Method 1600: Enterococci in Water by

Membrane Filtration Using membraneEnterococcus Indoxyl-$-D-Glucoside Agar

(mEI)

December 2009
U.S. Environmental Protection Agency

Office of Water (4303T)

1200 Pennsylvania Avenue, NW

Washington, DC 20460

EPA-821-R-09-016
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 
iii

Disclaimer

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 or OSTCWAMethods@epa.gov
iv

Table of Contents

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

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

3.0 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

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

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

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

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

15.0 Method Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

16.0 Pollution Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

17.0 Waste Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

18.0 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
v

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).
1 December 2009

Method 1600: Enterococci in Water by Membrane Filtration Using

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

December 2009

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 $-D-glucoside, a chromogenic

cellobiose analog used in place of esculin. In this procedure, $-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°C ± 0.5°C. 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.
Method 1600

December 2009 2

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. 

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)
Method 1600

3 December 2009

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

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 μm 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 41°C ± 0.5°C

6.23 Waterbath maintained at 50°C 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°C [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

December 2009 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°C (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 $-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°C (15 PSI) for 15 minutes and cool in a 50°C 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

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

completely. Autoclave at 121°C (15 PSI) for 15 minutes and cool in a 50°C waterbath. 

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.
Method 1600

5 December 2009

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°C (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

December 2009 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°C (15 PSI) for 15 minutes. Overheating

may cause darkening of the medium. Cool in a 50°C waterbath, 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°C (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 (bioMérieux Inc., Durham NC)

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

• Stock cultures of Escherichia coli (E. coli) ATCC #11775

• E. coli ATCC #11775 BioBalls (bioMérieux Inc., Durham NC) 
Method 1600

7 December 2009

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°C 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.
Method 1600

December 2009 8

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 (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% 85% - 106%

28% 14%

Ongoing precision and recovery (OPR) as percent

recovery

27% - 131% 78% - 113%
Method 1600

9 December 2009

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 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.

 
Method 1600

December 2009 10

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

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. 
Method 1600

11 December 2009

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) 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 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°C E. faecalis E. coli

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.
Method 1600

December 2009 12

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°C ± 0.5°C. 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°C ± 0.5°C. 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 single analyst. If no positive field samples are available, a 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 7.6.

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.
Method 1600

13 December 2009

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 phosphatebuffered 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 41°C ± 0.5°C for 24 ± 2 hours. 

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. 

Figure 1. Enterococci colonies on mEI produce blue halos.

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. 
Method 1600

December 2009 14

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°C ± 0.5°C.

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°C ± 0.5°C for 48 ± 3 hours.

12.3.2 Incubate BHIB at 45°C ± 0.5°C 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.

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°C ± 0.5°C and BHIB at 45°C ± 0.5°C 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. 
Method 1600

15 December 2009

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°C ± 3°C 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°C ± 3°C for 20 ± 4 hours. This culture is referred to as the

undiluted spiking suspension and should contain 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"
Method 1600

December 2009 16

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.

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°C ± 0.5°C 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.
Method 1600

17 December 2009

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.

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
Method 1600

December 2009 18

14.2.4.1 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) of spiked 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)

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%
Method 1600

19 December 2009

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 32 100 × (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

December 2009 20

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 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 BioBalls 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.
Method 1600

21 December 2009

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 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%
Method 1600

December 2009 22

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 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%
Method 1600

23 December 2009

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 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.
Method 1600

December 2009 24

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)
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 ground-glass 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). Screw-caps must not produce 

bacteriostatic or nutritive compounds upon sterilization. 

Figure 1. Suggested sample containers. 

1.2 Selection and Cleaning of Bottles: Samples 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. 

1 
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. Tightly stopper the bottle and place on ice (do 

not freeze) for transport to the laboratory. 

2
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
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 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. 

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. 

4
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 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. 

5
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. 

6
Appendix B:

Part II (General Operations), Sections C.3.5 (Counting Colonies)

and C.3.6 (Calculation of Results)
Counting Colonies1 

1.0 Counting Colonies 

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

appemdix 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. 

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. 

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. 

1 
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) 

= 30 CFU/ 100 mL

2 

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

carry counts to final reporting units, 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

 × 100 = 550 CFU /100 mL

10 

and 

30

 × 100 = 3000 CFU /100 mL 

1 

Calculate the arithmetic mean as in section 2.3.1 above: 

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

= 1775 CFU/100 mL

2 

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. 

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 
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

 × 100 = 180 CFU /100 mL

10 

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

 × 100 = 4 CFU /100 mL 

25 

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: 

110

 × 100 = 1,100,000 CFU /100 mL 

0.01 

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. 

3
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

 × 100 = 600,000 CFU /100 mL 

0.01 

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

 × 100 = 180 CFU /100 mL

10 

Report this as estimated count 180 CFU/100 mL. 

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. 

4