Office of Water EPA 821-R-14-011 

 

www.epa.gov September 2014 

 

 

Method 1600: Enterococci in 

Water by Membrane Filtration 

Using membrane-Enterococcus 

Indoxyl-β-D-Glucoside Agar (mEI)
U.S. Environmental Protection Agency

Office of Water (4303T)

1200 Pennsylvania Avenue, NW

Washington, DC 20460

EPA-821-R-14-011
Method 1600

i September 2014

Acknowledgments

This method was developed under the direction of James W. Messer and Alfred P. Dufour of the U.S. 

Environmental Protection Agency’s (EPA) Human Exposure Research Division, National Exposure Research 

Laboratory, Cincinnati, Ohio. 

The following laboratories are gratefully acknowledged for their participation in the validation of this method in 

wastewater effluents:

Volunteer Research Laboratories

 EPA Office of Research and Development, National Risk Management Research Lab: Mark C. Meckes 

 U.S. Army Corps of Engineers, Washington Aqueduct: Elizabeth A. Turner, Michael L. Chicoine, and Lisa 

Neal

Volunteer Verification Laboratories

 City of Los Angeles Bureau of Sanitation: Farhana Mohamed, Ann Dalkey, Ioannice Lee, Genevieve 

Espineda, and Zora Bahariance

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

Winckelmann, Kim Patton, Linda Kirchner, James Campbell, Arturo Diaz, and Lisa McMath

Volunteer Participant Laboratories

 City of Los Angeles Bureau of Sanitation: Farhana Mohamed, Ann Dalkey, Ioannice Lee, Genevieve 

Espineda, and Zora Bahariance

 County Sanitation Districts of Los Angeles County (JWPCP): Kathy Walker, Michele Padilla, and Albert 

Soof 

 County Sanitation Districts of Los Angeles County (SJC): Shawn Thompson and Julie Millenbach

 Environmental Associates (EA): Susan Boutros and John Chandler

 Hampton Roads Sanitation District (HRSD): Anna Rule, Paula Hogg, and Bob Maunz

 Hoosier Microbiological Laboratories (HML): Don Hendrickson, Katy Bilger, and Lindsey Shelton

 Massachusetts Water Resources Authority (MWRA): Steve Rhode and Mariya Gofhsteyn

 North Shore Sanitation District (NSSD): Robert Flood

 Texas A&M University: Suresh Pillai and Reema Singh

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

 Wisconsin State Laboratory of Hygiene (WSLH): Jon Standridge, Sharon Kluender, Linda Peterson, and 

Jeremy Olstadt

 Utah Department of Health: Sanwat Chaudhuri and Devon Cole 
Method 1600

September 2014 ii

Disclaimer

The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and approved 

this report for publication. The Office of Science and Technology directed, managed, and reviewed the work of 

DynCorp in preparing this report. Neither the United States Government nor any of its employees, contractors, 

or their employees make any warranty, expressed or implied, or assumes any legal liability or responsibility for 

any third party’s use of or the results of such use of any information, apparatus, product, or process discussed in 

this report, or represents that its use by such party would not infringe on privately owned rights. Mention of 

trade names or commercial products does not constitute endorsement or recommendation for use.

Questions concerning this method or its application 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

202-566-1053 (facsimile)
Method 1600

iii September 2014

Table of Contents

Acknowledgments........................................................................................................................................... i

Disclaimer......................................................................................................................................................ii

1.0 Scope and Application....................................................................................................................... 1

2.0 Summary of Method.......................................................................................................................... 1

3.0 Definitions ........................................................................................................................................ 1

4.0 Interferences...................................................................................................................................... 2

5.0 Safety................................................................................................................................................ 2

6.0 Equipment and Supplies .................................................................................................................... 2

7.0 Reagents and Standards..................................................................................................................... 3

8.0 Sample Collection, Handling, and Storage.......................................................................................... 7

9.0 Quality Control.................................................................................................................................. 7

10.0 Calibration and Standardization ....................................................................................................... 12

11.0 Procedure........................................................................................................................................ 12

12.0 Verification Procedure..................................................................................................................... 13

13.0 Data Analysis and Calculations........................................................................................................ 14

14.0 Sample Spiking Procedure............................................................................................................... 14

15.0 Method Performance ....................................................................................................................... 18

16.0 Pollution Prevention ........................................................................................................................ 21

17.0 Waste Management......................................................................................................................... 21

18.0 References........................................................................................................................................22
Method 1600

September 2014 iv

List of Appendices

Appendices A and B are taken from Microbiological Methods for Monitoring the Environment, Water and 

Wastes (Reference 18.7).

Appendix A: Part II (General Operations), Section A (Sample Collection, Preservation, and Storage).

Appendix B: Part II (General Operations), Sections C.3.5 (Counting Colonies) and C.3.6 (Calculation of 

Results).
Method 1600

1 September 2014

Method 1600: Enterococci in Water by Membrane Filtration Using 

membrane-Enterococcus Indoxyl-β-D-Glucoside Agar (mEI)

September 2014

1.0 Scope and Application

1.1 Method 1600 describes a membrane filter (MF) procedure for the detection and enumeration of the 

enterococci bacteria in water. This is a single-step method that is a modification of EPA Method 1106.1 

(mE-EIA). Unlike the mE-EIA method, it does not require the transfer of the membrane filter to 

another medium. The modified medium has a reduced amount of triphenyltetrazolium chloride (TTC) 

and includes indoxyl β-D-glucoside, a chromogenic cellobiose analog used in place of esculin. In this 

procedure, β-glucosidase-positive enterococci produce an insoluble indigo blue complex which diffuses 

into the surrounding media, forming a blue halo around the colony.

1.2 Enterococci are commonly found in the feces of humans and other warm-blooded animals. Although 

some strains are ubiquitous and not related to fecal pollution, the presence of enterococci in water is an 

indication of fecal pollution and the possible presence of enteric pathogens.

1.3 Epidemiological studies have led to the development of criteria which can be used to promulgate 

recreational water standards based on established relationships between health effects and water quality. 

The significance of finding enterococci in recreational fresh or marine water samples is the direct 

relationship between the density of enterococci and the risk of gastrointestinal illness associated with 

swimming in the water (References 18.1 and 18.2).

1.4 For method application please refer to Title 40 Code of Federal Regulations Part 136 (40 CFR Part 136).

2.0 Summary of Method

2.1 Method 1600 provides a direct count of bacteria in water based on the development of colonies on the 

surface of the membrane filter (Reference 18.4). A water sample is filtered through the membrane 

which retains the bacteria. Following filtration, the membrane containing the bacterial cells is placed on 

a selective medium, mEI agar, and incubated for 24 hours at 41 oC ± 0.5 oC. All colonies greater than or 

equal to (>) 0.5 mm in diameter (regardless of color) with a blue halo are recorded as enterococci 

colonies. A fluorescent lamp with a magnifying lens is used for counting to give maximum visibility of 

colonies.

3.0 Definitions

3.1 In Method 1600, enterococci are those bacteria which produce colonies greater than or equal to 0.5 mm 

in diameter with a blue halo after incubation on mEI agar. The blue halo should not be included in the 

colony diameter measurement. Enterococci include Enterococcus faecalis (E. faecalis), E. faecium, E. 

avium, E. gallinarium, and their variants. The genus Enterococcus includes the enterococci formerly 

assigned to the Group D fecal streptococci. 
Method 1600

September 2014 2

4.0 Interferences

4.1 Water samples containing colloidal or suspended particulate materials can clog the membrane filter and 

prevent filtration, or cause spreading of bacterial colonies which could interfere with enumeration and 

identification of target colonies.

5.0 Safety

5.1 The analyst/technician must know and observe the normal safety procedures required in a microbiology 

laboratory while preparing, using, and disposing of cultures, reagents, and materials, and while operating 

sterilization equipment.

5.2 The selective medium (mEI) and azide-dextrose broth used in this method contain sodium azide as well 

as other potentially toxic components. Caution must be exercised during the preparation, use, and 

disposal of these media to prevent inhalation or contact with the medium or reagents. 

5.3 This method does not address all of the safety issues associated with its use. It is the responsibility of 

the laboratory to establish appropriate safety and health practices prior to use of this method. A 

reference file of material safety data sheets (MSDSs) should be available to all personnel involved in 

Method 1600 analyses.

5.4 Mouth-pipetting is prohibited.

6.0 Equipment and Supplies

6.1 Glass lens with magnification of 2-5X or stereoscopic microscope

6.2 Lamp, with a cool, white fluorescent tube

6.3 Hand tally or electronic counting device

6.4 Pipet container, stainless steel, aluminum or borosilicate glass, for glass pipets

6.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, of appropriate volume

6.6 Sterile graduated cylinders, 100-1000 mL, covered with aluminum foil or kraft paper

6.7 Sterile membrane filtration units (filter base and funnel), glass, plastic or stainless steel, wrapped with 

aluminum foil or kraft paper

6.8 Ultraviolet unit for sanitization of the filter funnel between filtrations (optional)

6.9 Line vacuum, electric vacuum pump, or aspirator for use as a vacuum source (In an emergency or in the 

field, a hand pump or a syringe equipped with a check valve to prevent the return flow of air, can be 

used)

6.10 Flask, filter, vacuum, usually 1 L, with appropriate tubing
Method 1600

3 September 2014

6.11 A filter manifold to hold a number of filter bases (optional)

6.12 Flask for safety trap placed between the filter flask and the vacuum source

6.13 Forceps, straight or curved, with smooth tips to handle filters without damage

6.14 Ethanol, methanol or isopropanol in a small, wide-mouth container, for flame-sterilizing forceps

6.15 Burner, Bunsen or Fisher type, or electric incinerator unit for sterilizing loops and needles

6.16 Thermometer, checked against a National Institute of Standards and Technology (NIST) certified 

thermometer, or one that meets the requirements of NIST Monograph SP 250-23

6.17 Petri dishes, sterile, plastic, 9 x 50 mm, with tight-fitting lids; or 15 x 60 mm with loose fitting lids; or 15 

x 100 mm with loose fitting lids 

6.18 Bottles, milk dilution, borosilicate glass, screw-cap with neoprene liners, 125 mL volume 

6.19 Flasks, borosilicate glass, screw-cap, 250-2000 mL volume

6.20 Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45 m pore size

6.21 Platinum wire inoculation loops, at least 3 mm diameter in suitable holders; or sterile plastic loops

6.22 Incubator maintained at 41oC ± 0.5oC

6.23 Water bath maintained at 50 oC for tempering agar

6.24 Test tubes, 20 x 150 mm, borosilicate glass or plastic

6.25 Caps, aluminum or autoclavable plastic, for 20 mm diameter test tubes

6.26 Test tubes, screw-cap, borosilicate glass, 16 x 125 mm or other appropriate size

6.27 Autoclave or steam sterilizer capable of achieving 121 oC [15 lb pressure per square inch (PSI)] for 15 

minutes

7.0 Reagents and Standards

7.1 Purity of Reagents: Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, 

reagents shall conform to the specifications of the Committee on Analytical Reagents of the American 

Chemical Society (Reference 18.5). The agar used in preparation of culture media must be of 

microbiological grade.

7.2 Whenever possible, use commercial culture media as a means of quality control.

7.3 Purity of reagent water: Reagent-grade water conforming to specifications in: Standard Methods for the 

Examination of Water and Wastewater (latest edition approved by EPA in 40 CFR Part 136 or 141, as 

applicable), Section 9020 (Reference 18.6).
Method 1600

September 2014 4

7.4 Phosphate buffered saline (PBS)

7.4.1 Composition:

Sodium dihydrogen phosphate (NaH2PO4) 0.58 g

Disodium hydrogen phosphate (Na2HPO4) 2.5 g

Sodium chloride (NaCl) 8.5 g

Reagent-grade water 1.0 L

7.4.2 Dissolve the reagents in 1 L of reagent-grade water and dispense in appropriate amounts for 

dilutions in screw cap bottles or culture tubes, and/or into containers for use as rinse water. 

Autoclave after preparation at 121 oC (15 PSI) for 15 min. Final pH should be 7.4 ± 0.2.

7.5 mEI Agar

7.5.1 Composition:

Peptone 10.0 g

Sodium chloride (NaCl) 15.0 g

Yeast extract 30.0 g

Esculin 1.0 g

Actidione (Cycloheximide) 0.05 g

Sodium azide 0.15 g

Indoxyl β-D-glucoside 0.75 g

Agar 15.0 g

Reagent-grade water 1.0 L

7.5.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve completely. 

Autoclave at 121 oC (15 PSI) for 15 minutes and cool in a 50 oC water bath. 

7.5.3 After sterilization add 0.24 g nalidixic acid (sodium salt) and 0.02 g triphenyltetrazolium 

chloride (TTC) to the mEI medium and mix thoroughly.

Note: The amount of TTC used in this medium (mEI) is less than the amount used for mE 

agar in Method 1106.1.

7.5.4 Dispense mEI agar into 9 × 50 mm or 15 × 60 mm petri dishes to a 4-5 mm depth 

(approximately 4-6 mL), and allow to solidify. Final pH of medium should be 7.1 ± 0.2. 

Store in a refrigerator.

7.6 Tryptic soy agar (TSA)

7.6.1 Composition:

Pancreatic digest of casein 15.0 g

Enzymatic digest of soybean meal 5.0 g

Sodium chloride (NaCl) 5.0 g

Agar 15.0 g

Reagent-grade water 1.0 L
Method 1600

5 September 2014

7.6.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve completely. 

Autoclave at 121 oC (15 PSI) for 15 minutes and cool in a 50 oC water bath. Pour the medium 

into each 15 × 60 mm culture dish to a 4-5 mm depth (approximately 4-6 mL), and allow to 

solidify. Final pH should be 7.3 ± 0.2.

7.7 Brain heart infusion broth (BHIB)

7.7.1 Composition:

Calf brains, infusion from 200.0 g 7.7 g

Beef heart, infusion from 250.0 g 9.8 g

Proteose peptone 10.0 g

Sodium chloride (NaCl) 5.0 g

Disodium hydrogen phosphate (Na2HPO4) 2.5 g

Dextrose 2.0 g

Reagent-grade water 1.0 L

7.7.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve completely. 

Dispense in 10-mL volumes in screw cap tubes, and autoclave at 121 oC (15 PSI) for 15 

minutes. Final pH should be 7.4 ± 0.2.

7.8 Brain heart infusion broth (BHIB) with 6.5% NaC1

7.8.1 Composition:

BHIB with 6.5% NaC1 is the same as BHIB above (Section 7.7), but with additional NaC1.

7.8.2 Add NaCl to formula provided in Section 7.7 above, such that the final concentration is 6.5% 

(65 g NaCl/L). Typically, for commercial BHIB media, an additional 60.0 g NaCl per liter of 

medium will need to be added to the medium. Prepare as in Section 7.7.2.

7.9 Brain heart infusion agar (BHIA)

7.9.1 Composition: 

BHIA contains the same components as BHIB (Section 7.7), with the addition of 15.0 g agar per 

liter of BHIB.

7.9.2 Add agar to formula for BHIB provided in Section 7.7 above. Prepare as in Section 7.7.2. 

After sterilization, slant until solid. Final pH should be 7.4 ± 0.2.
Method 1600

September 2014 6

7.10 Bile esculin agar (BEA)

7.10.1 Composition:

Beef Extract 3.0 g

Pancreatic Digest of Gelatin 5.0 g

Oxgall 20.0 g

Esculin 1.0 g

Ferric Citrate 0.5 g

Bacto Agar 14.0 g

Reagent-grade water 1.0 L

7.10.2 Add reagents to 1 L reagent-grade water, heat with frequent mixing, and boil 1 minute to 

dissolve completely. Dispense 10-mL volumes in tubes for slants or larger volumes into flasks 

for subsequent plating. Autoclave at 121 oC (15 PSI) for 15 minutes. Overheating may cause 

darkening of the medium. Cool in a 50 oC water bath, and dispense into sterile petri dishes. 

Final pH should be 6.8 ± 0.2. Store in a refrigerator.

7.11 Azide dextrose broth (ADB)

7.11.1 Composition:

Beef extract 4.5 g

Pancreatic digest of casein 7.5 g

Proteose peptone No. 3 7.5 g

Dextrose 7.5 g

Sodium chloride (NaCl) 7.5 g

Sodium azide 0.2 g

Reagent-grade water 1.0 L

7.11.2 Add reagents to 1 L of reagent-grade water and dispense in screw cap bottles. Autoclave at 

121 oC (15 PSI) for 15 minutes. Final pH should be 7.2 ± 0.2.

7.12 Control cultures

7.12.1 Positive control and/or spiking organism (either of the following are acceptable)

 Stock cultures of Enterococcus faecalis (E. faecalis) ATCC 19433

 E. faecalis ATCC 19433 BioBalls (bioMerieux, Durham NC) 

7.12.2 Negative control organism (for each organism, either of the following are acceptable)

 Stock cultures of Escherichia coli (E. coli) ATCC 11775

 E. coli ATCC 11775 BioBalls (bioMerieux, Durham NC)

 Stock cultures of Enterobacter aerogenes (E. aerogenes) ATCC 13048

 E. aerogenes NCTC 10006 BioBalls (bioMerieux, Durham NC)
Method 1600

7 September 2014

8.0 Sample Collection, Handling, and Storage

8.1 Sampling procedures are briefly described below. Detailed sampling methods can be found in 

Reference 18.7 (see Appendix A). Adherence to sample preservation procedures and holding time 

limits is critical to the production of valid data. Samples not collected according to these rules should 

not be analyzed.

8.1.1 Sampling techniques

Samples are collected by hand or with a sampling device if the sampling site has difficult access 

such as a dock, bridge, or bank adjacent to a surface water. Composite samples should not be 

collected, since such samples do not display the range of values found in individual samples. 

The sampling depth for surface water samples should be 6-12 inches below the water surface. 

Sample containers should be positioned such that the mouth of the container is pointed away 

from the sampler or sample point. After removal of the container from the water, a small 

portion of the sample should be discarded to allow for proper mixing before analyses.

8.1.2 Storage temperature and handling conditions

Ice or refrigerate water samples at a temperature of <10 oC during transit to the laboratory. Do 

not freeze the samples. Use insulated containers to assure proper maintenance of storage 

temperature. Take care that sample bottles are not totally immersed in water during transit or 

storage.

8.1.3 Holding time limitations

Sample analysis should begin immediately, preferably within 2 hours of collection. The 

maximum transport time to the laboratory is 6 hours, and samples should be processed within 2 

hours of receipt at the laboratory.

9.0 Quality Control

9.1 Each laboratory that uses Method 1600 is required to operate a formal quality assurance (QA)

program that addresses and documents instrument and equipment maintenance and performance, 

reagent quality and performance, analyst training and certification, and records storage and retrieval. 

Additional recommendations for QA and quality control (QC) procedures for microbiological 

laboratories are provided in Reference 18.7.

9.2 The minimum analytical QC requirements for the analysis of samples using Method 1600 include

an initial demonstration of laboratory capability through performance of the initial precision and 

recovery (IPR) analyses (Section 9.3), ongoing demonstration of laboratory capability through 

performance of the ongoing precision and recovery (OPR) analysis (Section 9.4) and matrix spike (MS) 

analysis (Section 9.5, disinfected wastewater only), and the routine analysis of positive and negative 

controls (Section 9.6), filter sterility checks (Section 9.8), method blanks (Section 9.9), and media 

sterility checks (Section 9.11). For the IPR, OPR and MS analyses, it is necessary to spike samples with 

either laboratory-prepared spiking suspensions or BioBalls as described in Section 14.

Note: Performance criteria for Method 1600 are based on the results of the interlaboratory validation 

of Method 1600 in PBS and disinfected wastewater matrices. The IPR (Section 9.3) and OPR 
Method 1600

September 2014 8

(Section 9.4) recovery criteria (Table 1) are valid method performance criteria that should be 

met, regardless of the matrix being evaluated, the matrix spike recovery criteria (Section 9.5, 

Table 2) pertain only to disinfected wastewaters.

9.3 Initial precision and recovery (IPR)—The IPR analyses are used to demonstrate acceptable method 

performance (recovery and precision) and should be performed by each laboratory before the method is 

used for monitoring field samples. EPA recommends but does not require that an IPR be performed by 

each analyst. IPR samples should be accompanied by an acceptable method blank (Section 9.9) and

appropriate media sterility checks (Section 9.11). The IPR analyses are performed as follows:

9.3.1 Prepare four, 100-mL samples of PBS and spike each sample with E. faecalis ATCC 19433 

according to the spiking procedure in Section 14. Spiking with laboratory-prepared 

suspensions is described in Section 14.2 and spiking with BioBalls is described in Section 14.3. 

Filter and process each IPR sample according to the procedures in Section 11 and calculate the 

number of enterococci per 100 mL according to Section 13.

9.3.2 Calculate the percent recovery (R) for each IPR sample using the appropriate equation in 

Section 14.2.2 or 14.3.4 for samples spiked with laboratory-prepared spiking suspensions or 

BioBalls, respectively.

9.3.3 Using the percent recoveries of the four analyses, calculate the mean percent recovery and the 

relative standard deviation (RSD) of the recoveries. The RSD is the standard deviation divided 

by the mean, multiplied by 100.

9.3.4 Compare the mean recovery and RSD with the corresponding IPR criteria in Table 1, below. If 

the mean and RSD for recovery of enterococci meet acceptance criteria, system performance is 

acceptable and analysis of field samples may begin. If the mean recovery or the RSD fall 

outside of the required range for recovery, system performance is unacceptable. In this event, 

identify the problem by evaluating each step of the analytical process, media, reagents, and 

controls, correct the problem and repeat the IPR analyses.

Table 1. Initial and Ongoing Precision and Recovery (IPR and OPR) Acceptance Criteria

Performance test Lab-prepared spike 

acceptance criteria

BioBallTM 

acceptance criteria

Initial precision and recovery (IPR)

Mean percent recovery 

Precision (as maximum relative standard deviation)

31% - 127%

28%

85% - 106%

14%

Ongoing precision and recovery (OPR) as percent recovery 27% - 131% 78% - 113%

9.4 Ongoing precision and recovery (OPR)—To demonstrate ongoing control of the analytical system, the 

laboratory should routinely process and analyze spiked PBS samples. The laboratory should analyze 

one OPR sample after every 20 field and matrix spike samples or one per week that samples are 

analyzed, whichever occurs more frequently. OPR samples must be accompanied by an acceptable 

method blank (Section 9.9) and appropriate media sterility checks (Section 9.11). The OPR analysis is 

performed as follows:

9.4.1 Spike a 100-mL PBS sample with E. faecalis ATCC 19433 according to the spiking procedure 

in Section 14. Spiking with laboratory-prepared suspensions is described in Section 14.2 and 

spiking with BioBalls is described in Section 14.3. Filter and process each OPR sample 
Method 1600

9 September 2014

according to the procedures in Section 11 and calculate the number of enterococci per 100 mL 

according to Section 13.

9.4.2 Calculate the percent recovery (R) for the OPR sample using the appropriate equation in Section

14.2.2 or 14.3.4 for samples spiked with laboratory-prepared spiking suspensions or BioBalls, 

respectively.

9.4.3 Compare the OPR result (percent recovery) with the corresponding OPR recovery criteria in 

Table 1, above. If the OPR result meets the acceptance criteria for recovery, method 

performance is acceptable and analysis of field samples may continue. If the OPR result falls 

outside of the acceptance criteria, system performance is unacceptable. In this event, identify 

the problem by evaluating each step of the analytical process, media, reagents, and controls, 

correct the problem and repeat the OPR analysis.

9.4.4 As part of the laboratory QA program, results for OPR and IPR samples should be charted and 

updated records maintained in order to monitor ongoing method performance. The laboratory 

should also develop a statement of accuracy for Method 1600 by calculating the average percent 

recovery (R) and the standard deviation of the percent recovery (sr). Express the accuracy as a 

recovery interval from R - 2sr to R + 2sr.

9.5 Matrix spikes (MS)—MS analysis are performed to determine the effect of a particular matrix on 

enterococci recoveries. The laboratory should analyze one MS sample when disinfected wastewater 

samples are first received from a source from which the laboratory has not previously analyzed samples. 

Subsequently, 5% of field samples (1 per 20) from a given disinfected wastewater source should include 

a MS sample. MS samples must be accompanied by the analysis of an unspiked field sample 

sequentially collected from the same sampling site, an acceptable method blank (Section 9.9), and 

appropriate media sterility checks (Section 9.11). When possible, MS analyses should also be 

accompanied by an OPR sample (Section 9.4), using the same spiking procedure (laboratory-prepared 

spiking suspension or BioBalls). The MS analysis is performed as follows:

9.5.1 Prepare two, 100-mL field samples that were sequentially collected from the same site. One 

sample will remain unspiked and will be analyzed to determine the background or ambient 

concentration of enterococci for calculating MS recoveries (Section 9.5.3). The other sample 

will serve as the MS sample and will be spiked with E. faecalis ATCC 19433 according to the 

spiking procedure in Section 14.

9.5.2 Select sample volumes based on previous analytical results or anticipated levels of in the field 

sample in order to achieve the recommended target range of enterococci (20-60 CFU, including 

spike) per filter. If the laboratory is not familiar with the matrix being analyzed, it is 

recommended that a minimum of three dilutions be analyzed to ensure that a countable plate is 

obtained for the MS and associated unspiked sample. If possible, 100-mL of sample should be 

analyzed. 

9.5.3 Spike the MS sample volume(s) with a laboratory-prepared suspension as described in Section 

14.2 or with BioBalls as described in Section 14.3. Immediately filter and process the 

unspiked and spiked field samples according to the procedures in Section 11.

Note: When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS should be 

added to the funnel or an aliquot of sample should be dispensed into a 20-30 mL 
Method 1600

September 2014 10

dilution blank prior to filtration. This will allow even distribution of the sample on the 

membrane.

9.5.4 For the MS sample, calculate the number of enterococci (CFU/100 mL) according to Section 13 

and adjust the colony counts based on any background enterococci observed in the unspiked 

matrix sample.

9.5.5 Calculate the percent recovery (R) for the MS sample (adjusted based on ambient enterococci in 

the unspiked sample) using the appropriate equation in Section 14.2.2 or 14.3.4 for samples 

spiked with laboratory-prepared spiking suspensions or BioBalls, respectively.

9.5.6 Compare the MS result (percent recovery) with the appropriate method performance criteria in 

Table 2, below. If the MS recovery meets the acceptance criteria, system performance is 

acceptable and analysis of field samples from this disinfected wastewater source may continue. 

If the MS recovery is unacceptable and the OPR sample result associated with this batch of 

samples is acceptable, a matrix interference may be causing the poor results. If the MS 

recovery is unacceptable, all associated field data should be flagged.

9.5.7 Acceptance criteria for MS recovery (Table 2) are based on data from spiked disinfected

wastewater matrices and are not appropriate for use with other matrices (e.g., ambient waters).

Table 2. Matrix Spike Precision and Recovery Acceptance Criteria

Performance test Lab-prepared acceptance criteria BioBallTM acceptance criteria

Percent recovery for MS 29% - 122% 63% - 110%

9.5.8 Laboratories should record and maintain a control chart comparing MS recoveries for all

matrices to batch-specific and cumulative OPR sample results analyzed using Method 1600. 

These comparisons should help laboratories recognize matrix effects on method recovery and 

may also help to recognize inconsistent or sporadic matrix effects from a particular source. 

9.6 Culture Controls

9.6.1 Negative controls—The laboratory should analyze negative controls to ensure that the mEI 

agar is performing properly. Negative controls should be analyzed whenever a new batch of 

media or reagents is used. On an ongoing basis, the laboratory should perform a negative 

control every day that samples are analyzed.

9.6.1.1 Negative controls are conducted by filtering a dilute suspension of viable E. coli

(e.g., ATCC 11775) or E. aerogenes (e.g., ATCC 13048) and analyzing as described 

in Section 11. Viability of the negative controls should be demonstrated using a 

non-selective media (e.g., nutrient agar or tryptic soy agar).

9.6.1.2 If the negative control fails to exhibit the appropriate response, check and/or replace 

the associated media or reagents, and/or the negative control, and reanalyze the 

appropriate negative control.

9.6.2 Positive controls—The laboratory should analyze positive controls to ensure that the mEI agar 

is performing properly. Positive controls should be analyzed whenever a new batch of media 

or reagents is used. On an ongoing basis, the laboratory should perform a positive control 
Method 1600

11 September 2014

every day that samples are analyzed. An OPR sample (Section 9.4) may take the place of a 

positive control.

9.6.2.1 Positive controls are conducted by filtering a dilute suspension of viable E. faecalis

(e.g., ATCC 19433) and analyzing as described in Section 11. 

9.6.2.2 If the positive control fails to exhibit the appropriate response, check and/or replace 

the associated media or reagents, and/or the positive control, and reanalyze the 

appropriate positive control. 

9.6.3 Controls for verification media—All verification media should be tested with appropriate 

positive and negative controls whenever a new batch of media and/or reagents are used. On an 

ongoing basis, the laboratory should perform positive and negative controls on the verification 

media with each batch of samples submitted to verification. Examples of appropriate controls 

for verification media are provided in Table 3.

Table 3. Verification Controls

Medium Positive Control Negative Control

Bile esculin agar (BEA) E. faecalis E. coli

Brain heart infusion broth (BHIB) with 6.5% NaCl E. faecalis E. coli

Brain heart infusion broth (BHIB) incubated at 45 oC E. faecalis E. aerogenes

9.7 Colony verification—The laboratory should verify 10 typical colonies (positive) and 10 atypical

colonies (negative) per month or 1 typical colony and 1 atypical colony from 10% of all positive 

samples, whichever is greater. Verification procedures are provided in Section 12.0.

9.8 Filter sterility check—Place at least one membrane filter per lot of filters on a TSA plate, and incubate 

for 24 ± 2 hours at 35 oC ± 0.5 oC. Absence of growth indicates sterility of the filter. On an ongoing 

basis, the laboratory should perform a filter sterility check every day that samples are analyzed.

9.9 Method blank—Filter a 50-mL volume of sterile PBS and place the filter on a mEI agar plate and 

process according to Section 11.0. Absence of growth indicates freedom of contamination from the 

target organism. On an ongoing basis, the laboratory should perform a method blank every day that 

samples are analyzed.

9.10 Filtration blank—Filter a 50-mL volume of sterile PBS before beginning sample filtrations.

Place the filter on a TSA plate, and incubate for 24 ± 2 hours at 35 oC ± 0.5 oC. Absence of growth 

indicates sterility of the PBS buffer and filtration assembly. 

9.11 Media sterility check—The laboratory should test media sterility by incubating one unit (tube or plate) 

from each batch of medium (TSA, mEI agar, and verification media) as appropriate and observing for 

growth. Absence of growth indicates media sterility. On an ongoing basis, the laboratory should 

perform a media sterility check every day that samples are analyzed.

9.12 Analyst colony counting variability—Laboratories with two or more analysts should compare each 

analyst’s colony counts from one positive field sample per month. Colony counts should be within 10% 

between analysts. Laboratories with a single analyst should have that analyst perform duplicate colony 

counts of a single membrane filter each month. Duplicate colony counts should be within 5% for a 
Method 1600

September 2014 12

single analyst. If no positive field samples are available, an OPR sample may be substituted for these 

determinations.

10.0 Calibration and Standardization

10.1 Check temperatures in incubators twice daily with a minimum of 4 hours between each reading to ensure 

operation within stated limits.

10.2 Check thermometers at least annually against a NIST certified thermometer or one that meets the 

requirements of NIST Monograph SP 250-23. Check mercury columns for breaks.

10.3 Refrigerators used to store media and reagents should be monitored daily to ensure proper temperature 

control.

11.0 Procedure

11.1 Prepare the mEI agar as directed in Section 7.5.

11.2 Mark the petri dishes and report forms with sample identification and sample volumes.

11.3 Place a sterile membrane filter on the filter base, grid-side up and attach the funnel to the base so that the 

membrane filter is now held between the funnel and the base.

11.4 Shake the sample bottle vigorously about 25 times to distribute the bacteria uniformly, and measure the 

desired volume of sample or dilution into the funnel.

11.5 Select sample volumes based on previous knowledge of the enterococci level, to produce 20-60 

enterococci colonies on membranes. It is recommended that a minimum of three dilutions be analyzed 

to ensure that a countable plate (20-60 enterococci colonies) is obtained. 

11.6 Smaller sample size or sample dilutions can be used to minimize the interference of turbidity or for high 

bacterial densities. Multiple volumes of the same sample or sample dilutions may be filtered.

Note: When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS or 

phosphate-buffered dilution water should be added to the funnel or an aliquot of sample should 

be dispensed into a dilution blank prior to filtration. This will allow even distribution of the 

sample on the membrane.

11.7 Filter the sample, and rinse the sides of the funnel at least twice with 20-30 mL of sterile buffered rinse 

water. Turn off the vacuum and remove the funnel from the filter base.

11.8 Use sterile forceps to aseptically remove the membrane filter from the filter base, and roll it onto the mEI 

Agar to avoid the formation of bubbles between the membrane and the agar surface. Reseat the 

membrane if bubbles occur. Run the forceps around the edge of the filter outside the area of filtration, 

close to the edge of the dish, to be sure that the filter is properly seated on the agar. Close the dish, 

invert, and incubate at 41oC ± 0.5 oC for 24 ± 2 hours. 
Method 1600

13 September 2014

Note: If the medium is prepared in 15 × 60 mm loose lid petri dishes, they should be incubated in a 

tight fitting container (e.g., plastic vegetable crisper) containing a moistened paper towel to 

prevent dehydration of the membrane filter and medium.

11.9 After incubation, count and record colonies on those membrane filters containing, if practical, 20-60 

colonies ≥0.5 mm in diameter with a blue halo regardless of colony color as an enterococci (see Figure 

1). 

Note: When measuring colony size do not include the halo. Use magnification for counting and a 

small fluorescent lamp to give maximum visibility of colonies. 

12.0 Verification Procedure

12.1 Colonies ≥0.5 mm in diameter of any color having a blue halo after incubation on mEI agar are 

considered to be “typical” enterococci colonies. Verification of colonies may be required in evidence 

gathering and it is also recommended as a means of quality control. The verification procedure follows. 

Note: When evaluating wastewater using Method 1600, it is recommended that the false negative rate 

for each matrix be evaluated through biochemical confirmation and results adjusted 

accordingly, especially if large numbers of atypical colonies are observed in a particular matrix.

12.2 Using a sterile inoculating loop or needle, transfer growth from the centers of at least 10 well-isolated 

typical and at least 10 well-isolated atypical colonies into a BHIB tube and onto a BHIA slant. Incubate 

broth for 24 ± 2 hours and agar slants for 48 ± 3 hours at 35 oC ± 0.5 oC.

12.3 After a 24 hour incubation, transfer a loopful of growth from each BHIB tube to BEA, BHIB, and BHIB 

with 6.5% NaCl.

12.3.1 Incubate BEA and BHIB with 6.5% NaCl at 35 oC ± 0.5 oC for 48 ± 3 hours.

12.3.2 Incubate BHIB at 45 oC ± 0.5 oC for 48 ± 3 hours.

12.4 Observe all verification media for growth.

12.5 After 48 hour incubation, perform a Gram stain using growth from each BHIA slant.

Figure 1. Enterococci colonies on 

mEI produce blue halos.
Method 1600

September 2014 14

12.6 Gram-positive cocci that grow and hydrolyze esculin on BEA (i.e., produce a black or brown 

precipitate), and grow in BHIB with 6.5% NaCl at 35 oC± 0.5 oC and BHIB at 45 oC± 0.5 oC are verified 

as enterococci.

12.7 Alternately, commercially available multi-test identification systems (e.g., Vitek®) may be used to 

verify colonies. Such multi-test identification systems should include esculin hydrolysis and growth in 

6.5% NaCl.

13.0 Data Analysis and Calculations

Use the following general rules to calculate the enterococci count per 100 mL of sample:

13.1 If possible, select a membrane filter with 20-60 colonies ≥0.5 mm in diameter (regardless of colony 

color) with a blue halo. Calculate the number of enterococci per 100 mL according to the following 

general formula:

Enterococci/100 mL =

Number of enterococci colonies

× 100

Volume of sample filtered (mL)

13.2 See general counting rules in Reference 18.7 (see Appendix B).

13.3 Report results as enterococci per 100 mL of sample. 

14.0 Sample Spiking Procedure

14.1 Method 1600 QC requirements (Section 9.0) include the preparation and analysis of spiked reference 

(PBS) and matrix samples in order to monitor initial and ongoing method performance. For the IPR 

(Section 9.3), OPR (Section 9.4), and MS (Section 9.5) analyses it is necessary to spike samples with 

either laboratory-prepared spiking suspensions (Section 14.2) or BioBalls (Section 14.3) as described 

below.

14.2 Laboratory-Prepared Spiking Suspensions

14.2.1 Preparation

14.2.1.1 Stock Culture. Prepare a stock culture by inoculating a TSA slant (or other

non-selective media) with E. faecalis ATCC 19433 and incubating at 35 oC ± 3 oC

for 20 ± 4 hours. This stock culture may be stored in the dark at room temperature 

for up to 30 days.

14.2.1.2 Undiluted Spiking Suspension. Prepare a 1% solution of azide dextrose broth 

(ADB) by combining 99 mL of sterile phosphate buffered saline and 1 mL of sterile 

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

dilution water container. From the stock culture of E. faecalis ATCC 19433 in 

Section 14.2.1.1, transfer a small loopful of growth to the 1 % azide dextrose broth 

solution and vigorously shake a minimum of 25 times. Disperse the inoculum by 

vigorously shaking the broth culture and incubate at 35 oC ± 3 oC for 20 ± 4 hours. 

This culture is referred to as the undiluted spiking suspension and should contain 
Method 1600

15 September 2014

approximately 1.0 × 106- 1.0 × 107 E. faecalis colony forming units (CFU) per mL 

of culture.

14.2.1.3 Mix the undiluted spiking suspension (Section 14.2.1.2) thoroughly by shaking the 

bottle a minimum of 25 times and prepare a series of dilutions (4 total) in the 

following manner:

14.2.1.3.1 Dilution “A”—Aseptically transfer 1.0 mL of the undiluted spiking 

suspension to 99 mL of sterile PBS and mix thoroughly by shaking the 

bottle a minimum of 25 times. This is spiking suspension dilution “A”

and 1 mL contains 10-2 mL of the original undiluted spiking suspension.

14.2.1.3.2 Dilution “B”—Aseptically transfer 1.0 mL of dilution “A” to 99 mL of 

sterile PBS and mix thoroughly by shaking the bottle a minimum of 25 

times. This is spiking suspension dilution “B” and 1 mL contains 10-4

mL of the original undiluted spiking suspension.

14.2.1.3.3 Dilution “C”—Aseptically transfer 11.0 mL of dilution “B” to 99 mL of 

sterile PBS and mix thoroughly by shaking the bottle a minimum of 25 

times. This is spiking suspension dilution “C” and 1 mL contains 10-5

mL of the original undiluted spiking suspension.

14.2.1.3.4 Dilution “D”—Aseptically transfer 11.0 mL of dilution “C” to 99 mL 

of sterile PBS and mix thoroughly by shaking the bottle a minimum of 

25 times. This is spiking suspension dilution “D” and 1 mL contains 

10-6 mL of the original undiluted spiking suspension. 

14.2.2 Sample spiking

14.2.2.1 Add 3.0 mL of the spiking suspension dilution “D” (Section 14.2.1.3.4) to 100 mL or 

PBS or appropriate volume of sample and mix thoroughly by shaking the bottle a 

minimum of 25 times. The volume of undiluted spiking suspension added to each 

100 mL sample is 3.0 x 10-6 mL, which is referred to as Vspiked per 100 mL sample in Section 

14.2.4.1 below. Filter the spiked sample and analyze the filter according to the 

procedures in Section 11. 

14.2.3 Enumeration of spiking suspension

14.2.3.1 Prepare TSA spread plates, in triplicate, for spiking suspension dilutions “B”, “C”, 

and “D”. 

Note: Agar plates must be dry 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 using a laminar-flow hood.

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

dilution “B” onto the surface of each TSA plate in triplicate.

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

dilution “C” onto the surface of each TSA plate in triplicate.
Method 1600

September 2014 16

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

dilution “D” onto the surface of each TSA plate in triplicate.

14.2.3.5 Use a sterile bent glass rod or spreader to distribute the inoculum over the surface of 

plates by rotating the dish by hand or on a turntable. 

Note: Ensure that the inoculum is evenly distributed over the entire surface of the 

plate.

14.2.3.6 Allow the inoculum to absorb into the medium of each plate completely. Invert 

plates and incubate at 35 oC ± 0.5 oC for 20 ± 4 hours. 

14.2.3.7 Count and record number of colonies per plate. The number of enterococci 

(CFU/mL) in the undiluted spiking suspension will be calculated using all TSA 

plates yielding counts within the countable range of 30 to 300 CFU per plate.

14.2.4 Recovery calculations for spiked samples

14.2.4.1 Calculate the concentration of enterococci (CFU/mL) in the undiluted spiking 

suspension (Section 14.2.1.2) according to the following equation. Example 

calculations are provided in Table 4, below.

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

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

Where,

CFU = Number of colony forming units from TSA plates yielding counts within the

countable range of 30 to 300 CFU per plate

V = Volume of undiluted sample on each TSA plate yielding counts within the countable 

range of 30 to 300 CFU per plate

n = Number of plates with counts within the countable range of 30 to 300 CFU per plate

Note: The example calculated numbers provided in the tables below have been rounded at 

the end of each step for simplification purposes. Generally, rounding should only 

occur after the final calculation.
Method 1600

17 September 2014

Table 4. Example Calculations of Laboratory-prepared Enterococci Spiking Concentration

Examples

CFU/plate (triplicate analyses) from 

TSA plates

Enterococci CFU/mL in undiluted 

spiking suspension

(Enterococci undiluted spike)

a

10-5 mL plates 10-6 mL plates 10-7 mL plates 

Example 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

Example 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

a Enterococci undiluted spike is calculated using all plates yielding counts within the countable range of 30 to 300 CFU per plate

14.2.4.2 Calculate true concentration of spiked enterococci (CFU/100 mL) according to the 

following equation. Example calculations are provided in Table 5, 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

V spiked per 100 mL sample = mL of undiluted spiking suspension per 100 mL sample

Table 5. Example Calculations for Determination “True” Spiked Enterococci Concentration

Enterococci undiluted spike V spiked per 100 mL sample Tspiked Enterococci

9.6 x 106 CFU/mL 3.0 X 10-6 mL per 100 mL of 

sample

(9.6 x 106 CFU/mL) x (3.0 x 10-6 mL/100 mL) =

28.8 CFU/100 mL

5.3 x 106 CFU/mL 3.0 X 10-6 mL per 100 mL of 

sample

(2.8 x 106 CFU/mL) x (3.0 x 10-6 mL/100 mL) = 

8.4 CFU/100 mL

14.2.4.2 Calculate percent recovery (R) ofspiked enterococci (CFU/100 mL) according to the 

following equation. Example calculations are provided in Table 6, below.

R = 100 x 

(Ns - Nu)

T

Where,

R = Percent recovery

Ns = Enterococci (CFU/100 mL) in the spiked sample (Section 13)

Nu = Enterococci (CFU/100 mL) in the unspiked sample (Section 13)

T = True spiked enterococci (CFU/100 mL) in spiked sample (Section 14.2.4.1)
Method 1600

September 2014 18

Table 6. Example Percent Recovery Calculations for Lab-prepared Spiked Samples

Ns (CFU/100 mL) Nu (CFU/100 mL) Tspiked Enterococci (CFU/100 mL) Percent recovery (R)

42 <1 28.8

100 x (42 - 1)/28.8 

= 142%

34 10 28.8

100 x (34 - 10)/28.8

= 83%

10 <1 8.4

100 x (10 - 1)/8.4

= 107%

14.3 BioBallTM Spiking Procedure

14.3.1 Aseptically add 1 BioBallTM to 100 mL of PBS or appropriate volume of sample and mix by 

vigorously shaking the sample bottle a minimum of 25 times. Analyze the spiked sample 

according to the procedures in Section 11.

14.3.2 Recovery calculations for samples spiked with BioBalls—Calculate percent recovery (R) of 

spiked enterococci (CFU/100 mL) according to the following equation. Example calculations 

are provided in Table 7, below.

R = 100 x 

(Ns - Nu)

T

Where,

R = Percent recovery

Ns = Enterococci (CFU/100 mL) in the spiked sample (Section 13)

Nu = Enterococci (CFU/100 mL) in the unspiked sample (Section 13)

T = True spiked enterococci (CFU/100 mL) in spiked sample based on the lot mean 

value provided by manufacturer

Table 7. Example BioBallTM Percent Recovery Calculations

Ns (CFU/100 mL) Nu (CFU/100 mL) T (CFU/100 mL) Percent recovery (R)

24 <1 11.2 100 × (24 - 1)/32 = 72%

36 10 32 100 × (36 - 10)/32 = 81%

15.0 Method Performance

15.1 Performance Characteristics (Reference 18.4)

15.1.1 Precision - The degree of agreement of repeated measurements of the same parameter expressed 

quantitatively as the standard deviation or as the 95% confidence limits of the mean computed 

from the results of a series of controlled determinations. The precision among laboratories for 

marine water and surface water was 2.2% and 18.9%, respectively.

15.1.2 Bias - The persistent positive or negative deviation of the results from the assumed or accepted 

true value. The persistent positive or negative deviation of the results from the assumed or 

accepted true value was not significant.
Method 1600

19 September 2014

15.1.3 Specificity - The ability of a method to select and/or distinguish the target bacteria from other 

bacteria in the same water sample. The specificity characteristic of a method is usually 

reported as the percent of false positive and false negative results. The specificity for this 

medium as reported for various environmental water samples was 6.0% false positive and 6.5% 

false negative.

15.1.4 Multilaboratory variability - A collaborative study was conducted among fourteen collaborators 

at twelve laboratories to examine the interlaboratory reproducibility of the method. 

Reproducibility among laboratories (RSDR) for freshwater, marine water, chlorinated 

secondary effluent, and non-chlorinated primary effluent ranged from 2.2% for marine water to 

18.9% for freshwater with a low enterococcal density.

15.2 Interlaboratory Validation of Method 1600 in Disinfected Wastewater (Reference 18.3)

15.2.1 Twelve volunteer participant laboratories, two enterococci verification laboratories, and two 

research laboratories participated in the U.S. Environmental Protection Agency’s (EPA’s) 

interlaboratory validation study of EPA Method 1600. The purposes of the study were to 

characterize method performance across multiple laboratories and disinfected wastewater 

matrices and to develop quantitative quality control (QC) acceptance criteria. A detailed 

description of the study and results are provided in the validation study report (Reference 18.3). 

Results submitted by laboratories were validated using a standardized data review process to 

confirm that results were generated in accordance with study-specific instructions and the 

September 2002 version of EPA Method 1600.

15.2.2 Recovery - Method 1600 was characterized by mean laboratory-specific recoveries of 

enterococci from disinfected wastewater samples spiked with BioBallsTM ranging from 77.1% 

to 114.9%, with an overall mean recovery of 90.8%. Mean laboratory-specific recoveries of 

enterococci from PBS samples spiked with BioBallsTM ranged from 88.0% to 105.1%, with an 

overall mean recovery of 95.4%.

15.2.3 Precision - Method 1600 was characterized by laboratory-specific relative standard deviations 

(RSDs) from disinfected wastewater samples spiked with BioBallsTM ranging from 0% to 

69.5%, with an overall pooled, within-laboratory RSD of 22.6%. For PBS samples spiked 

with BioBalls, laboratory-specific RSDs ranged from 3.1% to 13.7%, with an overall pooled, 

within-laboratory RSD of 8.1%. 

15.2.4 False positive confirmation rates - Method 1600 laboratory-specific false positive confirmation 

rates for unspiked disinfected/secondary results combined, ranging from 0.0% to 10.0%. For 

secondary wastewater (excluding disinfected results), only 2 of 123 typical colonies submitted 

to verification were non-enterococci, resulting in a false positive confirmation rate of 1.6%. 

For disinfected wastewater (excluding secondary results), none of the 66 typical colonies 

submitted to verification were non-enterococci, resulting in a false positive confirmation rate of 

0.0%. Since all 2184 typical colonies observed during the study could not be submitted to 

confirmation, the percent of total colonies that would have resulted in a false positive result was 

estimated (see Table 6, Reference 18.3). It is estimated that 0.0% and 1.2% of the total 

colonies would have resulted in a false positive for disinfected wastewater and secondary 

wastewater, respectively.

15.2.5 False negative rates - Method 1600 laboratory-specific false negative rates laboratory-specific 

false negative confirmation rates for unspiked disinfected/secondary results combined, ranged 

from 28.6% to 100.0%. For secondary wastewater (excluding disinfected results), 62 of 79 
Method 1600

September 2014 20

atypical colonies submitted to verification were identified as enterococci, resulting in a false 

negative confirmation rate of 78.5% for secondary wastewater. For disinfected wastewater 

(excluding secondary results), eight of eight atypical colonies submitted to verification were 

identified as enterococci, resulting in a false negative confirmation rate of 100.0% for 

disinfected wastewater. Since all 839 atypical colonies observed during the study could not be 

submitted to confirmation, the percent of total colonies that would have resulted in a false 

negative result was estimated. It is estimated that 21.2% and 22.8% of the total colonies would 

have resulted in a false negative for disinfected wastewater and secondary wastewater, 

respectively. The false positive and negative assessments are provided in Table 8. 

Table 8. False Positive and False Negative Assessment for Unspiked Disinfected and Unspiked 

Secondary Wastewater Effluents

Matrix 

Total colonies False positive (FP) assessment False negative (FN) assessment

Typical Atypical

Typical 

colonies 

submitted

No. FP 

colonies

FP 

confirmation 

rate (%) a

Estimated 

% of total 

colonies 

that would 

have been 

a FP b

Atypical 

colonies 

submitted

No. FN 

colonies

FN 

confirmation 

rate (%) c

Estimated % 

of total 

colonies that 

would have 

been a FN d

Disinfected 391 105 66 0 0.0 0.0 8 8 100.0 21.2

Secondary 1793 734 123 2 1.6 1.2 79 62 78.5 22.8

Disinfected + 

Secondary

2184 839 189 2 1.1 0.8 87 70 80.5 22.3

a False positive confirmation rate = number of false positive colonies/number of typical colonies submitted 

b Percent of total colonies estimated to be false positives = [(total typical colonies FP confirmation rate)/(total number of 

typical and atypical colonies observed); e.g., [(1793 ×(2/123))/(1793+734)] × 100 = 1.2%

c False negative confirmation rate = number of false negative colonies/number of atypical colonies submitted 

d Percent of total colonies estimated to be false negatives = [(total atypical colonies* FN confirmation rate)/(total number of 

typical and atypical colonies observed)] × 100; e.g., [(734 ×(62/79))/(1793+734)] × 100 = 22.8%

15.2.6 During evaluation of the study results, it was noted that many of the false negatives (atypical 

colonies submitted to verification which were identified as enterococci) were pink to red in 

color but simply lacked a blue halo. The predecessor to EPA Method 1600 for enterococci is 

EPA Method 1106.1 which uses mE and EIA media. For EPA Method 1106.1, pink to red 

colonies on mE, which produce a brown precipitate after transfer to EIA are considered positive 

for enterococci. Tetrazolium chloride (TTC), the reagent responsible for producing pink to red 

enterococci colonies on mE, is also included as a reagent in mEI. A follow-on study was 

conducted, for which pink to red colonies without halos from unspiked secondary wastewaters 

were submitted to verification. For pink to red colonies without halos that were ≥0.5 mm 

colony size, 54 of 90 colonies submitted were identified as enterococci, resulting in a 60.0% 

verification rate. 

Results of the verification analyses from the initial study were assessed with pink to red colonies 

without halos being counted as enterococci. When pink to red colonies without halos are 
Method 1600

21 September 2014

counted as enterococci, the estimated percent of total colonies that would have resulted in false 

positives increases slightly from 0.8% to 2.7%, for combined disinfected and secondary results. 

More importantly, the estimated percent of total colonies that would have resulted in false 

negatives decreased from 22.3% to 7.0% for combined disinfected and secondary results and 

from 21.2% to 2.9% for disinfected wastewater. The re-assessment of false positive and false 

negative initial study results with pink to red colonies without halos counted as enterococci are 

provided in Table 9. 

Table 9. Re-Assessment of False Positive and False Negative Initial Study Results with Pink to Red 

Colonies without Halos Counted as Enterococci

Matrix 

(sample no.) 

Total colonies False positive (FP) assessment False negative (FN) assessment

Typical Atypical

Typical 

colonies 

submitted

No. FP 

colonies

FP 

confirmation 

rate (%) a

Estimated 

% of total 

colonies 

that would 

have been 

a FP b

Atypical 

colonies 

submitted

No. FN 

colonies

FN 

confirmation 

rate (%) c

Estimated % 

of total 

colonies that 

would have 

been a FN d

Disinfected 

(Samples 1-4) 477 19 69 0 0.0 0.0 4 3 75.0 2.9

Secondary 

(Samples 5, 

6)

2291 236 166 7 4.2 3.8 32 27 84.4 7.9

Disinfected & 

Secondary 

(Samples 1-6)

2768 255 235 7 3.0 2.7 36 30 83.3 7.0

a False positive confirmation rate = number of false positive colonies/number of typical colonies submitted 

b Percent of total colonies estimated to be false positives = [(total typical colonies × FP confirmation rate)/(total number of 

typical and atypical colonies observed)] × 100; e.g., [(2291×(7/166))/(2291+236)] × 100 = 3.8%

c False negative confirmation rate = number of false negative colonies/number of atypical colonies submitted 

d Percent of total colonies estimated to be false negatives = [(total atypical colonies × FN confirmation rate)/(total number of 

typical and atypical colonies observed)] × 100; e.g., [(236 ×(27/32))/(2291+236)] × 100 = 7.9%

16.0 Pollution Prevention

16.1 The solutions and reagents used in this method pose little threat to the environment when recycled and 

managed properly.

16.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize the 

volume of expired materials to be disposed.

17.0 Waste Management

17.1 It is the laboratory’s responsibility to comply with all federal, state, and local regulations governing 

waste management, particularly the biohazard and hazardous waste identification rules and land disposal 

restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fume 
Method 1600

September 2014 22

hoods and bench operations. Compliance with all sewage discharge permits and regulations is also 

required.

17.2 Samples, reference materials, and equipment known or suspected to have viable enterococci attached or 

contained must be sterilized prior to disposal.

17.3 Samples preserved with HCl to pH <2 are hazardous and must be neutralized before being disposed, or 

must be handled as hazardous waste.

17.4 For further information on waste management, consult “The Waste Management Manual for Laboratory 

Personnel” and “Less Is Better: Laboratory Chemical Management for Waste Reduction,” both available 

from the American Chemical Society’s Department of Government Relations and Science Policy, 1155 

16th Street NW, Washington, DC 20036. 

18.0 References

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

Microbial Indicators to Health Effects at Marine Bathing Beaches. Amer. Jour. Public Health. 

69:690-696.

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

of Research and Development, USEPA.

18.3 USEPA. 2004. Results of the Interlaboratory Validation of EPA Method 1600 (mEI) for Enterococci in 

Wastewater Effluent. December 2004. EPA 821-R-04-008. 

18.4 Messer, J.W. and A.P. Dufour. 1998. A Rapid, Specific Membrane Filtration Procedure for 

Enumeration of Enterococci in Recreational Water. Appl. Environ. Microbiol. 64:678-680.

18.5 ACS. 2000. Reagent Chemicals, American Chemical Society Specifications. American Chemical 

Society, New York. For suggestions of the testing of reagents not listed by the American Chemical 

Society, see AnalaR Standards for Laboratory Chemicals, BDH, Poole, Dorset, UK and the United 

States Pharmacopeia.

18.6 APHA. 1998. Standard Methods for the Examination of Water and Wastewater. 20th Edition. American 

Public Health Association, Washington D.C.

18.7 Bordner, R., J.A. Winter, and P.V. Scarpino (eds.). Microbiological Methods for Monitoring the 

Environment: Water and Wastes, EPA-600/8-78-017. Cincinnati, OH: U.S. Environmental Protection 

Agency, 1978.
Appendix A

Part II (General Operations)

Section A (Sample Collection, Preservation, and Storage)
1

Sample Collection1

1.0 Sample Containers

1.1 Sample Bottles: bottles must be resistant to sterilizing conditions and the solvent 

action of water. Wide-mouth borosilicate glass bottles with screw-cap or groundglass stopper or heat-resistant plastic bottles may be used if they can be sterilized 

without producing toxic materials (see examples A and C in Figure 1). Screwcaps must not produce bacteriostatic or nutritive compounds upon sterilization.

Figure 1. Suggested sample containers.

1.2 Selection and Cleaning of Bottles: Sample bottles should be at least 125 mL 

volume for adequate sampling and for good mixing. Bottles of 250 mL, 500 mL, 

and 1000 mL volume are often used for multiple analyses. Discard bottles which 

have chips, cracks, and etched surfaces. Bottle closures must be water-tight. 

Before use, thoroughly cleanse bottles and closures with detergent and hot water, 

followed by a hot water rinse to remove all trace of detergent. Then rinse them 

three times with laboratory-pure water.

1.3 Dechlorinating Agent: The agent must be placed in the bottle when water and 

wastewater samples containing residual chlorine are anticipated. Add sodium 

thiosulfate to the bottle before sterilization at a concentration of 0.1 mL of a 10% 

solution for each 125 mL sample volume. This concentration will neutralize 

approximately 15 mg/L of residue chlorine.

1.4 Chelating Agent: A chelating agent should be added to sample bottles used to 

collect samples suspected of containing >0.01 mg/L concentrations of heavy 

metals such as copper, nickel or zinc, etc. Add 0.3 mL of a 15% solution of 

ethylenediaminetetraacetic acid (EDTA) tetrasodium salt, for each 125 mL 

sample volume prior to sterilization.

 

1 The text is taken from Part II, Section A, of the EPA publication "Microbiological Methods for Monitoring the 

Environment" EPA-600/8-78-017, December 1978.
2

1.5 Wrapping Bottles: Protect the tops and necks of glass stoppered bottles from 

contamination by covering them before sterilization with aluminum foil or kraft 

paper.

1.6 Sterilization of Bottles: Autoclave glass or heat-resistant plastic bottles at 121°C 

for 15 minutes. Alternatively, dry glassware may be sterilized in a hot oven at 

170°C for not less than two hours. Ethylene oxide gas sterilization is acceptable 

for plastic containers that are not heat-resistant. Sample bottles sterilized by gas 

should be stored overnight before being used to allow the last traces of gas to 

dissipate.

1.7 Plastic Bags: The commercially available bags (Whirl-pak) (see example B in 

Figure 1) are a practical substitute for plastic or glass samples bottles in sampling 

soil, sediment, or biosolids. The bags are sealed in manufacture and opened only 

at time of sampling. The manufacturer states that such bags are sterilized.

2.0 Sampling Techniques

Samples are collected by hand or with a sampling device if the sampling site has difficult 

access such as a bridge or bank adjacent to a surface water.

2.1 Chlorinated Samples: When samples such as treated waters, chlorinated 

wastewaters or recreational waters are collected, the sample bottle must contain a 

dechlorinating agent (see section 1.3 above).

2.2 Composite Sampling: In no case should a composite sample be collected for 

bacteriologic examination. Data from individual samples show a range of values. 

A composite sample will not display this range. Individual results will give 

information about industrial process variations in flow and composition. Also, one 

or more portions that make up a composite sample may contain toxic or nutritive 

materials and cause erroneous results.

2.3 Surface Sampling by Hand: A grab sample is obtained using a sample bottle 

prepared as described in (1) above. Identify the sampling site on the bottle label 

and on a field log sheet. Remove the bottle covering and closure and protect from 

contamination. Grasp the bottle at the base with one hand and plunge the bottle 

mouth down into the water to avoid introducing surface scum (Figure 2). Position 

the mouth of the bottle into the current away from the hand of the collector and, if 

applicable, away from the side of the sampling platform. The sampling depth 

should be 15-30 cm (6-12 inches) below the water surface. If the water body is 

static, an artificial current can be created, by moving the bottle horizontally in the 

direction it is pointed and away from the sampler. Tip the bottle slightly upwards 

to allow air to exit and the bottle to fill. After removal of the bottle from the 

stream, pour out a small portion of the sample to allow an air space of 2.5-5 cm 

(1-2 inches) above each sample for proper mixing of the sample before analyses. 
3

Tightly stopper the bottle and place on ice (do not freeze) for transport to the 

laboratory.

Figure 2. Grab sampling technique for surface waters.

3.0 Selection of Sampling Sites and Frequency

These will be described for streams, rivers, estuarine, marine, and recreational waters as 

well as domestic and industrial wastewaters.

3.1 Stream Sampling: The objectives of the initial survey dictate the location, 

frequency and number of samples to be collected.

3.1.1 Selection of Sampling Sites: A typical stream sampling program includes 

sampling locations upstream of the area of concern, upstream and 

downstream of waste discharges, upstream and downstream from tributary 

entrances to the river and upstream of the mouth of the tributary. For more 

complex situations, where several waste discharges are involved, sampling 

includes sites upstream and downstream from the combined discharge area 

and samples taken directly from each industrial or municipal waste 

discharge. Using available bacteriological, chemical and discharge rate 

data, the contribution of each pollution source can be determined.

3.1.2 Small Streams: Small streams should be sampled at background stations 

upstream of the pollution sources and at stations downstream from 

pollution sources. Additional sampling sites should be located downstream 

to delineate the zones of pollution. Avoid sampling areas where stagnation 

may occur (e.g., backwater of a tributary) and areas located near the inside 

bank of a curve in the stream which may not be representative of the main 

channel.

3.1.3 Large Streams and Rivers: Large streams are usually not well mixed 

laterally for long distances downstream from the pollution sources. 

Sampling sites below point source pollution should be established to 
4

provide desired downstream travel time and dispersal as determined by 

flow rate measurements. Particular care must be taken to establish the 

proper sampling points. Occasionally, depth samples are necessary to 

determine vertical mixing patterns.

3.2 Estuarine and Marine Sampling: Sampling estuarine and marine waters 

requires the consideration of other factors in addition to those usually recognized 

in fresh water sampling. They include tidal cycles, current patterns, bottom 

currents and counter-currents, stratification, seasonal fluctuations, dispersion of 

discharges and multi-depth samplings.

The frequency of sampling varies with the objectives. When a sampling program 

is started, it may be necessary to sample every hour around the clock to establish 

pollution loads and dispersion patterns. The sewage discharges may occur 

continuously or intermittently.

When the sampling strategy for a survey is planned, data may be available from 

previous hydrological studies done by the Coast Guard, Corps of Engineers, 

National Oceanic and Atmospheric Administration (NOAA), U.S. Geological 

Survey, or university and private research investigations. In a survey, float studies 

and dye studies are often carried out to determine surface and undercurrents. 

Initially depth samples are taken on the bottom and at five feet increments 

between surface and bottom. A random grid pattern for selecting sampling sites is 

established statistically.

3.2.1 Estuarine Sampling: When a survey is made on an estuary, samples are 

often taken from a boat, usually making an end to end traverse of the 

estuary. Another method involves taking samples throughout a tidal cycle, 

every hour or two hours from a bridge or from an anchored boat at a 

number of fixed points.

In a large bay or estuary where many square miles of area are involved, a 

grid or series of stations may be necessary. Two sets of samples are 

usually taken from an area on a given day, one at ebb or flood slack water, 

and the other three hours earlier, or later, at the half tidal interval. 

Sampling is scheduled so that the mid-sampling time of each run coincides 

with the calculated occurrence of the tidal condition.

In location sampling sites, one must consider points at which tributary 

waters enter the main stream or estuary, location of shellfish beds and 

bathing beaches. The sampling stations can be adjusted as data 

accumulate. For example, if a series of stations half mile apart consistently 

show similar values, some of these stations may be dropped and other 

stations added in areas where data shows more variability.
5

Considerable stratification can occur between the salt water from the sea 

and the fresh water supplied by a river. It is essential when starting a 

survey of an unknown estuary to find out whether there is any marked 

stratification. This can be done by chloride determinations at different 

locations and depths. It is possible for stratification to occur in one part of 

an estuary and not in another.

On a flood tide, the more dense salt water pushing up into the less dense 

fresh river water will cause an overlapping with the fresh water flowing on 

top. A phenomenon called a salt water wedge can form. As a result, 

stratification occurs. If the discharge of pollution is in the salt water layer, 

the contamination will be concentrated near the bottom at the flood tide. 

The flow or velocity of the fresh water will influence the degree of 

stratification which occurs. If one is sampling only at the surface, it is 

possible that the data will not show the polluted underflowing water which 

was contaminated at the point below the fresh water river. Therefore, 

where stratification is suspected, samples at different depths will be 

needed to measure vertical distribution.

3.2.2 Marine Sampling: In ocean studies, the environmental conditions are most 

diverse along the coast where shore, atmosphere and the surf are strong 

influences. The shallow coastal waters are particularly susceptible to daily 

fluctuations in temperature and seasonal changes.

Sampling during the entire tidal cycle or during a half cycle may be 

required. Many ocean studies such as sampling over the continental shelf 

involve huge areas and no two areas of water are the same.

Selection of sampling sites and depths are most critical in marine waters. 

In winter, cooling of coastal waters can result in water layers which 

approach 0°C. In summer, the shallow waters warm much faster than the 

deeper waters. Despite the higher temperature, oxygen concentrations are 

higher in shallow than in deeper waters due to greater water movement, 

surf action and photosynthetic activity from macrophytes and the 

plankton.

Moving from the shallow waters to the intermediate depths, one observes 

a moderation of these shallow water characteristics. In the deeper waters, 

there is a marked stabilization of conditions. Water temperatures are lower 

and more stable. There is limited turbulence, little penetration of light, 

sparse vegetation and the ocean floor is covered with a layer of silts and 

sediments.

3.3 Recreational Waters (Bathing Beaches): Sampling sites at bathing beaches or 

other recreational areas should include upstream or peripheral areas and locations 

adjacent to natural drains that would discharge stormwater, or run-off areas 
6

draining septic wastes from restaurants, boat marinas, or garbage collection areas. 

Samples of bathing beach water should be collected at locations and times of 

heaviest use. Daily sampling, preferably in the afternoon, is the optimum 

frequency during the season. Weekends and holidays which are periods of highest 

use must be included in the sampling program. Samples of estuarine bathing 

waters should be obtained at high tide, ebb tide and low tide in order to determine 

the cyclic water quality and deterioration that must be monitored during the 

swimming season.

3.4 Domestic and Industrial Waste Discharges: It is often necessary to sample 

secondary and tertiary wastes from municipal waste treatment plants and various 

industrial waste treatment operations. In situations where the plant treatment 

efficiency varies considerably, grab samples are collected around the clock at 

selected intervals for a three to five day period. If it is known that the process 

displays little variation, fewer samples are needed. In no case should a composite 

sample be collected for bacteriological examination. The National Pollution 

Discharge Elimination System (NPDES) has established wastewater treatment 

plant effluent limits for all dischargers. These are often based on maximum and 

mean values. A sufficient number of samples must be collected to satisfy the 

permit and/or to provide statistically sound data and give a fair representation of 

the bacteriological quality of the discharge.
Appendix B

Part II (General Operations)

Sections C.3.5 (Counting Colonies) and C.3.6 (Calculation of Results)
1

Counting Colonies1

1.0 Counting Colonies

Colonies should be counted using a fluorescent lamp with a magnifying lens. The 

fluorescent lamp should be nearly perpendicular to the membrane filter. Count colonies 

individually, even if they are in contact with each other. The technician must learn to 

recognize the difference between two or more colonies which have grown into contact 

with each other and single, irregularly shaped colonies which sometimes develop on 

membrane filters. The latter colonies are usually associated with a fiber or particulate 

material and the colonies conform to the shape and size of the fiber or particulates. 

Colonies which have grown together almost invariably show a very fine line of contact.

2.0 Calculation of Results

2.1 Select the membrane filter with the number of colonies in the acceptable range 

and calculate count per 100 mL according to the general formula:

Count per 100 mL = (No. of colonies counted/Volume of sample filtered, in mL) x 100

2.2 Counts Within the Acceptable Limits

The acceptable range of colonies that are countable on a membrane is a function 

of the method. Different methods may have varying acceptable count ranges. All 

examples in this appendix assume that the acceptable range of counts is between 

20-60 colonies per membrane.

For example, assume that filtration of volumes of 50, 15, 5, 1.5, and 0.5 mL 

produced colony counts of 200, 110, 40, 10, and 5, respectively.

An analyst would not actually count the colonies on all filters. By inspection the 

analyst would select the membrane filter with the acceptable range of target 

colonies, as defined by the method, and then limit the actual counting to such 

membranes.

After selecting the best membrane filter for counting, the analyst counts colonies 

and applies the general formula as in section 2.1 above to calculate the count/100 

mL.

 

1 The text is largely taken from Part II, Section C, of the EPA publication "Microbiological Methods for Monitoring 

the Environment" EPA-600/8-78-017, December 1978. Some examples were kindly provided by Kristen Brenner, 

US EPA.
2

2.3 More Than One Acceptable Count

2.3.1 If there are acceptable counts on replicate plates, carry counts 

independently to final reporting units, then calculate the arithmetic mean 

of these counts to obtain the final reporting value.

Example, if the counts are 24 and 36 for replicate plates of 100 mL each, 

then the arithmetic mean is calculated as follows:

(24 CFU/100 mL + 36 CFU/100 mL)

2

= 30 CFU / 100 mL

2.3.2 If there is more than one dilution having an acceptable range of counts, 

independently carry counts to final reporting units, and then average for 

final reported value.

For example, if volumes of 100, 10, 1 and 0.1 mL produced colony counts 

of Too Numerous To Count (TNTC), 55, 30, and 1, respectively, then two 

volumes, 10 mL and 1 mL, produced colonies in the acceptable counting 

range.

Independently carry each MF count to a count per 100 mL:

55

10

x 100 = 550 CFU/100 mL

and

30

1

x 100 = 3000 CFU/100 mL

Calculate the arithmetic mean as in section 2.3.1 above:

(550 CFU/100 mL + 3000 CFU/100 mL)

2

= 1775 CFU / 100 mL

Report this as 1775 CFU/100 mL.

2.4 If all MF counts are below the lower acceptable count limit, select the most nearly 

acceptable count.

2.4.1 For example, sample volumes of 100, 10 and 1 mL produced colony 

counts of 17, 1 and 0, respectively.
3

Here, no colony count falls within recommended limits. Calculate on the 

basis of the most nearly acceptable plate count, 17, and report as 17 

CFU/100 mL.

Note that in this case, because no calculations were done (i.e. this is the 

count for 100 mL), the count is reported as 17 CFU/100 mL rather than an 

“estimated count of 17 CFU/100 mL”

2.4.2 As a second example, assume a count in which sample volumes of 10 and 

1 mL produced colony counts of 18 and 0, respectively.

Here, no colony count falls within recommended limits. Calculate on the 

basis of the most nearly acceptable plate count, 18, and calculate as in 

section 2.3.2 above.

18

10

x 100 = 180 CFU/100 mL

Report this as an estimated count of 180 CFU/100 mL

.

2.5 If counts from all membranes are zero, calculate using count from largest 

filtration volume.

For example, sample volumes of 25, 10, and 2 mL produced colony counts of 0, 

0, and 0, respectively, and no actual calculation is possible, even as an estimated 

report. Calculate the number of colonies per 100 mL that would have been 

reported if there had been one colony on the filter representing the largest 

filtration volume. In this example, the largest volume filtered was 25 mL and thus 

the calculation would be:

1

25

x 100 = 4 CFU/100 mL

Report this as < (less than) 4 CFU/100 mL.

2.6 If all membrane counts are above the upper acceptable limit, calculate count using 

the smallest volume filtered.

For example, assume that the volumes 1, 0.3, and 0.01 mL produced colony 

counts of TNTC, 150, and 110 colonies, respectively. Since all colony counts are 

above the acceptable limit, use the colony count from the smallest sample volume 

filtered and estimate the count as:
4

110

0.01

x 100 = 1,100,000 CFU/100 mL

Report this as estimated count 1.1 × 106 CFU/100 mL

2.7 If typical colonies are too numerous to count (TNTC), use upper limit count with 

smallest filtration volume.

For example, assume that the volumes 1, 0.3, and 0.01 mL all produced too many 

typical colonies, and that the laboratory bench record indicated TNTC.

Use the upper acceptable count for the method (60 colonies in this example) as 

the basis of calculation with the smallest filtration volume and estimate the count 

as:

60

0.01

x 100 = 600,000 CFU/100 mL

Report this as > (greater than) 6 ×105 CFU/100 mL

2.8 If colonies are both above and below the upper and lower acceptable limits (i.e., 

no counts are within the acceptable limits), select the most nearly acceptable 

count.

2.8.1 For example, sample volumes of 100, 10 and 1 mL produced colony 

counts of 64, 6 and 0, respectively.

Here, no colony count falls within recommended limits. Calculate on the 

basis of the most nearly acceptable plate count, 64, and report as 64 

CFU/100 mL

Note that in this case, because no calculations were done (i.e. this is the 

count for 100 mL), the count is reported as 64 CFU/100 mL rather than an 

“estimated count of 64 CFU/100 mL”

2.8.2 As a second example, assume a count in which sample volumes of 100, 10 

and 1 mL produced colony counts of 98, 18, and 0, respectively.

Here, no colony count falls within recommended limits. Calculate on the 

basis of the most nearly acceptable plate count, 18, and calculate as in 

section 2.3.2 above.

18

10

x 100 = 180 CFU/100 mL

Report this as estimated count 180 CFU/100 mL.
5

2.9 If there is no result because of a confluent growth, > 200 atypical colonies 

(TNTC), lab accident, etc., report as No Data and specify the reason.