Charles McGee, Michael von Winckelmann, Kim Patton, Linda Kirc hner, James Campbell, and Arturo Diaz

• State of Hawaii Department of Health, Envi ronmental Microbiology Section: Myron Honda, Kristine Kang, Ginger Cachero, Lori Suya ma, Tiffany Lau, and Remedios Peralta

• University of Iowa Hygienic Laboratory: Nancy Hall and Cathy Lord

• Utah Department of Health: Sanwat Chaudhur i, Clarice Evans, and Michael Mortensen

• Wisconsin State Laboratory of Hygiene: Sharon Kluender and Jeremy Olstadtii 

## Disclaimer

This document has been reviewed and approved by the EPA/EAD. Mention of company names, trade names, or commercial products does not cons titute 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 202.566.1075 202.566.1053 (facsimile)iii 

## Table of Contents

Section 1.0 Introduction....................................................................................................... ..................1 1.1 Summary of EPA Method 1106.1 for Enterococci .............................................................1 1.2 Summary of EPA Method 1600 for Enterococci................................................................1 1.3 Summary of EPA Method 1103.1 for E. coli ......................................................................1 1.4 Summary of EPA Method 1603 for E. coli .........................................................................1 Section 2.0 Study Objectives................................................................................................... ..............2

Section 3.0 Technical Approach................................................................................................. ...........3

3.1 Identification of Laboratories ............................................................................................. 3 3.2 Sample Collection, Storage Conditions, and Holding Times .............................................3 3.3 Methods .................................................................................................................... ..........3 3.4 Study Design and Analyses ................................................................................................3 3.5 Preliminary Analyses....................................................................................................... ...4 3.6 Range-Findi ng Analyses..................................................................................................... 4 3.7 Study Analyses ............................................................................................................. ......4 3.8 Quality Control (QC) Analyses ..........................................................................................5 Section 4.0 Study Implementation............................................................................................... ..........6 4.1 Study Management ........................................................................................................... ..6 4.2 Study Schedule ............................................................................................................. ......6 4.3 Participant Laboratories................................................................................................... ...6 Section 5.0 Data Repor ting and Validation ...................................................................................... .....8 5.1 Data Reporting............................................................................................................. .......8 5.2 Data Validation............................................................................................................ .......8 5.3 Censored Data.............................................................................................................. .......8 Section 6.0 Results, Data Analysis, an d Discussion............................................................................. .9 6.1 Initial Precision and Recovery (IPR) Holding Time Study Results for Enterococci (Methods 1106.1 and 1600) and E. coli (1103.1 and 1603)................................................9 6.2 Enterococci (Methods 1106.1 and 1600) Holding Time Study Results for Marine and Fresh Water.................................................................................................................... ...10 6.3 E. coli (Methods 1103.1 and 1603) Holding Time Study Results for Fresh Water..........13

Section 7.0 Conclusions........................................................................................................ ...............15

Section 8.0 Refere nces......................................................................................................... ................16

Section 9.0 Acronyms........................................................................................................... ...............17iv 

## List of Tables

Table 1. Study Analysis Summary................................................................................................ ................ 4

Table 2. Participant Laboratories and Methods/Mat rices each Laboratory Evaluated during the Holding

Time Study ..................................................................................................................... ................ 7

Table 3. Initial Precision and Recovery Results ................................................................................ ........... 9

Table 4. Enterococci Mean Results (CFU/ 100 mL) for Marine Water Samples (n=10) ............................ 11

Table 5. Enterococci Mean Results (CFU/100 mL) for Fresh Water Samples (n=10)............................... 12

Table 6. E. coli Mean Results (CFU/100 mL) for Fresh Water Samples (n=10)........................................ 14v 

## List of Appendices

Appendix A: Laboratory Capabilities Checklist

Appendix B: Instructions for the Beach Holding Time Study for Enterococci (Methods 1106.1 and 1600)

Appendix C: Instructions for the Beach Holding Time Study for E. coli (Methods 1103.1 and 1603)

Appendix D: Enterococci Holding Time Study Spiking Protocol (Methods 1106.1 and 1600)

Appendix E: E. coli Holding Time Study Spiking Protocol (Methods 1103.1 and 1603)vi 

## Executive Summary

This report presents the results of the U.S. Enviro nmental Protection Agency’s (EPA’s) Beach Holding Time Study (the “Study”) of membrane filtration (MF) procedures for the analysis of enterococci (using EPA Methods 1106.1 and 1600) concentrations in fresh and marine recreational waters and Escherichia coli (using EPA Methods 1103.1 and 1603) concen trations in fresh recreational water.

The purpose of this study was to determine whether recreational water samples may be held for 24 hours without significant change in bacterial densities for these analytes.

Holding time study samples were spik ed if the ambient enterococci or Escherichia coli ( E. coli) concentration was ≤ 150 CFU/100 mL. Samples were analyzed at 0, 6, and 24 hours after sample collection or sample spiking. Time zero samples were analyzed within two hours of sample collection or sample spiking.

Based on the results of this study, neither enterococci nor E. coli recreational water samples may be analyzed 24 hours after sample collection and still gene rate data comparable to those generated at 6 hours after sample collection. In general, enterococci and E. coli concentrations had a tendency to decrease with extended sample holding times.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 1 

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## 1.0 I NTRODUCTION

Enterococci and Escherichia coli ( E. coli ) analyses are recommended as a measure of recreational water quality. Epidemiological studies have led to the de velopment of criteria that have been used to promulgate recreational water standards based on est ablished relationships between health effects and water quality. Methods for enteroco cci (Methods 1106.1 and 1600) and E. coli (Methods 1103.1 and 1603) have recently been approved for monitoring ambi ent waters. Bacterial analysis of non-potable waters for compliance monitoring requires that sample analysis begin within 6 hours of sample collection. The purpose of this study is to determine whether marine and fresh ambient recreational water samples analyzed for enterococci and fresh ambien t recreational water samples analyzed for E. coli could be held for 24 hours prior to sample analysis without a signifi cant change in bacterial concentrations using the above methods. Samples were analyzed at 0, 6, and 24 hours after sample collection or sample spiking. Time zero samples were analyzed within two ho urs of sample collection or sample spiking.

This study was collaboratively designed by US EPA and the CSC Microbiology and Biochemistry Studies Group. Study management, data review, a nd data analysis activities were performed by CSC under contract to the EPA Office of Water’s Engineering and Analysis Division (OW/EAD).

1.1 Summary of EPA Method 1106.1 for Enterococci (Reference 8.1)

Water samples are filtered through a 0.45 μ m pore-size membrane. After filtration, membranes are placed on a selective medium, mE agar, and incubate d at 41.0°C ± 0.5°C for 48 ± 3 hours. Following incubation, filters are transferred to a differential medium, EIA agar, and incubated at 41.0°C ± 0.5°C for an additional 20-30 minutes. Pink to red colonies that develop a black or reddish-brown precipitate on the underside of the filter are considered enterococci.

1.2 Summary of EPA Method 1600 for Enterococci (Reference 8.2)

Water samples are filtered through a 0.45 μ m pore-size membrane. After filtration, membranes are placed on a selective medium, mEI agar, and incubated at 41.0°C ± 0.5°C for 24 ± 2 hours. All colonies greater than 0.5 mm in size that produce a blue ha lo (regardless of color) are considered enterococci.

1.3 Summary of EPA Method 1103.1 for E. coli (Reference 8.3)

Water samples are filtered through a 0.45 μ m pore-size membrane. After filtration, membranes are placed on a selective medium, mTEC agar, incubated at 35.0°C ± 0.5°C for 2 ± 0.5 hours to resuscitate injured or stressed bacteria, and then incubated at 44.5°C ± 0.2°C for 22 ± 2 hours . Following incubation, filters are transferred to pads saturated with urea s ubstrate. After 15-20 minutes, all yellow, yellow-green or yellow-brown colonies are considered E. coli

. 1.4 Summary of EPA Method 1603 for E. coli (Reference 8.4)

Water samples are filtered through a 0.45 μ m pore-size membrane. After filtration, membranes are placed on a selective medium, modified mTEC agar, incubated at 35.0°C ± 0.5°C for 2 ± 0.5 hours to resuscitate injured or stressed bacteria, and then in cubated at 44.5°C ± 0.2°C for 22 to 24 hours. All red and magenta colonies are considered E. coli

.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 2 

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## 2.0 S TUDY 

## O BJECTIVES

The following study objectives were established for the holding time study:

• Evaluate the effect of holding time across multiple laboratories and fresh and marine recreational water matrices on enterococci concentrations us ing EPA Methods 1106.1 (mE-EIA) and 1600 (mEI).

• Evaluate the effect of holding time across multiple laboratories and fresh recreational water matrices

on E. coli concentrations using EPA Methods 1 103.1 (mTEC) and 1603 (modified mTEC).

To accomplish these objectives, qualified volunteer laboratories analyzed recreational water samples for enterococci and E. coli at 0, 6, and 24 hours from either sample collection or sample spiking.

The following data quality objective was established for this study:

• Data produced under this study was generated acco rding to the analytical and QA/QC procedures as described in each of the analytical methods or a pproved changes to these pr ocedures to ensure that the data are of known and reliable quality.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 3 

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## 3.0 T ECHNICAL 

## A PPROACH

3.1 Identification of Laboratories

Participant laboratories were representative of th e general user community, had experience analyzing marine and/or fresh recreational water samples for enterococci and E. coli using membrane filtration techniques, and had access to marine and/or fresh water matrices. A detailed laboratory capabilities checklist (Appendix A) was used to collect info rmation from laboratories and to screen potential participants and to ensure that laboratories were qua lified. Laboratory availability was also considered.

To reduce study costs, volunteer laboratories were recruited. To reduce the burden on the participant laboratories and encourage volunteer participants, EPA provided media, reagents, and disposable supplies for the holding time study.

3.2 Sample Collection, Storage Conditions, and Holding Times

A single bulk sample of at least five liters of each matrix was collected 6-12 inches below the water surface and transported to the laboratory on ice and maintained at <10°C and above freezing. At the laboratory, the bulk sample was split into replicates, and spiked (if necessar y), and stored in the refrigerator at <10°C and above freezing. Time zero was defined as two hours after sample collection or sample spiking. After 0, 6, and 24 hours from sample collection or spiking, aliquots of each replicate were analyzed by the appropriate procedure ( Table 1 ). Sample volumes were selected based on anticipated background levels to obtain countable pl ates with 20 to 60 enterococci colonies or 20 to 80 E. coli colonies.

3.3 Methods

The holding time for enterococci was evaluated in bo th marine and fresh water matrices using Methods 1106.1 and 1600. Holding time for E. coli was only evaluated in fresh water matrices using Methods 1103.1 and 1603.

3.4 Study Design and Analyses

During the holding time study, all four methods (1106.1, 1600, 1103.1, and 1603) were used to analyze unspiked and spiked samples at multiple laboratori es. Table 1 summarizes the number and type of samples that were evaluated during the holding time study to meet the objectives listed in Section 2.0.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 4 Table 1. Study Analysis Summary a No. of Replicates per Holding Time b Matrix Analyte Method 0 c hours 6 hours 24 hours
1106.1 (mE-EIA) 10 10 10 Marine Water Enterococci 1600 (mEI) 10 10 10

1106.1 (mE-EIA) 10 10 10 Enterococci 1600 (mEI) 10 10 10
1103.1 (mTEC) 10 10 10 Fresh Water

E. coli 1603 (modified mTEC) 10 10 10

a Range finding analyses were conducted one day prior to analyzing study samples in an effort to assess ambient concentrations of the target analyte.

b If it was necessary to spike the matrix due to low ambient concentrations of the target analyte, two additional unspiked samples were analyzed for each holding time.

c Time zero samples were anal yzed within two hours of sa mple collection or spiking

3.5 Preliminary Analyses
Preliminary analyses were conducted on unspiked fresh and marine recreational waters, as appropriate, prior to the start of the holding time study. Th e time between preliminary and holding time study analyses was used to resolve issues that were observed during preliminary analyses.

3.6 Range-Finding Analyses

Range-finding analyses were conducted prior to the st art of the holding time study to determine ambient concentrations of enterococci and E. coli . Range-finding results were used to determine whether spiking was necessary. Holding time samples were spiked if the ambient E. coli and enterococci concentrations

were ≤ 150 CFU/100 mL.

3.7 Study Analyses

In most cases, study analyses were conducted during the same week of range-finding analyses. Due to the 48 hour incubation time in Method 1106.1, par ticipant laboratories using Method 1106.1 analyzed range-finding samples the week prior to the holding time study or used historical data to determine if spiking was necessary.

3.7.1 Enterococci

Since range-finding analyses indicated low ambient ente rococci concentrations for all the matrices being evaluated, all holding time samples were spiked. La boratories measured 36, 100-mL replicates from the bulk marine or fresh water sample into separate ster ile bottles. Six replicates remained unspiked (2 per holding time) and 30 replicates (10 per holding time) were spiked. The spiked replicates were spiked with a laboratory-prepared Enterococcus faecalis (ATCC #19433) spiking suspension according to the Enterococci Holding Time Study Spiking Protocol (Appendix D). After spiking, all replicates, except 0 hour, were immediately stored in the refrigerator at <10°C and above freezi ng. Two unspiked and 10 spiked replicates were analyzed by EPA Methods 1106 .1 and 1600 at 0, 6, and 24 hours after sample spiking. The unspiked replicates were analy zed to determine ambient concentrations.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 5 3.7.2 E. coli

Five of the seven laboratories range-finding results indicated low ambient E. coli concentrations. As a result, laboratories 4 and 12 analyzed unspiked replicates during the holding time study and the remaining laboratories analyzed spiked holding time study replicates.

Laboratories 4 and 12 . A single bulk fresh water sample (~5 L) was collected and transported to the laboratory on ice and maintained at <10°C and above freezing. At the laboratory, the bulk sample was split into 10 replicates (labeled 1-10) of approximate ly 450 mL. At 0, 6, and 24 hours after sample collection, aliquots of each replicate were anal yzed by each method (Methods 1103.1 and 1603).

Laboratories 1, 2, 8, 9, and 11

. Laboratories measured 36, 100-mL replicates from the bulk fresh water sample and dispensed each replicate in a separate sterile bottle. Six replicates remained unspiked (2 per holding time) and 30 replicates (10 per holding time) were spiked. The spiked replicates were spiked with a laboratory-prepared Escherichia coli (ATCC #11775) spiking suspension according to the E. coli Holding Time Study Spiking Protocol (Appendix E). After sample spiking, all replicates, except 0 hour, were immediately stored in the refrigerator at < 10°C and above freezing. Tw o unspiked and 10 spiked replicates were analyzed by EPA Methods 1103.1 and 1603 at 0, 6, and 24 hours after sample spiking. The unspiked samples were analyzed to determine ambient concentrations.

3.8 Quality Control (QC) Analyses

Participating laboratories completed the following QC analyses: media sterility checks, dilution water sterility checks, filter sterility check, filtration bl anks, positive controls, and negative controls.

• EPA Methods 1106.1 and 1600: E. faecalis (ATCC #19433) served as the positive control and E. coli (ATCC #11775) as the negative control.

• EPA Methods 1103.1 and 1603: E. coli (ATCC #11775) served as the positive control and E. faecalis (ATCC #19433) as the negative control.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

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## 4.0 S TUDY 

## I MPLEMENTATION

4.1 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 Pr otection Agency’s (EPA’s) Office of Water (OW). The EPA technical lead was Robin K. Oshiro. Coor dination of activities for the Study was performed by the CSC Microbiology and Biochemistry Studies Group.

4.2 Study Schedule

Both enterococci and E. coli analyses were conducted the Spring of 2006. Availability, scheduling, and weather determined when samples were analyzed at each participant laboratory. Some of the fresh water matrices were frozen until late spring, requiring samp le collection once these waters were accessible. Prior to analyzing holding time study samples each laboratory was required to analyze four initial precision and recovery (IPRs) and two matrix spike sa mples to demonstrate method proficiency. Some laboratories repeated IPR analyses due to problems enumerating the spiking suspension using the spread plate technique.

4.3 Participant Laboratories

The twelve participating laboratories involved in the Study are shown in Table 2

.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 7 Table 2. Participant Laboratories and Methods/Matrices each Laboratory Evaluated during the Holding Time Study a

City of Los Angeles Bureau of Sanitation Stan Asato, Ioannice Lee, Hung Pham, Marieta Ravelo, Nicanor Arzadon, Amber Kuhn 12000 Vista del Mar, Playa del Rey, CA 90293 Narragansett Bay Commission Nora Lough, Cynthia Walker, Walter Palm 1 Service Road, Providence, RI 02905

County Sanitation Districts of Los Angeles County (JWPCP) Kathy Walker, Michele Padilla, Albert Soof 24501 S. Figueroa Street, Carson, CA 90745 Orange County Sanitation District Charles McGee, Michael von Winckelmann, Kim Patton, Linda Kirchner, James Campbell, Arturo Diaz 10844 Ellis Avenue, Fountain Valley, CA 92708

East Bay Municipal Utility District Laboratory Daniel Mills, Wanda Poerwantoro 2020 Wake Avenue, Oakland, CA 94607 State of Hawaii Dept. of Health, Environmental Microbiology Myron Honda, Kristine Kang, Ginger Cachero, Lori Suyama, Tiffany Lau, Remedios Peralta 2725 Waimamo Home Road, Pearl City, HI 96782

Hampton Roads Sanitation District Robin Parnell, Paula Hogg, Bob Maunz 1432 Air Rail Avenue, Virginia Beach, VA 23455 University of Iowa Hygenic Laboratory Nancy Hall, Cathy Lord 102 Oakdale Campus # H101 HO, Iowa City, IA 52242

King County Environmental Laboratory Colin Elliott, Despina Strong, Joe Clark, Bo bbie Anderson 322 West Ewing Street, Seattle, WA 98119 Utah Department of Health Sanwat Chaudhuri, Clarice Evans, Michael Mortensen 46 North Medical Drive, Salt Lake City, UT 84113

Massachusetts Water Resources Authority Steve Rhodie, Mariya Gofshteyn, Laura Ducott 190 Tafts Avenue, Winthrop, MA 02152 Wisconsin State Laboratory of Hygiene Sharon Kluender, Jeremy Olstadt 2601 Agriculture Drive, Madison, WI 53718

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.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

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## 5.0 D ATA 

## R EPORTING AND 

## V ALIDATION

5.1 Data Reporting

Laboratories submitted the following data to CSC for review and validation:

• Completed cover sheet with sample collection and QC information

• Completed sample-specific reporting forms

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

5.2 Data Validation

Data review checklists were used to ensure that each data package was complete and to ensure that each sample result met the study-specific and method-specific requirements. Items reviewed for each sample included the following:

• Confirmation that original forms were submitted

• Confirmation that all QC checks were perform ed and exhibited the appropriate response

• Confirmation that all method-specific inc ubation times and temperatures were met

• Confirmation that all media and reagents were used within expiration dates

• Confirmation that samples were spiked with appropriate dilution

• Confirmation that all calculations are correct

This process was performed independently by two data reviewers, each of whom entered the results into separate spreadsheets designed for data review and valid ation for this study. The results were compared to verify consistency and identif y potential data entry errors.

Laboratories 8, 12, and 12 did not meeting IPR crite ria for Method 1103.1 during preliminary analyses. In addition, laboratory 8 did not meet IPR criteria for Method 1106.1 and labo ratory 12 did not meet criteria for Method 1600. However, unless specifically noted in this section all of the laboratories that failed the IPR criteria did meet the OPR criteria during the holding time study. While it would have been preferable for the laboratories to repeat the IPR analyses prior to moving forward with the holding time study, it was not possible due to scheduling constraint s. Since the laboratories met OPR criteria, data from these laboratories was considered valid a nd included in subseque nt data analyses.

Laboratory 12 did not meet IPR or OPR criteria for method 1103.1 during the validation study. As a result, data from Laboratory 12 was considered invalid and not included in sub sequent data analyses.

Laboratory 4 indicated that they may have spiked replicate 10 (6 hour) incorrectly resulting in significantly higher concentration of enterococci compared to all of the replicates analyzed at 0, 6, and 24 hours. As a result, data for replicate 10 at 6 hours we re considered invalid and not included in subsequent data analyses.

5.3 Censored Data

During the evaluation of holding time study samples, results below the analytical range of the method (less-than results, also referred to as left-censore d) were observed for all of the unspiked samples analyzed for enterococci, regardless of matrix. In addition, two of th e replicates analyzed using mTEC were below the analytical range of the method. A ll censored values were set to one-half the detection limit (less-than value) prior to data analyses.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

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## 6.0 R ESULTS 

## , D ATA 

## A NALYSIS 

## , AND 

## D ISCUSSION

This section includes initial precision and recovery (IPR ) results for PBS samples spiked with enterococci

or E. coli . Holding time study results for enterococci samples in fresh and marine waters and E. coli in fresh water are provided in Sections 6.2 and 6.3, resp ectively. Only valid results are included in this section; a detailed description of data invalida tion information is incl uded in Section 5.0.

6.1 Initial Precision and Recovery (IPR) Results for Enterococci (Methods 1106.1 and 1600) and E. coli (1103.1 and 1603)

Initial precision and recovery (IPR) analyses were pe rformed by each laboratory prior to the analyses of holding time study samples. IPR results for Methods 1106.1, 1600, 1103.1, and 1603 are summarized in Table 3

. Table 3. Initial Precision and Recovery Results

Method 1106.1 Acceptance Criteria Mean % Recovery (15 - 36) RSD ≤ 21% Method 1600 Acceptance Criteria Mean % Recovery (31-127) RSD ≤ 28% Method 1103.1 Acceptance Criteria Mean % Recovery (76 -124) RSD ≤ 41% Method 1603 Acceptance Criteria Mean % Recovery (46 -119) RSD ≤ 36% Lab

Mean % Recovery RSD a Mean % Recovery RSD a Mean % Recovery RSD a Mean % Recovery RSD a

1 44 11 34 15 84 14 85 12
2 77 4 88 17
3 111 18 112 12
4 49 6 46 16 85 34 103 20
5 76 16 91 12
6 33 4
7 45 3
8 64 b 23 63 19 66 b 27 110 15
9 45 12 45 8 98 14 75 8
10 83 9
11
12 82 16 61 7 45 b 32 84 9
13 115 19 128 b 15 3 b,c 0 107 16 Shading indicates laboratories did not conduct sample analyses

a Relative standard deviation b Laboratory did not m eet IPR criteria

c Laboratory did not meet IPR or OPR criteriaHolding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

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6.2 Enterococci (Methods 1106.1 and 1600) Holding Time Study Results for Marine and Fresh Water

All enterococci results were natural log-transformed prior to performing any statistical analyses as a result of the skewed distribution of the results, as reco mmended in Statistical Methods for Environmental Pollution Monitoring (Reference 8.5). Results were stratified by EPA Method (1106.1 and 1600) and by matrix (marine and freshwater). For each method a nd matrix, a two-way Analysis of Variance (ANOVA) model was fit to assess the effect of holding time and laboratory on the log-tran sformed concentrations, and to assess whether there was a significant inte raction between holding time and laboratory ( i.e ., the effect of holding time on the enterococci counts diffe red significantly between the laboratories). For Method 1600 in fresh water, there was no significan t interaction between holding time and laboratory, and, as a result, pairwise comparisons were performed in a single ANOVA model using data from all laboratories. For all other method/matrix combin ations, there was a significant interaction between holding time and laboratory, and therefore an ANOVA model was fit for each laboratory separately.

Within each ANOVA model, comparisons between each of the three holding times were performed using the Tukey-Kramer method for pairwise comparisons.

Enterococci results for marine and fr esh water matrices are summarized in Tables 4 and 5 , respectively. Significant decreases in bacterial concentrations at 24 compared to 0 hour and/or 6 hour results are indicated by a “ D ”. For the 24 hour holding time, a footnot e is provided for si gnificant differences to indicate whether the difference is compared to the 0 hour or the 6 hour holding time. For Method 1600 in fresh water, Tukey-Kramer results are provided in the mean results row because there was no significant interaction between holding time and laboratory. Si nce significant interactions between laboratory and holding time were observed for all other method/matr ix combinations, laboratory-specific Tukey-Kramer results are provided.

A significant interaction between holding time and laboratory means that the effect of holding time on the fecal coliform counts differed significantly between the laboratories.

Based on statistical analysis of the enteroco cci data, the following observations were made:

Marine Samples Analyzed Using Method 1106.1

• Enterococci concentrations were significantly decr eased by the 6 hour holding time compared to the 0 hour holding time at Laboratory 1

• Enterococci concentrations were significantly d ecreased by the 24 hour holding time compared to both the 0 hour and 6 hour holding time at Laboratories 1, 2, and 11.

• Enterococci concentrations were significantly d ecreased by the 24 hour holding time compared to the 0 hour holding time at Laboratory 3.

Marine Samples Analyzed Using Method 1600

• Enterococci concentrations were significantly decr eased by the 6 hour holding time compared to the 0 hour holding time at Laboratories 1 and 3.

• Enterococci concentrations were significantly d ecreased by the 24 hour holding time compared to both the 0 hour and 6 hour holding time at Laboratory 1.

• Enterococci concentrations were significantly d ecreased by the 24 hour holding time compared to the 0 hour holding time at Laboratory 3.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 11 Fresh water Samples Analyzed Using Method 1106.1

• Enterococci concentrations were significantly d ecreased by the 24 hour holding time compared to both the 0 hour and 6 hour holding time at Laboratory 9.

Fresh water Samples Analyzed Using Method 1600

• Enterococci concentrations were significantly d ecreased by the 24 hour holding time compared to both the 0 hour and 6 hour holding time.

Table 4. Enterococci Mean Results (CFU/100 mL) for Marine Water Samples (n=10) Method 1106.1 (mE-EIA) Method 1600 (mEI) Lab 0 hours 6 hours 24 hours 0 hours 6 hours 24 hours

1 294 186 D a 123 D a,b 234 170 D a 110 D a,b

2 110 106 92 D a,b

3 223 201 185 D a 222 197 D a 189 D a

4
5
6 333 362 373
7 272 250 230
8 c 319 335 315 349 377 350
8 c 481 494 530 510 523 538
9
10 140 137 135
11 277 278 215 D a,b 252 277 230
12 109 96 112 118 121 102
Mean 261 243 225 270 270 253
RSD d 17 19 13 17 17 16 Shading indicates laboratories did not conduct sample analyses “D” indicates a significant decrease in enterococci concentra tion at 24 hours compared to 0 and/or 6 hour results

a Results were significantly differ ent compared to “0” hour results b Results were significantly different compared to “6” hour results

c Laboratory analyzed samples from two different marine sites d Pooled within-lab relative standard deviation was determined by calculating the square root of the weighted mean of the squared lab RSDsHolding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 12 Table 5. Enterococci Mean Results (CFU/100 mL) for Fresh Water Samples (n= 10) Method 1106.1 (mE-EIA) Method 1600 (mEI) Lab 0 hours 6 hours 24 hours 0 hours 6 hours 24 hours

1 214 217 222 218 224 207
2 149 143 120
3
4 179 168 150 180 177 143
5 397 428 407
6
7
8 277 287 288 300 273 271
9 141 140 109 D a,b 135 134 119
10
11 135 156 161 136 154 145
12
Mean 182 185 175 228 232 215 D a,b

RSD c 18 20 14 15 18 18 Shading indicates laboratories did not conduct sample analyses “D” indicates a significant decrease in enterococci concentra tion at 24 hours compared to 0 and/or 6 hour results

a Results were significantly differ ent compared to “0” hour results b Results were significantly differ ent compared to “6” hour results

c Pooled within-lab relative standard deviation was determin ed by calculating the square r oot of the weighted mean of

the squared lab RSDsHolding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 13 6.3 E. coli (Methods 1103.1 and 1603) Holding Time Study Results for Fresh Water

All E. coli results were natural log-transformed prior to performing any statistical analyses as a result of the skewed distribution of the results, as recommended in Statistical Methods for Environmental Pollution Monitoring (Reference 8.5). Results were stratif ied by EPA Method (1103.1 and 1603). For each method, a two-way Analysis of Variance (ANOVA) model was fit to assess the effect of holding time and laboratory on the log-transformed c oncentrations, and to assess whether there was a significant interaction between holding time and laboratory ( i.e ., the effect of holding time on the E. coli counts differed significantly between the laboratories). For Method 1103.1, there was no significant interaction between holding time and laboratory, and, as a result, pair wise comparisons were performed in a single ANOVA model using data from all laboratories. For Met hod 1603, there was a significant interaction between holding time and laboratory, and therefore an ANOVA model was fit for each laboratory separately. Within each ANOVA model, comparisons between each of the three holding times were performed using the Tukey-Kramer method for pairwise comparisons.

E. coli results for freshwater matrices are summarized in Table 6 . Significantly different holding times are indicated by an “ I ” or “ D ,” with an “ I ” indicating a significant increase compared to 0 hour and/or 6 hour results and a “ D ” indicating a significant decrease in concentration compared to 0 hour and/or 6 hour results. For the 24-hour holding time, a footnote is provided for significant differences to indicatewhether the difference is compared to the 0 hour or the 6 hour holding time. For Method 1103.1, TukeyKramer results are provided in the mean results ro w because there was no significant interaction between holding time and laboratory. Since significant in teractions between laboratory and holding time were observed for Method 1603, laboratory-specifi c Tukey-Kramer results are provided.

Based on statistical analysis of the E. coli data, the following observations were made:

Fresh water Samples Analyzed Using Method 1103.1

• E. coli concentrations were significantly decreased by the 24 hour holding time compared to the 0-hour and 6-hour holding time.

Fresh water Samples Analyzed Using Method 1603

• E. coli concentrations were significantly decreased by the 24 hour holding time compared to both the 0 hour and 6 hour holding times at Laboratories 1 and 5.

• E. coli concentrations were significantly decreased by the 24 hour holding time compared the 0 hour holding time at Laboratory 9.

• E. coli concentrations were significantly decreased by the 24 hour holding time compared to the 6 hour holding time at Laboratory 4.

• E. coli concentrations were significantly increased by the 24 hour holding time compared to both the 0 hour and 6 hour holding times at Laboratory 8.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 14 Table 6. E. coli Mean Results (CFU/100 mL) for Fresh Water Samples (n=10) Method 1103.1 (mTEC) Method 1603 (modified mTEC) Lab 0 hours 6 hours 24 hours 0 hours 6 hours 24 hours

1 146 103 92 181 149 D a 119 D a,b

2 221 204 202
3
4 c 14 13 11 7 10 4 D b

5 234 241 196 D a,b

6
7
8 187 147 169 61 66 158 I a,b

9 569 556 363 563 512 444 D a

10
11 259 155 160 229 231 202
12 c 64 64 55
Mean 197 176 166 D a 191 182 168
RSD d 52 31 29 40 32 42 Shading indicates laboratories did not conduct sample analyses “D” indicates a significant decrease in E. coli concentration at 24 hours compar ed to 0- and/or 6-hour results “I” indicates a significant increase in E. coli concentration at 24 hours compared to 0- and/or 6-hour results

a Results were significantly differ ent compared to “0” hour results b Results were significantly differ ent compared to “6” hour results

c Laboratory analyzed unspiked samples, range-finding analyses indicated spiking was not necessary d Pooled within-lab relative standard deviation was determined by calculating the square root of the weighted mean of the squared lab RSDsHolding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 15 

## S ECTION 

## 7.0 C ONCLUSIONS

Enterococci and E. coli analyses are recommended as a measure of recreational water quality. Epidemiological studies have led to the development of criteria that have been used to promulgate recreational water standards based on established relati onships between health effects and water quality. Methods for enterococci (Methods 1106.1 and 1600) and E. coli (Methods 1103.1 and 1603) have been approved for monitoring ambient waters. The purpose of this study was to determine whether marine and fresh water samples analyzed for enteroco cci and fresh water samples analyzed for E. coli can be held for longer than 6 hours prior to sample analysis without a significant change in bacterial concentrations.

Based on the results of this study, neither enterococci nor E. coli recreational water samples may be analyzed 24 hours after sample collection and still gene rate data comparable to those generated at 6 hours after sample collection. In general, enterococci and E. coli concentrations had a tendency to decrease with extended sample holding times.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 16 

## S ECTION 

## 8.0 R EFERENCES

8.1 US EPA. 2005. EPA Method 1106.1: Enteroco cci in Water by Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE-EIA), EPA-821-R-04-022, April 2005.

8.2 US EPA. 2005. EPA Method 1600: Enteroco cci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl- β -D Glucoside Agar (mEI), EPA-821-R-02-023, April 2005.

8.3 US EPA. 2005. EPA Method 1103.1: Escherichia coli ( E. coli ) in Water by Membrane Filtration Using membrane-Thermotolerant Escherichia coli Agar (mTEC), EPA-821-R-04-024, April 2005.

8.4 US EPA. 2005. EPA Method 1603: Escherichia coli ( E. coli ) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC), EPA-821-R02-025, April 2005.

8.5 Gilbert, R.O. 1987. Statistical Methods for E nvironmental Pollution Monitoring. Van Nostrand Reinhold, New York.Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water

September 2006 17 

## S ECTION 

## 9.0 A CRONYMS

ADB Azide dextrose broth CFU Colony forming unit EIA Esculin iron agar IPR Initial precision and recovery LTB Lauryl tryptose broth MF Membrane filtration mTEC membrane-Thermotolerant Escherichia coli Agar mEI membrane-Enterococcus Indoxyl- β -D Glucoside Agar OPR Ongoing precision and recovery PBS Phosphate buffered saline QC Quality control RSD Relative standard deviation SAS Statistical analysis software SD Standard deviation TSA Tryptic soy agar

## Appendix A: Laboratory Capabilities Checklist1 

## Laboratory Capabilities Checklist Beach Holding Time Study

(January 4, 2006)

EPA plans to invite up to 20 laboratories (10-marine and 10 -fresh water) to participate in a study for the detection and enumeration of E. coli and enterococci in marine and/or fresh water at 0, 6, and 24 hour holding times. The methods to be used include EPA Methods 1603 (modified mTEC) and 1103.1 (mTEC) for E. coli, and Methods 1600 (mEI) and 1106.1 (mE/EIA) for enterococci. Each of these methods will require filtration of diluted samples and subsequent incubation on selective media prior to target analyte detection and enumeration. EPA will provide all media and disposable materials for the st udy and will also cover all shipping costs. Volunteer laboratories will be acknowledged in the holding time study re ports. The study is tentatively scheduled to begin the end of January 2006.

If your laboratory is interested in participating in th e holding time study, please provide the requested information below and fax the signed, completed checklist to Katr ina Keller at 703.461.8056 by Monday, January 8th. In addition, please send the form electronically to kkeller5@csc.com . Katrina will confirm the receipt of the checklist. If you have any questions pertaining to th e information requested below or the holding time study, please do not hesitate to contact Yildiz Chambers at 703.461.2165 or ychambers@csc.com.

Section 1. Laboratory Ca pabilities and Experience

A. Please complete the requested capabilities and experien ce information below, if this information has not been previously provided to CSC. The information requested in Table 1 pertains to experience with a given method, regardless of matrix (e.g., marine, fresh water) analyzed.

Table 1. Analyst Experience
Years of experience or estimated number of samples analyzed

Methods to be used Analyst

1603 (modified mTEC) 1103.1 (mTEC) 1106.1 (mE/EIA) 1600 (mEI)

B. Primary analyst’s name:

C. Primary analyst's years of experience performing microbial water analyses:

D. What certifications does your laboratory have for microbial analyses?

E. Additional comments:2 Section 2. Background Information

A Does your laboratory have access to marine and/or fresh water samples? Yes No Please indicate in Table 2, below.

B. Has your laboratory ever participated in a holding time study? Yes No

C. Does your laboratory have experience analyzi ng samples using membrane filtration procedure?

Yes No

D. If your laboratory has experience analyzing marine and/or fresh water samples for E. coli and/or enterococci, please place a check A / @ next to the water type(s) which you have analyzed and indicate the method(s) used for analysis and typical concentrations of each analyte. Please indicate in Table 2, below.

Table 2. E. coli and Enterococci

E. coli Enterococci Access? Water type Monitoring frequency Methods Typical range Methods Typical range

Example Primary treated 1 per month SM 9221B/F 300 1106.1 120

Marine

Fresh

E. If you indicated (Table 2) that your laboratory has access to and/or experience analyzing water samples, please provide as much information regarding these matr ices as possible (below). This information will assist in the evaluation matrix suitability.

F. If available, please provide historical bacterial data (e.g. E. coli , enterococci) from the marine and/or fresh water matrices.

G. Will your laboratory be prepared to collect and analyze water samples within the 0, 6, and 24-hour holding times? Yes No H. Additional comments:3 Section 3. General Information.

A. How many membrane filtration funnels w ill be available for use during the study?

B. How many funnels may be used at one time (i.e., th e size of the manifold that will be used to analyze samples 3, 6, etc.)?

C. How does your laboratory disinfect filtration assemblies/funnels between sample filtrations?

I certify that the information provide d above is accurate and complete:

Primary Analyst or Lab Manager (please print)

Laboratory Name

Signature and Date

## Appendix B: Instructions for the Beach Hold ing Time Study for Enterococci (Methods 1106.1 and 1600)February 2006 1 

## Instructions for the Beach Holding Time Study for Enterococci (Methods 1106.1 and 1600)

February 2, 2006

The purpose of this study is to determine whether marine and fresh water samples can be held for longer than 6 hours prior to sample analysis without a signifi cant change in enterococci concentrations.

During the holding time study, each laboratory will analyze ma rine and fresh water samples for enterococci at 0, 6, and 24 hours after sample collection or spiking. Samp les will be analyzed using EPA Methods 1600 (mEI) and 1106.1 (mE-EIA). Range-finding analysis will be performed on Day 1 to determine enterococci concentrations. Option A or B will be selected depending on ambient enterococci concentration.

The following samples will be analyzed for each method:

Option A - Ambient enterococci concentrations > 150 CFU/100 mL 30 unspiked replicates (10 per holding time) 6 spiked matrix samples (MS) (2 per holding time) 3 unspiked PBS replicates (1 per holding time) 6 spiked PBS (OPR) replicates (2 per holding time)

Option B

- Ambient enterococci concentrations # 150 CFU/100 mL 6 unspiked replicates (2 per holding time) 30 spiked replicates (10 per holding time) 3 unspiked PBS replicates (1 per holding time) 6 spiked PBS (OPR) replicates (2 per holding time)

Day One: Sample Analyses

Range-finding: Collect a 1L fresh or marine water bulk sample and transport to the laboratory. Filter 1.0, 10, and 100 mL volumes of sample and place filters on mEI (Method 1600) plates and incubate at 41°C ± 0.5°C for 24 ± 2 hours.

Spiking suspension: Inoculate enterococci spiking suspension accord ing to the spiking protocol and incubate at 35°C ± 0.5°C for 24 ± 4 hours.

OPR samples: Prepare a total of 18 PBS replicates (100 mL each) for the holding time study analyses.

Note: Method 1106.1 is not being used to evaluate range -finding samples because it requires a 48-hour incubation.February 2006 2 Day Two: Sample Analyses

Range-finding analyses

Method 1600 (mEI) • After 24 ± 2 hours, count and record all colonies with blue halos.

Fax range-finding results to Ruth Grunerud at 703-461-8056. Results will be used to determine if matrix spiking is necessary.

Holding time study analyses • Dilute and plate spiking suspension according to spiking protocol and incubate TSA plates at 35°C ± 0.5°C for 24 ± 4 hours. • For each Method (1600 and 1106.1), spike six, 100 mL PBS replicates (OPRs) with E. faecalis ATCC #19433 according to the spiking protocol (Section 2.1.2.2). • Collect two, 4L fresh and/or marine water bulk sam ples (one for each Method: 1106.1 and 1600) and transport to the laboratory. Split each bulk sample into 36, 100 mL volumes. • If enterococci levels are greater than 150 CFU/100 mL, please use option A. If enterococci levels are less than or equal to 150 CFU/100 mL, please use option B.

Sample spiking

Spike the following replicates according to the spiking protocol (Sections 2.1.2.1 and 2.1.2.2).

Option A Method 1106.1 • 6 spiked matrix (100 mL) replicates (2 matrix spikes per holding time) • 6 spiked PBS (100 mL) replicates (2 OPR replicates per holding time)
Method 1600 • 6 spiked matrix (100 mL) replicates (2 matrix spikes per holding time) • 6 spiked PBS (100 mL) replicates (2 OPR replicates per holding time) Option B
Method 1106.1 • 30 spiked matrix (100 mL) replicates (10 replicates per holding time) • 6 spiked PBS (100 mL) replicates (2 OPR replicates per holding time)
Method 1600 • 30 spiked matrix (100 mL) replicates (10 replicates per holding time) • 6 spiked PBS (100 mL) replicates (2 OPR replicates per holding time)

Label replicates with replicate number and holding time (0 , 6, or 24) and refrigerate 6 and 24 hour replicates at <10°C and above freezing.February 2006 3 0 hour replicates (Analyze within two hours of sample collection or spiking)

Filter the following volumes of each replicate for each method: Unspiked PBS replicate (1 replicate)
100 mL Spiked PBS (OPR) replicates (2 replicates)
100 mL Unspiked matrix replicates (Option A: 10 replicates or Option B: 2 replicates) 10, 25, and 50 mL Spiked matrix replicates (Option A: 2 replicates or Option B: 10 replicates) 1.0, 10, and 25 mL

After filtration, incubate plates at 41°C ± 0.5°C for 24 ± 2 hour (mEI) or 41°C ± 0.5°C for 48 ± 3 hours (mE).

6 hour replicates (Analyze six hours after sample collection or spiking)

Remove 6 hour replicates from refrigerator. Filter volumes (as shown for 0 hour replicates) for each method and incubate plates at 41°C ± 0.5°C for 24 ± 2 hour (mEI) or 41°C ± 0.5°C for 48 ± 3 hours (mE).

Day Three: Sample Analyses

• Read TSA plates prepared on Day 2 and record results of spike enumeration.

0 hour replicates

Method 1600 (mEI) • Count and record all colonies with a blue halo.

24 hour replicates (Analyze twenty four hours after sample collection or spiking)

Remove 24 hour replicates from refrigerator. Filter aliquots (as shown for 0 hour replicates) for each method with the exception of the matrix replicates (provided belo w), which have been increased to account for potential bacterial die-off.

Spiked matrix replicates 10, 25, and 50 mL

After filtration, incubate plates at 41°C ± 0.5°C for 24 ± 2 hour (mEI) or 41°C ± 0.5°C for 48 ± 3 hours (mE).

6 hour replicates

Method 1600 (mEI) • Count and record all colonies with a blue halo.February 2006 4 Day Four: Sample Analyses

0 hour replicates

Method 1106.1 (mE-EIA) • Transfer filters from mE plates to EIA and incubate for 20 to 30 minutes. Count and record colonies that are pink to dark red on mE and produce a reddi sh-brown to black precipitate on the underside of the filter when placed on EIA.

24 hour replicates

Method 1600 (mEI) • Count and record all colonies with a blue halo.

6 hour replicates

Method 1106.1 (mE-EIA) • Transfer filters from mE plates to EIA and incuba te for 20 to 30 minutes. Count and record colonies that are pink to dark red on mE and produce a reddi sh-brown to black precipitate on the underside of the filter when placed on EIA.

Day Five: Sample Analyses

24 hour replicates

Method 1106.1 (mE-EIA) • Transfer filters from mE plates to EIA and incuba te for 20 to 30 minutes. Count and record colonies that are pink to dark red on mE and produce a reddi sh-brown to black precipitate on the underside of the filter when placed on EIA.

Fax data reporting sheets for study analysis to Ruth Grunerud at 703-461-8056. After the study, FedEx all hard copy study data to CSC.

## Appendix C: Instructions for the Beach Holding Time Study for E. coli (Methods 1103.1 and 1603)February 2006 1 

## Instructions for the Beach Holding Time Study for E. coli (Methods 1103.1 and 1603)

February 2, 2006

The purpose of this study is to determine whether fresh water samples can be held for longer than 6 hours prior to sample analysis without a significant change in E. coli concentrations. During the holding time study, each laboratory will analyze freshwater samples for E. coli at 0, 6, and 24 hours after sample collection or spiking. Samples will be anal yzed using EPA Methods 1603 (modified mTEC) and 1103.1 (mTEC). Range-finding analysis will be performed on Day 1 to determine ambient E. coli concentrations. Option A

or B will be selected based on ambient E. coli concentration.
The following samples will be analyzed for each method:

Option A - Ambient E. coli concentrations > 150 CFU/100 mL 30 unspiked replicates (10 per holding time) 6 matrix spikes (MS) (2 per holding time) 3 unspiked PBS replicates (1 per holding time) 6 spiked PBS (OPR) replicates (2 per holding time)

Option B

- Ambient E. coli concentrations # 150 CFU/100 mL 6 unspiked replicates (2 per holding time) 30 spiked replicates (10 per holding time) 3 unspiked PBS replicates (1 per holding time) 6 spiked PBS (OPR) replicates (2 per holding time)

Day One: Sample Analyses

Range-finding : Collect a 1L bulk freshwater sample and transport to the laboratory. Filter 1.0, 10, and 100 mL sample volumes for each Method (1103.1 and 1603). Place filte rs on plates and incubate 35°C ± 0.5°C for 2 ± 0.5 hours. After a 2 ± 0.5 hour incubation at 35°C ± 0.5°C, transfer plates to Whirl-Pak® bags, seal the bag, and submerge in a 44.5°C ± 0.2°C waterbath for 22 ± 2 hours. Spiking suspension: Inoculate E. coli spiking suspension according to the spiking protocol and incubate at 35°C ± 0.5°C for 24 ± 4 hours. OPR samples: Prepare a total of 18 PBS replicates (100 mL each) for the holding time study analyses.

Day Two: Sample Analyses
Range-finding analyses

Method 1603 (modified mTEC) • After 22 ± 2 hours, remove the plates from the waterbat h, count and record the number of red or magenta colonies. Method 1103.1 (mTEC) • After 22 ± 2 hours incubation, remove the plates from th e waterbath, transfer filter to an absorbent pad saturated with urea substrate medium. Incubate plates for 15-20 minutes at room temperature, count, and record the number of yellow, yellow-green, or yellow-brown colonies.
Fax range-finding results to Ruth Grunerud at 703-461-8056. Results will be used to determine if matrix spiking is necessary.February 2006 2 Holding time study analyses • Dilute and plate spiking suspension according to spiking protocol and incubate TSA plates at 35°C ± 0.5°C for 24 ± 4 hours. • For each Method (1603 and 1103.1), spike six, 100 mL PBS replicates (OPRs) with E. coli ATCC #11775 according to the spiking protocol (Section 2.1.2.2). • Collect two 4L freshwater bulk samples (one for each Method 1103.1 and 1603) and tran sport to the laboratory. Split each bulk sample into 36, 100 mL volumes.
• If E. coli levels are greater than 150 CFU/100 mL please use option A. If E. coli levels are less than or equal to 150 CFU/100 mL please use option B.

Sample spiking

Spike the following replicates according to the spiking protocol (Sections 2.1.2.1 and 2.1.2.2).

Option A Method 1103.1

• 6 spiked matrix (100 mL) replicates (2 matrix spikes per holding time) • 6 spiked PBS (100 mL) replicates (2 OPR replicates per holding time)
Method 1603

• 6 spiked matrix (100 mL) replicates (2 matrix spikes per holding time) • 6 spiked PBS (100 mL) replicates (2 OPR replicates per holding time) Option B
Method 1103.1

• 30 spiked matrix (100 mL) replicates (10 replicates per holding time) • 6 spiked PBS (100 mL) replicates (2 OPR replicates per holding time)
Method 1603

• 30 spiked matrix (100 mL) replicates (10 replicates per holding time) • 6 spiked PBS (100 mL) replicates (2 OPR replicates per holding time)

Label replicates with replicate number and holding time (0 , 6, or 24) and refrigerate 6 and 24 hour replicates at <10°C and above freezing.

0 hour replicates (Analyze within two hours of sample collection or spiking)

Filter the following volumes of each replicate for each method: Unspiked PBS replicate (1 replicate)
100 mL Spiked PBS (OPR) replicates (2 replicates)
100 mL Unspiked matrix replicates (Option A: 10 replicates or Option B: 2 replicates) 10, 25, and 50 mL Spiked matrix replicates (Option A: 2 replicates or Option B: 10 replicates) 1.0, 10, and 25 mLFebruary 2006 3 After filtration, incubate plates at 35°C ± 0.5°C for 2 ± 0. 5 hours. After the 2 ± 0.5 hour incubation, transfer the plates to a 44.5°C ± 0.2°C waterbath for 22 ± 2 hours.

6 hour replicates (Analyze six hours after sample collection or spiking)

Remove 6 hour replicates from refrigerator. Filter volumes (as shown for 0 hour replicates) for each Method (1103.1 and 1603) and incubate plates at 35°C ± 0.5°C for 2 ± 0.5 hours. After the 2 ± 0.5 hour incubation, transfer the plates to a 44.5°C ± 0.2°C waterbath for 22 ± 2 hours.

Day Three : Sample Analyses

• Read TSA plates prepared on Day 2 and record results of spike enumeration.

0 hour replicates

Method 1603 (modified mTEC) • Count and record the number of red or magenta colonies.

Method 1103.1 (mTEC) • After 22 ± 2 hours, remove plates from the waterbath, tr ansfer the filter to an absorbent pad saturated with urea substrate medium, and incubate at room temper ature for 15-20 minutes. Count and record the number of yellow, yellow-green, or yellow-brown colonies.

24 hour replicates (Analyze twenty four hours after sample collection or spiking)

Remove 24 hour replicates from refrigerator. Filter volumes (as shown for 0 hour replicates) for each method with the exception of the matrix replicates (provided below) wh ich have been increased to account for potential bacterial die-off.

Spiked matrix replicates 10, 25, and 50 mL

After filtration, incubate plates at 35°C ± 0.5 °C for 2 ± 0.5 hours. After the 2 ± 0.5 hour incubation, transfer the plates to a 44.5°C ± 0.2°C waterbath for 22 ± 2 hours.

6 hour replicates

Method 1603 (modified mTEC) • Count and record the number of red or magenta colonies.

Method 1103.1 (mTEC) • After 22 ± 2 hours, remove plates from the waterbath, tr ansfer the filter to an absorbent pad saturated with urea substrate medium, and incubate at room temper ature for 15-20 minutes. Count and record the number of yellow, yellow-green, or yellow-brown colonies.February 2006 4 Day Four: Sample Analyses

24 hour replicates

Method 1603 (modified mTEC) • Count and record the number of red or magenta colonies.

Method 1103.1 (mTEC) • After 22 ± 2 hours, remove plates from the waterbath, tr ansfer the filter to an absorbent pad saturated with urea substrate medium, and incubate at room temper ature for 15-20 minutes. Count and record the number of yellow, yellow-green, or yellow-brown colonies.

Fax data reporting sheets for study analysis to Ruth Grunerud at 703-461-8056. After the study, FedEx all hard copy study data to CSC.

## Appendix D: Enterococci Holding Time Study Spiking Protocol (Methods 1106.1 and 1600)February 2006 1 

## Enterococci Spiking Protocol Beach Holding Time Study Using Method s 1106 .1 (mE- EIA ) and 1600 ( mEI)

February

2, 2006

The purpose of this protocol is to provide laborato ries with enterococci spik ing procedures for th e beach holding time stu dy using Me thods 1106.1 a nd 1600. During th is stu dy, labo rator ies wil l spike s amples us ing labo rator yprepared spiking

solutions. The following sections are incl

uded in this pr otocol:

Laboratory -Prepared Spik ing Solutions Section 1: Preparation of Laboratory -Prepared Spik

ing Suspensions Section 2: Laboratory- Prepared Sample Spiking and Spiking S uspension Enum eration Section 3: Calcula

tion of “True” C oncentr

ation of Spik ed Ente rococci

1.0 Preparation of Laboratory -Prepared Spik ing Suspensio ns

1.1 Stock Cultur

e. Prepare a stock culture by inoculating a trypticase soy

agar (TSA) slant (or o ther nonselective media) with Enteroc occus fa ecalis ATCC # 19433 a

nd incu bating at 35° C ± 3° C for 2 0 ± 4 hours. This stock cu

lture may b e stored in the dark at room tem

perature for up to 30 d

ays.

1.2 Phosp hate B uffere d Salin e (PBS ). Prepare PBS accord ing to Method 1106 .1, Section 7.4 o r Method 1600, Section 7 .4.

1.3 1% A

zide D extros e Brot h. Prepare a 1% solution of az ide dextrose broth by

combining

99 mL of
ste ril e pho spha te bu ffe red s ali ne (M eth od 11 06.1 , Sec tio n 7.4 or Me thod 1600 , Sec tio n 7.4 ) and 1.0 mL of sterile single streng

th azide dextrose bro th in a sterile screw cap bottle or re-sealable dilution water container. Shake

to mix.

1.4 Spikin g Susp ension (Undi luted). From the sto ck cul

ture of E. faec alis ATCC #19433 in Section 1.1, transfer a sm

all loopful of grow

th to the 1% azide d

extrose broth solution and vigorously shake a minimum of 25 times. Incubate at 35°C ± 3°C for 20 ± 4 hours. The resulting spiking suspension contains approxima

tely 1.0 × 10 6 to 1.0 × 10 7 enteroc occi co lony f ormin g unit s (CFU

) per m L. This is referred to as the “undiluted spiking suspen sion.” Note: During the holding time study , growth of spikin g susp ensions

should begin one da y prio r to the

day t hat sam ples wi ll be sp iked. For ex ample , if samples will be spiked on Tuesd ay, growth of spiki ng suspe nsion s will b egin on M onday.

1.5 Proceed to Section 2.0 for sample spik

ing and enum

eration of spiking

suspension.

2.0 Laboratory -Prepared Samp le Spiking and Spiking Sus pension Enume ration

The objective of spiking

the sample is to

ensure sufficient bacterial conc entrations to assess the effect o f holding time . This section provid es instructions for sam

ple spiking (S ection 2.1) and spik ing suspension enumeration (Section

2.2). Note: Please be sure to thorou ghly m ix the spiking

suspensions in the steps below, as this is critical for accurate sample spik ing and spik

ing suspension enu

meration.February 2006 2 2.1 Sample spiking 2.1.1 Dilute spiking suspension
2.1.1.1 Mix the undiluted spik ing suspension by

vigorously

shaking the

bottle a minim

um of
25 times. U se a sterile pipette to transfer 1. 0 mL of the undiluted spiking suspension to 99 mL of sterile phosphate buffered saline (Method 1106.1, Section 7.4 or Method 1600, Section 7.4), cap, and m ix by vig orously shakin

g the bottle a m

inimum o f 25 times . This is

spiki ng su spensio n diluti on “A” . A 1. 0-m L vol ume o f dilutio

n “A” i s 10 -2 mL of the o

riginal undiluted spik

ing suspension. 2.1.1.2 Use a sterile pipette to transfer 1.0 mL of spiking suspen sion dilution “A” (from

Section 2.1.1.1 ab ove) to 99 m L of sterile phosphate bu ffered saline, cap, and mix by vigorously

shaking the

bottle a minim

um of 25 tim

es. This is spiking

suspension dilution “B”. A 1 .0-m

L volume of dilution “B” is 10 -4 mL of the o riginal undiluted spiking suspen sion. 2.1.1.3 Use a sterile pipette to transfer 11.0 mL o f spiking susp ension dilution “B” (from

Section 2.1.1.2 ab ove) to 99 m L of sterile phosphate bu ffered saline, cap, and mix by vigorously

shaking the

bottle a minim

um of 25 tim

es. This is spiking

suspension dilution “C”. A 1 .0-m

L volume of dilution “C” is 10 -5 mL of the o riginal undiluted spiking suspen sion. 2.1.1.4 Use a sterile pipette to transfer 11.0 mL o f spiking susp ension dilution “C” (from

Section 2.1.1.3 ab ove) to 99 m L of sterile phosphate bu ffered saline, cap, and mix by vigorously

shaking the

bottle a minim

um of 25 tim

es. This is spiking

suspension dilution “D”. A 1 .0-m

L volume of dilution “D” is 10 -6 mL of the o riginal undiluted spiking suspen sion. 2.1.2 Spike

samp le(s)
2.1.2.1 Matrix. To spike sample, add 1 .5 mL of sp iking suspensio n dilution “C” (from
Section 2.1.1.3 ab ove) to 100- mL of unspik

ed sample and mix by v

igorously shak

ing the bottle a minim

um of 25 tim

es. This is the “spike d” sample. The volume (m L) of undiluted spiking

suspension added to each 100 mL of sample is 1 .5 x 10 -5 mL per 100 mL, w

hich is referred to as V spiked p er 100 mL sam ple in Section 3.2 below . Analyz e the spiked sam

ple according to the

instructions provided in Section 11 of Methods 1106.1 and 1600.

2.1.2.2 OPRs (P BS). Add 3.0 m L of the spiking

suspension dilution “D”

(Section 2.1.1.4 above) to 100 m

L (or appropriate vo lume) of sam ple and mix tho

roughly by

shaking the bottle a minim

um of 25 tim

es. The volum

e (mL) of und

iluted spiking s

uspension added to each 100 m

L sample is 3. 0 x 10 -6 mL, which is referr ed to as V sp ik ed pe r 1 00 mL

samp le in Sect

ion 3.2 below. Filter the spik ed sam ple and analy ze the filter a ccordin

g to the procedures in Section 11 of Methods 1106.1 and 1600.

2.2 Enumeration of U ndiluted Spiking Suspension (prepared in S ection 1.3)
2.2.1 Prepare trypticase soy agar (TSA) according

to manufacturer’s d

irections, add 10 -

15 mL of TSA per 100 x 15 m

m petri dish, an

d allow to solidify. Ensure that ag ar surface is dry. Note: Agar plates m

ust be dry and free from condensa tion prior to use. To ensu re that the agar surface is dry, plates shou

ld be made sev

eral days in adv

ance and stored inv erted at room tem perature or dried for 5 to 10 m

inutes in a laminar- flow hood.February 2006 3 2.2.2 Each of the following will be conducted in triplicate, resulting in the preparation aid evaluatio n of nine spread plates:

• Mix dilution “B” by

vigorously

shaking the

bottle a minim

um of 25 tim

es. Pipet 0.1 m

L of dilution “B” onto surface of a pre-dried TSA plate [10 -5 (0.00001) m L of the original spik

ing
suspens ion].

• Mix dilution “C” by

vigorously

shaking the

bottle a minim

um of 25 tim

es. Pipet 0.1 m

L of dilution “C” onto surface of a pre-dried TSA plate [10 -6 (0.000001) m L of the original spikin g susp ension]

.

• Mix dilution “D” by

vigorously

shaking the

bottle a minim

um of 25 tim

es. Pipet 0.1 m

L of dilution “D” onto surface of a pre-dried TSA plate [10 -7 (0.0000001) m L of the original spikin g susp ension]

. 2.2.3 For each spread plate, using a sterile bent g lass rod or spreader, distribu te inoculum ov

er the
surface of medium

by rotating the dish by hand or on a turntable. Note: Please ensure that the inoculum is ev

enly distributed ov

er the entire surface of the plate. 2.2.4 Allow i nocul um to abso rb int o the medi um complet ely.
2.2.5 Invert pla

tes and incubate at 35° C ± 0.5°C for 20 ± 4 hours.
2.2.6 Count and record num ber of colonies per plate. Refer to Section 3.0 for calculation of spik

ing suspension concentration.

3.0 Calcul ation o f “Tru e” Con centra tion of Spike d Ente rococc i

Calculation of the “true” co ncentration of spiked enterococci will be calculated in two steps as indicated in Sections 3.1 and 3.2 below. Note: The exa mple cal culat ed number s provid ed in th e tabl es belo w have been rounded at the en

d of each step. I f your laboratory recalculates the exam

ples using a spread sheet and rou nds onl y afte r the fin al calcu lation ( Step 2) , the “t

rue” sp iked e

nteroco cci m ay be slight

ly differen t.

3.1 Step 1: Calcula te Concentration of Enterococ ci (CFU / mL) in Undiluted Spikin g Suspension

3.1.1 The number of entero cocci (CFU / m L) in the undiluted spik

ing suspension (prep ared in Section 1.3 above) will be calculated using all TSA plates from Section 2.2 yieldin g counts within th e ideal range of 30 to 300 CFU per plate. 3.1.2 If the num

ber of colonies exceeds th e upper range (i.e ., >300) or if the co lonies are not discrete,
results should be reco rded as “too nu merou s to cou

nt” (TNTC ). 3.1.3 Calculate the concentration o f enterococci (CFU / m L) in the undiluted spik

ing suspension according to the follo

wing equation. (Example calculatio ns are provided in Table 1 below.)



## Enterococci undiluted spi ke 

## = (CFU 1 

## + CFU 2 

## + ... + CFU
n 

## ) / (V 1 

## + V 2 

## + ... + V
n 

## )

Where, Enterococci undil uted spi ke = Enterococci (C FU /mL) in un diluted spi king suspension CFU = Number of colony forming units from TSA pla tes yieldi ng counts wi thin the id eal range of 30 to 300 CFU p er pla te V = Volum e of und ilute d sampl e on e ach TSA plate yieldi ng counts wi thin the id eal range of 30 to 300 CFU p er pla teFebruary 2006 4 n = Numb er of pla tes w ith coun ts w ithi n the ide al r ange

T ABLE 1. E XAMPLE CALCULATIONS OF E NTE ROCOC CI SP IK IN G SUS PEN SI ON CON CEN TRA TI ON

Examples CFU / pl at e ( t r i pl i ca t e a na l y se s) f r om TS A plat es in Sec tion 2.2.5 En tero co cci C FU / mL in undi lut ed spi ki ng suspe nsi on (En tero co cci undiluted spike )* 10 -5 mL plates 10 -6 mL plates 10 -7 mL plates

Ex am ple 1 94, 106, 89 9, 11, 28 1, 0, 4 (94+106+89) / ( 10 -5 +10 -5 +10 -5 ) =
289 / ( 3.0 x 10 -5 ) = 9, 633,333 =

9.6 x 10 6 CF U / mL

Ex am ple 2 32, 55, 72 8, 5, 3 0, 0, 0 (32+55+72) / ( 10 -5 +10 -5 +10 -5 ) =
159 / ( 3.0 x 10 -5 ) =5, 300,000 =

5.3 x 10 6 CF U / mL

*E nte ro co cc i undiluted spike is cal culat ed usin g all plat es yi eldi ng counts wi thi n the i deal r ange of 30 t o 300 CFU per plate

3.2 Step 2: Calcula te “True” S piked Enteroco cci (CFU / 10 0 mL)

3.2.1 Calculate the “true” concen tration of spiked entero cocci (CFU / 100 mL) according to the following equation. Example calc ulations are provided in Table 2 below.



## T Spiked En terococci 

## = (Ente rococci undil uted s pike 

## ) x (V spik ed pe r 10 0 mL sam ple 

## )

Where, T Spiked Enter ococci = Number of spiked En terococci (CFU / 100 mL) Enterococci undil uted spi ke = Enterococci (C FU / mL) in u ndiluted spiking suspension (calculated in Section 3.1.3) V sp ike d p er 10 0 m L s am ple = mL of undiluted spiking suspensi on per 100 mL sampl e (Section 2 .1.2.1)

Table 2. Exampl e Calc ulation s of Sp iked E nteroc occi

En tero co cci undiluted spike (Table 1 above) V spiked per 100 mL sample (Secti on 2.1.2 .1 above) T Spiked En terococci

9.6 x 10 6 CFU /

mL 1.5 X 10 -5 mL per 100 mL of

sample (9.6 x 10 6 CFU / mL) x (1.5 x 10 -5 mL / 100 mL) = 144 CFU / 100

mL

5.3 x 10 6 CFU /

mL 1.5 X 10 -5 mL per 100 mL of

sample (5.3 x 10 6 CFU / mL) x (1.5 x 10 -5 mL / 100 mL) = 79.5 CFU / 100 mL

## Appendix E: E. coli Holding Time Study Spiking Protocol (Methods 1103.1 and 1603)February 2006 1 

## E. coli Spiking Protocol Beach Holding Time Study Using Methods 1103.1 (mTEC) and 1603 (modified mTEC)

February

2, 2006

The purpose of this protocol is to provide laborato ries with E. coli spi king p roce dure s for the b each hold ing t ime study using Methods 1103.1 and 1603. During t his st udy, lab orato ries wi ll spi ke samples using la borat oryprepare d spik

ing so lutions. The follo

wing section s are in cluded in this p rotocol :
Laboratory -Prepared Spik ing Solutions Section 1: Preparation of Laboratory -Prepared Spik

ing Suspensions Section 2: Laboratory- Prepared Sample Spiking and Spiking S uspension Enum eration Section 3: Calculation of “True” Conc entration of Spiked E. coli

1.0 Preparation of Laboratory -Prepared Spik ing Suspensio ns

1.1 Stock Culture. Prepare a stock culture by inoculatin

g a trypticase soy agar (TSA) slant (or other nonselective media) with Escheri chia co li ATCC #11775 and incubating at 35° C ± 3°C for 20 ± 4 hours. This stock culture m

ay be stored in th e dark at room temperature for up

to 30 days.

1.2 Phosp hate B uffere d Salin e (PBS ). Prepare PBS according to Method 1103.1, Section 7.4 or Method 1603, Section 7 .4.

1.3 1% La ury l Try ptose Broth (LTB ). Prepare a 1% solut ion of LTB by combining 99 mL of sterile PBS (Meth od 1103. 1, Sect ion 7.4 or Meth od 1603, Sectio n 7.4) an d 1.0 mL of st erile single s trengt h lauryl tryptose broth in a sterile screw cap bottle or re- sealable dilution water contain er. Shake to m ix.

1.4 Spikin g Susp ension (Undi luted). From the sto ck cul

ture of E. coli

ATCC #11775 in Section 1.1, transfer a small loopfu

l of growth to th e 1 % LTB solution and vigorously shake a m

inimum o f 25 times. Incubate at 35 °C ± 3°C for 20 ± 4 hours. The resu lting spiking

suspension contains app roximately 1. 0 × 10 7 to 1.0 × 10 8 E. coli colony form ing units (CFU) per mL. This is refe rred to as the “undiluted spiking suspension.” Note: During the holding time study , growth of sp iking suspensio ns should begin one day prior to the day that sa mples will be spike d. For exam

ple, if sampl es will be spik ed on Tu esday , gro wth of spiki ng suspe nsion s will b egin on M onday.

1.5 Proceed to Section 2.0 for sample spik

ing and enum

eration of spiking

suspension.

2.0 Laboratory -Prepared Samp le Spiking and Spiking Sus pension Enume ration

The objective of spiking

the sample is to

ensure sufficient bacterial conc entrations to assess the effect o f holding time . This section provid es instructions for sam

ple spiking (S ection 2.1) and spik ing suspension enumeration (Section

2.2). Note : Please be sure to thorou ghly m ix the spiking

suspensions in the steps below, as this is critical for accurate sample spik ing and spik

ing suspension enu

meration.February 2006 2 2.1 Sample Spiking 2.1.1 Dilute spiking suspension
2.1.1.1 Mix the undiluted spik ing suspension by

vigorously

shaking the

bottle a minim

um of
25 times. Use

a sterile pipette to transfer 1. 0 mL of the undiluted spiking suspension (Method 1103.1 , Secti on 7.4 or Metho d 1603, Section 7.4) to 99 m L of ste rile phosphate buffered saline, cap, and mix by vig orously shakin g the bottle a m

inimum of 25 times. This is spiking sus pension dilution “A”. A 1.0-m L volume of dilution “A” is 10 -2 mL of the o

riginal undiluted spik

ing suspension. 2.1.1.2 Use a sterile pipette to transfer 1.0 mL of spiking suspen sion dilution “A” (from

Section 2.1.1.1 ab ove) to 99 m L of sterile phosphate bu ffered saline, cap, and mix by vigorously

shaking the

bottle a minim

um of 25 tim

es. This is spiking

suspension dilution “B”. A 1. 0-mL

volume of dilution “B” is 10 -4 mL of the o riginal undiluted spiking suspen sion. 2.1.1.3 Use a sterile pipette to transfer 11.0 mL o f spiking susp ension dilution “B” (from

Section 2.1.1.2 ab ove) to 99 m L of sterile phosphate bu ffered saline, cap, and mix by vigorously

shaking the

bottle a minim

um of 25 tim

es. This is spiking

suspension dilution “C”. A 1 .0-m

L volume of dilution “C” is 10 -5 mL of the o riginal undiluted spiking suspen sion. 2.1.1.4 Use a sterile pipette to transfer 11.0 mL o f spiking susp ension dilution “C” (from

Section 2.1.1.3 ab ove) to 99 m L of sterile phosphate bu ffered saline, cap, and mix by vigorously

shaking the

bottle a minim

um of 25 tim

es. This is spiking

suspension dilution “D”. A 1 .0-m

L volume of dilution “D” is 10 -6 mL of the o riginal undiluted spiking suspen sion. 2.1.2 Spike

samp le(s)
2.1.2.1 Matrix. To spike sam ple, add 1.5 mL of

spiking suspension dilution “D” (from
Section 2.1.1.4 ab ove) to 100- mL of unspik

ed sample and mix by v

igorously shak

ing the bottle a minim

um of 25 tim

es. This is the “spike d” sample. The volume (mL) of undiluted spiking

suspension added to each 100 mL of sample is 1 .5 x 10 -6 mL per 100 mL of sample, which is referred to as V spiked p er 100 mL sam ple in Section 3.2 below

. Analyze the spiked sam

ple according to the

instructions provided in Section 11 of Methods 1103.1 and 1 603.

2.1.2.2 OPR (P BS). Add 0.3 mL of the spiking suspension dil ution "D" to 100 mL of PBS

and mix thoroug

hly by shak

ing the bottle a m

inimum o f 25 times. The volume (m L) of undiluted spiking

suspension added to each 100 mL sample is 3.0

x 10 -7 mL [ ( 0.3 mL x 10 -6 mL) per 100 mL of sample] which is referred to as V spiked p er 100 mL sam ple in Section 3.2 below. Filter the spiked sa mple and analy

ze the filter according

to the procedu res in S ection 1 1 of Methods 1103.1 and 1 603

.

2.2 Enumeration of U ndiluted Spiking Suspension (prepared in S ection 1.3)
2.2.1 Prepare trypticase soy agar (TSA) according

to manufacturer’s d

irections, add 10 -

15 mL of TSA per 100 x 15 m

m petri dish, an

d allow to solidify. Ensure that agar surface is dry. Note: Agar plates m

ust be dry and free from condensa tion prior to use. To ensure that the agar surface is dry, plates shou

ld be made sev

eral days in adv

ance and stored inv erted at room tem perature or dried for 5 to 10 m

inutes in a laminar- flow hood.February 2006 3 2.2.2 Each of the following will be conducted in triplicate, resulting in the preparation and ev aluation
of nine spread plates:

• Mix dilution “B” by

vigorously

shaking the

bottle a minim

um of 25 tim

es. Pipet 0.1 m

L of dilution “B” onto surface of a pre-dried TSA plate [10 -5 (0.00001) m L of the original s

piking
suspens ion].

• Mix dilu

tion “C ” by v

igorou sly sh aking

the bot tle a m inimu m of 2 5 tim es. Pip et 0.1 mL of dilution “C” onto surface of a pre-dried TSA plate [10 -6 (0.000001) m L of the original spikin g susp ension]

.

• Mix dilution “D” by

vigorously

shaking the

bottle a minim

um of 25 tim

es. Pipet 0.1 m

L of dilution “D” (Section 2. 1.1.4) onto surface o f a pre-dried TSA plate [10 -7 (0.0000001) m L of the orig inal spi king suspen sion]. 2.2.3 For each spread plate, using a sterile bent g lass rod or spreader, distribu te inoculum ov

er the
surface of medium

by rotating the dish by hand or on a turntable. Note: Please ensure that the inoculum is ev

enly distributed ov

er the entire surface of the plate. 2.2.4 Allow i nocul um to abso rb int o the medi um complet ely.
2.2.5 Invert pla

tes and incubate at 35° C ± 0.5°C for 20 ± 4 hours.
2.2.6 Count and record num ber of colonies per plate. Refer to Section 3.0 for calculation of spikin g suspension concentration.

3.0 Calculate “T rue” Concentra tion of Spiked E. coli

Calculation of the “true” co ncentration of spiked E. coli will be calcula ted in t wo step s as ind

icated i n Sections 3.1 and 3. 2 below. Note: The exa mple cal culat ed number s provid ed in th e tabl es belo w have been rounded at the en

d of each step. I f your laboratory recalculates the exam

ples using a spread sheet and rounds only after the final calculation (Step 2), the “true” spike d E. coli may be slig htly d ifferent

.

3.1 Step 1 : Calcu late Co ncentr ation o f E. coli (

CFU / mL) in Undiluted Sp iking Suspens ion

3.1.1 The num

ber of E. coli

(CFU / m

L) in the undiluted spik

ing suspension (prep ared in Section 1.3 above) will be calcu

lated using all TSA p lates from Section 2.2 yielding counts within the ideal range of 30 to 3 00 CFU per plate. 3.1.2 If the num

ber of colonies exceeds th e upper range (i.e ., >300) or if the co lonies are not discrete,
results should be reco rded as “too nu merou s to cou

nt” (TNTC ). 3.1.3 Calcula

te the c oncentr

ation of E. coli

(CFU / m

L) in the undiluted spik

ing suspension acco

rding to the following eq

uation. (Examp le calculations are prov ided in Table 1 below.)



## E. coli undiluted spi ke 

## = (CFU 1 

## + CFU 2 

## + ... + CFU
n 

## ) / (V 1 

## + V 2 

## + ... + V
n 

## )

Where, E. coli undil uted spi ke = E. coli (CFU / mL) in undiluted spiking suspens ion CFU = Number of colony forming units from TSA pla tes yieldi ng counts wi thin the id eal range of 30 to 300 CFU per pl ate V = Volum e of und ilute d sampl e on e ach TSA plate yieldi ng counts wi thin the id eal range of 30 to 300 CFU per pl ate

n = Numb er of pla tes w ith coun ts w ithi n the ide al r angeFebruary 2006 4 T ABLE 1. E XAMPLE CALCULATIONS OF E. CO LI SP IK IN G SUS PEN SI ON CON CEN TRA TI ON

Examples CFU / pl at e ( t r i pl i ca t e a na l y se s) f r om TS A plat es in Sec tion 2.2.5 E. co li CFU / mL i n undi lut ed spi ki ng suspe nsi on (EC undiluted spike )* 10 -5 mL plates 10 -6 mL plates 10 -7 mL plates

Ex am ple 1 TNTC, TNTC,

TNTC 94, 106, 89 10, 0 , 4 (94+106+89) / ( 10 -6 +10 -6 +10 -6 ) =
289 / ( 3.0 x 10 -6 ) = 96, 333,333 =

9.6 x 10 7 CF U / mL

Ex am ple 2 269, 289, 304 24, 30, 28 0, 2, 0 (269+289+30) / ( 10 -5 +10 -5 +10 -6 ) =
588 / ( 2.1 x 10 -5 ) =28, 000,000 =

2.8 x 10 7 CF U / mL

*EC undiluted spike is calculated using al l plates yielding counts within the ideal range of 30 to 300 CF U per plate

3.2 Step 2: Calcula te “True” S piked E. coli (CFU / 100 mL)

3.2.1 Calculate “true” concentration of spiked E. coli (CFU / 100 m

L) according to the

following equation. Exam

ple calculations are prov

ided in Table 2 below.



## T Spiked E. coli 

## = ( E. coli undiluted spi ke 

## ) x (V spiked per 100 mL sample 

## )

Where, T Spik ed E. c oli = Number of spiked E. coli (CFU / 100 mL) E. coli undil uted spi ke = E. coli (CFU / mL) in undiluted spiking suspens ion (calculated in Section 3.1.3) V sp ike d p er 10 0 m L s am ple = mL of und ilute d spi king sus pensi on per 100 mL sample (Secti on 2.1.2.1)

Table 2. Example Calcula tions of Spiked E. coli

EC undiluted spike (Table 1 above) V spiked per 100 mL sample (Secti on 2.1.2 .1 above) T Spiked E. coli

9.6 x 10 7 CF U /

mL 1.5 X 10 -6 mL p e r 1 0 0 mL

of sa m ple (9.6 x 10 7 CFU / mL) x (1.5 x 10 -6 m L / 100 m L) = 144 CF U / 10 0 mL

2.8 x 10 7 CF U /

mL 1.5 X 10 -6 mL p e r 1 0 0 mL

of sa m ple (2.8 x 10 7 CFU / mL) x (1.5 x 10 -6 mL / 100 mL) = 42 CF U / 10 0 mL