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-06-019
i

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 Technology’s Engineering and Analysis Division within 

the U.S. Environmental Protection Agency (EPA’s) Office of Water. 

The contributions of the following volunteer participants 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 Laboratory: Daniel Mills, Wanda Poerwantoro, and 

Microbiology Staff 

• Hampton Roads Sanitation District: Robin 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 Mazengia, Robin Revelle, and 

Jodeen Wieser 

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

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

• Orange County Sanitation District, Environmental Sciences Laboratory: Charles McGee, Michael 

von Winckelmann, Kim Patton, Linda Kirchner, James Campbell, and Arturo Diaz 

• State of Hawaii Department of Health, Environmental Microbiology Section: Myron Honda, 

Kristine Kang, Ginger Cachero, Lori Suyama, Tiffany Lau, and Remedios Peralta 

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

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

• Wisconsin State Laboratory of Hygiene: Sharon Kluender and Jeremy Olstadt 
ii

Disclaimer

This document has been reviewed and approved by the EPA/EAD. 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 

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-Finding 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 Reporting and Validation ...........................................................................................8 

5.1 Data Reporting....................................................................................................................8 

5.2 Data Validation...................................................................................................................8 

5.3 Censored Data.....................................................................................................................8 

Section 6.0 Results, Data Analysis, and 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 References.........................................................................................................................16 

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

List of Tables 

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

Table 2. Participant Laboratories and Methods/Matrices 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)........................................ 14 
 v

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. Environmental 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) concentrations 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 spiked 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 generate 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 

1 September 2006 

SECTION 1.0 INTRODUCTION

Enterococci and Escherichia coli (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 relationships between health effects and 

water quality. Methods for enterococci (Methods 1106.1 and 1600) and E. coli (Methods 1103.1 and 

1603) have recently been approved for monitoring ambient 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 ambient recreational water samples analyzed for E. coli could be held 

for 24 hours prior to sample analysis without a significant 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 hours 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, and 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 μm pore-size membrane. After filtration, membranes are 

placed on a selective medium, mE agar, and incubated 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 μm 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 halo (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 μm 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 substrate. 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 μm 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 incubated 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

SECTION 2.0 STUDY OBJECTIVES

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 using 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 1103.1 (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 according to the analytical and QA/QC procedures as 

described in each of the analytical methods or approved changes to these procedures to ensure that 

the data are of known and reliable quality. 
Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water 

3 September 2006 

SECTION 3.0 TECHNICAL APPROACH

3.1 Identification of Laboratories 

Participant laboratories were representative of the 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 information from laboratories and to screen potential 

participants and to ensure that laboratories were qualified. 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 necessary), 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 plates with 20 to 60 enterococci colonies or 20 to 80 E. 

coli colonies. 

3.3 Methods 

The holding time for enterococci was evaluated in both 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 laboratories. 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 Summarya

No. of Replicates per Holding Timeb

Matrix Analyte Method 

0c

 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 analyzed within two hours of sample 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. The 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 start 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, participant 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 enterococci concentrations for all the matrices being 

evaluated, all holding time samples were spiked. Laboratories measured 36, 100-mL replicates from the 

bulk marine or fresh water sample into separate sterile 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 freezing. 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 analyzed to determine ambient concentrations. 
Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water 

5 September 2006 

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 approximately 450 mL. At 0, 6, and 24 hours after sample 

collection, aliquots of each replicate were analyzed 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. Two 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 blanks, 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

September 2006 6

SECTION 4.0 STUDY IMPLEMENTATION

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 Protection Agency’s (EPA’s) Office of Water (OW). 

The EPA technical lead was Robin K. Oshiro. Coordination 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 sample 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 samples 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 

7 September 2006 

Table 2. Participant Laboratories and Methods/Matrices each Laboratory Evaluated during the 

 Holding Time Studya

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, Bobbie 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

September 2006 8

SECTION 5.0 DATA REPORTING AND VALIDATION

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 performed and exhibited the appropriate response 

• Confirmation that all method-specific incubation 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 validation for this study. The results were compared 

to verify consistency and identify potential data entry errors. 

Laboratories 8, 12, and 12 did not meeting IPR criteria for Method 1103.1 during preliminary analyses. 

In addition, laboratory 8 did not meet IPR criteria for Method 1106.1 and laboratory 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 constraints. Since the laboratories met OPR criteria, data 

from these laboratories was considered valid and included in subsequent 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 subsequent 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 were 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-censored) were observed for all of the unspiked samples 

analyzed for enterococci, regardless of matrix. In addition, two of the replicates analyzed using mTEC 

were below the analytical range of the method. All 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 

9 September 2006 

SECTION 6.0 RESULTS, DATA ANALYSIS, AND DISCUSSION

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, respectively. Only valid results are included in this 

section; a detailed description of data invalidation information is included 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 performed 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 RSDa Mean 

% Recovery RSDa Mean % Recovery RSDa Mean % Recovery RSDa

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 64b 23 63 19 66b 27 110 15 

9 45 12 45 8 98 14 75 8 

10 83 9 

11 

12 82 16 61 7 45b 32 84 9 

13 115 19 128b 15 3b,c 0 107 16 

Shading indicates laboratories did not conduct sample analyses 

a

 Relative standard deviation 

b

 Laboratory did not meet IPR criteria c

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

 

 September 2006 10 

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 recommended 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 and matrix, a two-way Analysis of Variance (ANOVA) 

model was fit to assess the effect of holding time and laboratory on the log-transformed concentrations, 

and to assess whether there was a significant interaction between holding time and laboratory (i.e., the 

effect of holding time on the enterococci counts differed significantly between the laboratories). For 

Method 1600 in fresh water, there was no significant 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 combinations, 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 fresh 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 footnote is provided for significant 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. Since significant interactions between laboratory and 

holding time were observed for all other method/matrix 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 enterococci data, the following observations were made: 

Marine Samples Analyzed Using Method 1106.1

• Enterococci concentrations were significantly decreased by the 6 hour holding time compared to 

the 0 hour holding time at Laboratory 1 

• Enterococci concentrations were significantly decreased 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 decreased 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 decreased by the 6 hour holding time compared to 

the 0 hour holding time at Laboratories 1 and 3. 

• Enterococci concentrations were significantly decreased by the 24 hour holding time compared to 

both the 0 hour and 6 hour holding time at Laboratory 1. 

• Enterococci concentrations were significantly decreased 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 

11 September 2006 

Fresh water Samples Analyzed Using Method 1106.1

• Enterococci concentrations were significantly decreased 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 decreased 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 Da

 123 Da,b 234 170 Da 110 Da,b

2 110 106 92 Da,b 

3 223 201 185 Da 222 197 Da

 189 Da

4 

5 

6 333 362 373 

7 272 250 230 

8c 319 335 315 349 377 350 

8c 481 494 530 510 523 538 

9 

10 140 137 135 

11 277 278 215 Da,b 252 277 230 

12 109 96 112 118 121 102 

Mean 261 243 225 270 270 253 

RSDd 17 19 13 17 17 16 

Shading indicates laboratories did not conduct sample analyses 

“D” indicates a significant decrease in enterococci concentration at 24 hours compared to 0 and/or 6 hour results a Results were significantly different 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 RSDs 
Holding 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 Da,b 135 134 119 

10 

11 135 156 161 136 154 145 

12 

Mean 182 185 175 228 232 215 Da,b

RSDc 18 20 14 15 18 18 

Shading indicates laboratories did not conduct sample analyses 

“D” indicates a significant decrease in enterococci concentration at 24 hours compared to 0 and/or 6 hour results a Results were significantly different compared to “0” hour results b

 Results were significantly different compared to “6” hour results c Pooled within-lab relative standard deviation was determined by calculating the square root of the weighted mean of 

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

13 September 2006 

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 stratified 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 concentrations, 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, pairwise comparisons were performed in a single ANOVA 

model using data from all laboratories. For Method 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 indicate 

whether 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 row because there was no significant interaction between 

holding time and laboratory. Since significant interactions between laboratory and holding time were 

observed for Method 1603, laboratory-specific 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 Da

 119 Da,b

2 221 204 202 

3 

4c 14 13 11 7 10 4 Db

5 234 241 196 Da,b 

6 

7 

8 187 147 169 61 66 158 I

a,b

9 569 556 363 563 512 444 Da

10 

11 259 155 160 229 231 202 

12c 64 64 55 

Mean 197 176 166 Da 191 182 168 

RSDd 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 compared 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 different compared to “0” hour results b

 Results were significantly different 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 RSDs 
Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water 

15 September 2006 

SECTION 7.0 CONCLUSIONS 

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 relationships 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 enterococci 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 generate 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 

SECTION 8.0 REFERENCES

8.1 US EPA. 2005. EPA Method 1106.1: Enterococci 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: Enterococci 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 Environmental Pollution Monitoring. Van Nostrand 

Reinhold, New York. 
Holding Time Effects on Enterococci and E. coli Concentrations in Recreational Water 

17 September 2006 

SECTION 9.0 ACRONYMS

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 Checklist 
1 

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 study and will also cover all shipping costs. Volunteer 

laboratories will be acknowledged in the holding time study reports. The study is tentatively scheduled to begin 

the end of January 2006. 

If your laboratory is interested in participating in the holding time study, please provide the requested information 

below and fax the signed, completed checklist to Katrina 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 the 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 Capabilities and Experience

A. Please complete the requested capabilities and experience 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 analyzing 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 matrices 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 will be available for use during the study? 

B. How many funnels may be used at one time (i.e., the 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 provided above is accurate and complete: 

Primary Analyst or Lab Manager (please print) 

Laboratory Name 

Signature and Date 
Appendix B: 

Instructions for the Beach Holding Time Study for Enterococci 

(Methods 1106.1 and 1600) 
1 February 2006 

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 significant change in enterococci concentrations. 

During the holding time study, each laboratory will analyze marine and fresh water samples for enterococci at 0, 6,

and 24 hours after sample collection or spiking. Samples 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 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.

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

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 samples (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.
3 February 2006 

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 below), 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.
4 February 2006 

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 reddish-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 incubate for 20 to 30 minutes. Count and record colonies

that are pink to dark red on mE and produce a reddish-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 incubate for 20 to 30 minutes. Count and record colonies

that are pink to dark red on mE and produce a reddish-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) 
1 February 2006 

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 freshwater 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 analyzed 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 filters 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 waterbath, count and record the number of red or magenta

colonies. 

Method 1103.1 (mTEC)

• After 22 ± 2 hours incubation, remove the plates from the 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.
2 February 2006 

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 transport 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 mL
3 February 2006

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, transfer the filter to an absorbent pad saturated with

urea substrate medium, and incubate at room temperature 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) which 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, transfer the filter to an absorbent pad saturated with

urea substrate medium, and incubate at room temperature for 15-20 minutes. Count and record the number

of yellow, yellow-green, or yellow-brown colonies.
4 February 2006

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, transfer the filter to an absorbent pad saturated with

urea substrate medium, and incubate at room temperature 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)
1 February 2006

Enterococci Spiking Protocol

Beach Holding Time Study Using Methods 1106.1 (mE-EIA) and 1600 (mEI)

February 2, 2006

The purpose of this protocol is to provide laboratories with enterococci spiking procedures for the beach holding

time study using Methods 1106.1 and 1600. During this study, laboratories will spike samples using laboratoryprepared spiking solutions. The following sections are included in this protocol:

Laboratory-Prepared Spiking Solutions

Section 1: Preparation of Laboratory-Prepared Spiking Suspensions

Section 2: Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration

Section 3: Calculation of “True” Concentration of Spiked Enterococci

1.0 Preparation of Laboratory-Prepared Spiking Suspensions

1.1 Stock Culture. Prepare a stock culture by inoculating a trypticase soy agar (TSA) slant (or other nonselective media) with Enterococcus faecalis ATCC #19433 and incubating at 35°C ± 3°C for 20 ± 4

hours. This stock culture may be stored in the dark at room temperature for up to 30 days.

1.2 Phosphate Buffered Saline (PBS). Prepare PBS according to Method 1106.1, Section 7.4 or

Method 1600, Section 7.4.

1.3 1% Azide Dextrose Broth. Prepare a 1% solution of azide dextrose broth by combining 99 mL of

sterile phosphate buffered saline (Method 1106.1, Section 7.4 or Method 1600, Section 7.4) and 1.0 mL

of sterile single strength azide dextrose broth in a sterile screw cap bottle or re-sealable dilution water

container. Shake to mix. 

1.4 Spiking Suspension (Undiluted). From the stock culture of E. faecalis ATCC #19433 in Section

1.1, transfer a small loopful of growth to the 1% azide dextrose 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 approximately 1.0 × 106

 to 1.0 × 107

 enterococci colony forming units (CFU) per mL. This is

referred to as the “undiluted spiking suspension.” Note: During the holding time study, growth of

spiking suspensions should begin one day prior to the day that samples will be spiked. For example, if

samples will be spiked on Tuesday, growth of spiking suspensions will begin on Monday.

1.5 Proceed to Section 2.0 for sample spiking and enumeration of spiking suspension.

2.0 Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration

The objective of spiking the sample is to ensure sufficient bacterial concentrations to assess the effect of

holding time. This section provides instructions for sample spiking (Section 2.1) and spiking suspension

enumeration (Section 2.2). Note: Please be sure to thoroughly mix the spiking suspensions in the steps

below, as this is critical for accurate sample spiking and spiking suspension enumeration.
2 February 2006

2.1 Sample spiking

2.1.1 Dilute spiking suspension

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 (Method 1106.1, Section 7.4 or Method

1600, Section 7.4), 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

10-2 mL of the original undiluted spiking suspension.

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

Section 2.1.1.1 above) 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 10-4 mL of the original undiluted

spiking suspension.

2.1.1.3 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution “B” (from

Section 2.1.1.2 above) 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 10-5 mL of the original undiluted

spiking suspension. 

2.1.1.4 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution “C” (from

Section 2.1.1.3 above) 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 10-6 mL of the original undiluted

spiking suspension.

2.1.2 Spike sample(s) 

2.1.2.1 Matrix. To spike sample, add 1.5 mL of spiking suspension dilution “C” (from

Section 2.1.1.3 above) to 100-mL of unspiked sample and mix by vigorously shaking

the bottle a minimum of 25 times. This is the “spiked” sample. The volume (mL) of

undiluted spiking suspension added to each 100 mL of sample is 1.5 x 10-5 mL per

100 mL, which is referred to as Vspiked p er 100 mL sam ple in Section 3.2 below. Analyze the

spiked sample according to the instructions provided in Section 11 of Methods 1106.1

and 1600.

2.1.2.2 OPRs (PBS). Add 3.0 mL of the spiking suspension dilution “D” (Section 2.1.1.4

above) to 100 mL (or appropriate volume) of sample and mix thoroughly by shaking

the bottle a minimum of 25 times. The volume (mL) of undiluted spiking suspension

added to each 100 mL sample is 3.0 x 10-6 mL, which is referred to as V spiked per 100 mL

sample in Section 3.2 below. Filter the spiked sample and analyze the filter according to

the procedures in Section 11 of Methods 1106.1 and 1600. 

2.2 Enumeration of Undiluted Spiking Suspension (prepared in Section 1.3)

2.2.1 Prepare trypticase soy agar (TSA) according to manufacturer’s directions, add 10 - 15 mL of

TSA per 100 x 15 mm petri dish, and allow to solidify. Ensure that agar surface is dry. Note:

Agar plates must be dry and free from condensation prior to use. To ensure that the agar surface

is dry, plates should be made several days in advance and stored inverted at room temperature or

dried for 5 to 10 minutes in a laminar-flow hood.
3 February 2006

2.2.2 Each of the following will be conducted in triplicate, resulting in the preparation aid evaluation

of nine spread plates:

• Mix dilution “B” by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of

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

suspension].

• Mix dilution “C” by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of

dilution “C” onto surface of a pre-dried TSA plate [10-6 (0.000001) mL of the original

spiking suspension].

• Mix dilution “D” by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of

dilution “D” onto surface of a pre-dried TSA plate [10-7 (0.0000001) mL of the original

spiking suspension].

2.2.3 For each spread plate, using a sterile bent glass rod or spreader, distribute inoculum over the

surface of medium by rotating the dish by hand or on a turntable. Note: Please ensure that the

inoculum is evenly distributed over the 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. 

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 “True” Concentration of Spiked Enterococci 

Calculation of the “true” concentration of spiked enterococci will be calculated in two steps as indicated

in Sections 3.1 and 3.2 below. Note: The example calculated numbers 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 “true” spiked enterococci may be slightly

different.

3.1 Step 1: Calculate Concentration of Enterococci (CFU / mL) in Undiluted Spiking

Suspension

3.1.1 The number of enterococci (CFU / mL) in the undiluted spiking suspension (prepared in Section

1.3 above) will be calculated using all TSA plates from Section 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 enterococci (CFU / mL) in the undiluted spiking suspension

according to the following equation. (Example calculations are provided in Table 1 below.)

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

Where,

Enterococci undiluted spike = Enterococci (CFU /mL) in undiluted spiking

suspension

CFU = Number of colony forming units from TSA plates

yielding counts within the ideal range of 30 to 300

CFU per plate

V = Volume of undiluted sample on each TSA plate

yielding counts within the ideal range of 30 to 300

CFU per plate
4 February 2006

n = Number of plates with counts within the ideal range

TABLE 1. EXAMPLE CALCULATIONS OF ENTEROCOCCI SPIKING SUSPENSION CONCENTRATION

Examples

CFU / plate (triplicate analyses) from 

TSA plates in Section 2.2.5

Enterococci CFU / mL in

undiluted 

spiking suspension

(Enterococci undiluted spike 10 )* -5 mL plates 10-6 mL plates 10-7 mL plates 

Exam 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 106 CFU / mL

Exam 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 106 CFU / mL

*Enterococci undiluted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per

plate

3.2 Step 2: Calculate “True” Spiked Enterococci (CFU / 100 mL)

3.2.1 Calculate the “true” concentration of spiked enterococci (CFU / 100 mL) according to the

following equation. Example calculations are provided in Table 2 below. 

TSpiked Enterococci = (Enterococci undiluted spike) x (V spiked per 100 mL sample)

Where,

TSpiked Enterococci = Number of spiked Enterococci (CFU

/ 100 mL)

Enterococci undiluted spike = Enterococci (CFU / mL) in undiluted

spiking suspension (calculated in

Section 3.1.3)

V spiked per100 m L sam ple = mL of undiluted spiking suspension

per 100 mL sample (Section 2.1.2.1)

Table 2. Example Calculations of Spiked Enterococci 

Enterococci undiluted spike

(Table 1 above)

V spiked per 100 mL sample

(Section 2.1.2.1 above) TSpiked Enterococci 

 9.6 x 106 CFU / mL 1.5 X 10-5 mL per 100 mL of

sample

(9.6 x 106

 CFU / mL) x (1.5 x 10-5 mL / 100 mL) =

144 CFU / 100 mL

5.3 x 106 CFU / mL 1.5 X 10-5 mL per 100 mL of

sample

(5.3 x 106

 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) 
1 February 2006

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 laboratories with E. coli spiking procedures for the beach holding time

study using Methods 1103.1 and 1603. During this study, laboratories will spike samples using laboratoryprepared spiking solutions. The following sections are included in this protocol:

Laboratory-Prepared Spiking Solutions

Section 1: Preparation of Laboratory-Prepared Spiking Suspensions

Section 2: Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration

Section 3: Calculation of “True” Concentration of Spiked E. coli

1.0 Preparation of Laboratory-Prepared Spiking Suspensions

1.1 Stock Culture. Prepare a stock culture by inoculating a trypticase soy agar (TSA) slant (or other nonselective media) with Escherichia coli ATCC #11775 and incubating at 35°C ± 3°C for 20 ± 4 hours.

This stock culture may be stored in the dark at room temperature for up to 30 days.

1.2 Phosphate Buffered Saline (PBS). Prepare PBS according to Method 1103.1, Section 7.4 or

Method 1603, Section 7.4. 

1.3 1% Lauryl Tryptose Broth (LTB). Prepare a 1% solution of LTB by combining 99 mL of sterile PBS

(Method 1103.1, Section 7.4 or Method 1603, Section 7.4) and 1.0 mL of sterile single strength lauryl

tryptose broth in a sterile screw cap bottle or re-sealable dilution water container. Shake to mix. 

1.4 Spiking Suspension (Undiluted). From the stock culture of E. coli ATCC #11775 in Section 1.1,

transfer a small loopful of growth to the 1 % LTB solution and vigorously shake a minimum of 25 times.

Incubate at 35°C ± 3°C for 20 ± 4 hours. The resulting spiking suspension contains approximately 1.0 ×

107 to 1.0 × 108 E. coli colony forming units (CFU) per mL. This is referred to as the “undiluted spiking

suspension.” Note: During the holding time study, growth of spiking suspensions should begin one day

prior to the day that samples will be spiked. For example, if samples will be spiked on Tuesday, growth

of spiking suspensions will begin on Monday.

1.5 Proceed to Section 2.0 for sample spiking and enumeration of spiking suspension.

2.0 Laboratory-Prepared Sample Spiking and Spiking Suspension Enumeration

The objective of spiking the sample is to ensure sufficient bacterial concentrations to assess the effect of

holding time. This section provides instructions for sample spiking (Section 2.1) and spiking suspension

enumeration (Section 2.2). Note: Please be sure to thoroughly mix the spiking suspensions in the steps

below, as this is critical for accurate sample spiking and spiking suspension enumeration.
2 February 2006

2.1 Sample Spiking

2.1.1 Dilute spiking suspension

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

(Method 1103.1, Section 7.4 or Method 1603, Section 7.4) 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 10-2 mL of the original undiluted spiking suspension.

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

Section 2.1.1.1 above) 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 10-4 mL of the original undiluted

spiking suspension.

2.1.1.3 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution “B” (from

Section 2.1.1.2 above) 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 10-5 mL of the original undiluted

spiking suspension. 

2.1.1.4 Use a sterile pipette to transfer 11.0 mL of spiking suspension dilution “C” (from

Section 2.1.1.3 above) 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 10-6 mL of the original undiluted

spiking suspension.

2.1.2 Spike sample(s) 

2.1.2.1 Matrix. To spike sample, add 1.5 mL of spiking suspension dilution “D” (from

Section 2.1.1.4 above) to 100-mL of unspiked sample and mix by vigorously shaking

the bottle a minimum of 25 times. This is the “spiked” 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 Vspiked p er 100 mL sam ple in Section 3.2 below. 

Analyze the spiked sample according to the instructions provided in Section 11 of

Methods 1103.1 and 1603.

2.1.2.2 OPR (PBS). Add 0.3 mL of the spiking suspension dilution "D" to 100 mL of PBS

and mix thoroughly by shaking the bottle a minimum of 25 times. The volume (mL)

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 Vspiked p er 100 mL sam ple in

Section 3.2 below. Filter the spiked sample and analyze the filter according to the

procedures in Section 11 of Methods 1103.1 and 1603. 

2.2 Enumeration of Undiluted Spiking Suspension (prepared in Section 1.3)

2.2.1 Prepare trypticase soy agar (TSA) according to manufacturer’s directions, add 10 - 15 mL of

TSA per 100 x 15 mm petri dish, and allow to solidify. Ensure that agar surface is dry. Note:

Agar plates must be dry and free from condensation prior to use. To ensure that the agar surface

is dry, plates should be made several days in advance and stored inverted at room temperature or

dried for 5 to 10 minutes in a laminar-flow hood.
3 February 2006

2.2.2 Each of the following will be conducted in triplicate, resulting in the preparation and evaluation

of nine spread plates: 

• Mix dilution “B” by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of

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

suspension].

• Mix dilution “C” by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of

dilution “C” onto surface of a pre-dried TSA plate [10-6 (0.000001) mL of the original

spiking suspension].

• Mix dilution “D” by vigorously shaking the bottle a minimum of 25 times. Pipet 0.1 mL of

dilution “D” (Section 2.1.1.4) onto surface of a pre-dried TSA plate [10-7 (0.0000001) mL of

the original spiking suspension].

2.2.3 For each spread plate, using a sterile bent glass rod or spreader, distribute inoculum over the

surface of medium by rotating the dish by hand or on a turntable. Note: Please ensure that the

inoculum is evenly distributed over the 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. 

2.2.6 Count and record number of colonies per plate. Refer to Section 3.0 for calculation of spiking

suspension concentration.

3.0 Calculate “True” Concentration of Spiked E. coli

Calculation of the “true” concentration of spiked E. coli will be calculated in two steps as indicated in

Sections 3.1 and 3.2 below. Note: The example calculated numbers 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 “true” spiked E. coli may be slightly different.

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

3.1.1 The number of E. coli (CFU / mL) in the undiluted spiking suspension (prepared in Section 1.3

above) will be calculated using all TSA plates from Section 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. coli (CFU / mL) in the undiluted spiking suspension according

to the following equation. (Example calculations are provided in Table 1 below.)

E. coli undiluted spike = (CFU1

 + CFU2

 + ... + CFUn

) / (V1

 + V2

 + ... + Vn

)

Where,

E. coli undiluted spike = E. coli (CFU / mL) in undiluted spiking suspension

CFU = Number of colony forming units from TSA plates

yielding counts within the ideal range of 30 to 300 CFU

per plate

V = Volume of undiluted sample on each TSA plate

yielding counts within the ideal range of 30 to 300 CFU

per plate

n = Number of plates with counts within the ideal range
4 February 2006

TABLE 1. EXAMPLE CALCULATIONS OF E. COLI SPIKING SUSPENSION CONCENTRATION

Examples

CFU / plate (triplicate analyses) from 

TSA plates in Section 2.2.5

E. coli CFU / mL in undiluted 

spiking suspension

(EC undiluted spike)* 10-5 mL plates 10-6 mL plates 10-7 mL plates 

Exam 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 107

 CFU / mL

Exam 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 107 CFU / mL

*EC undiluted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per plate

3.2 Step 2: Calculate “True” Spiked E. coli (CFU / 100 mL)

3.2.1 Calculate “true” concentration of spiked E. coli (CFU / 100 mL) according to the following

equation. Example calculations are provided in Table 2 below. 

TSpiked E. coli = (E. coli undiluted spike) x (V spiked per 100 mL sample)

Where,

TSpiked E. coli = Number of spiked E. coli (CFU / 100 mL)

E. coli undiluted spike = E. coli (CFU / mL) in undiluted spiking suspension

(calculated in Section 3.1.3)

V spiked per100 m L sam ple = mL of undiluted spiking suspension per 100 mL

sample (Section 2.1.2.1)

Table 2. Example Calculations of Spiked E. coli 

EC undiluted spike

(Table 1 above)

V spiked per 100 mL sample

(Section 2.1.2.1 above) TSpiked E. coli

 9.6 x 107 CFU / mL 1.5 X 10-6 mL per 100 mL

of sample

(9.6 x 107

 CFU / mL) x (1.5 x 10-6 mL / 100 mL) =

144 CFU / 100 mL

2.8 x 107 CFU / mL 1.5 X 10-6 mL per 100 mL

of sample

(2.8 x 107 CFU / mL) x (1.5 x 10-6 mL / 100 mL) = 

42 CFU / 100 mL