United States

Environmental Protection Agency

Office of Water

(4303-T)

EPA-821-R-14-010

September 2014

Method 1603: Escherichia coli (E. coli) 

in Water by Membrane Filtration Using 

Modified membrane-Thermotolerant 

Escherichia coli Agar (Modified mTEC)
U.S. Environmental Protection Agency

Office of Water (4303T)

1200 Pennsylvania Avenue, NW

Washington, DC 20460
Method 1603

Acknowledgments

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

wastewater effluents:

Volunteer Research Laboratory

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

Volunteer Verification Laboratory

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

Espineda, and Zora Bahariance

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 

i September 2014
Method 1603

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 

CSC Biology Studies Group 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)

ii September 2014
Method 1603

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 and Contamination.........................................................................................................2

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

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

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

8.0 Sample Collection, Handling, and Storage...........................................................................................9

9.0 Quality Control...................................................................................................................................9

10.0 Calibration and Standardization.........................................................................................................14

11.0 Procedure .........................................................................................................................................14

12.0 Verification Procedure......................................................................................................................15

13.0 Data Analysis and Calculations.........................................................................................................16

14.0 Sample Spiking Procedure ................................................................................................................16

15.0 Method Performance ........................................................................................................................21

16.0 Pollution Prevention .........................................................................................................................22

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

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

iii September 2014
Method 1603

List of Appendices

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

Wastes (Reference 18.5).

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).

iv September 2014
Method 1603

Method 1603: Escherichia coli (E. coli) in Water by Membrane Filtration 

Using Modified membrane- Thermotolerant Escherichia coli Agar 

(modified mTEC)

September 2014

1.0 Scope and Application

1.1 Method 1603 describes a membrane filter (MF) procedure for the detection and enumeration of

Escherichia coli bacteria in ambient waters and disinfected wastewaters. This method is a single-step 

modification of EPA Method 1103.1 (mTEC). Unlike the mTEC media method, it does not require the 

transfer of the membrane filter to another substrate. The modified medium contains a chromogen 

(5-bromo-6-chloro-3-indolyl-β-D-glucuronide), which is catabolized to glucuronic acid and a red- or 

magenta-colored compound by E. coli that produces the enzyme β-D-glucuronidase. The apparatus and 

equipment, and sampling, filtration, and verification procedures for the modified mTEC method are 

identical to those of the original mTEC method.

1.2 E. coli is a common inhabitant of the intestinal tract of warm-blooded animals, and its presence in water 

samples 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 E. coli in recreational fresh water samples is the direct relationship between 

the density of E. coli and the risk of gastrointestinal illness associated with swimming in the water 

(Reference 18.1).

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 1603 provides a direct count of E. coli in ambient water or wastewater based on the 

development of colonies that grow on the surface of a membrane filter. A sample is filtered through the 

membrane, which retains the bacteria. After filtration, the membrane is placed on a selective and 

differential medium, modified mTEC agar, incubated at 35°C ± 0.5°C for 2 ± 0.5 hours to resuscitate 

injured or stressed bacteria, and then incubated at 44.5°C ± 0.2°C for 22 ± 2 hours. The target colonies 

on modified mTEC agar are red or magenta in color after the incubation period. 

3.0 Definitions

3.1 In Method 1603, E. coli are those bacteria which produce red or magenta colonies on the modified 

mTEC agar.

1 September 2014
Method 1603

4.0 Interferences and Contamination

4.1 Water samples containing colloidal or suspended particulate material 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 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 Mouth-pipetting is prohibited.

5.3 This method does not address all safety issues associated with its use. The laboratory is responsible for 

maintaining a safe work environment and a current awareness file of OSHA regulations regarding the 

safe handling of the chemicals specified in this method. A reference file containing material safety data 

sheets (MSDSs) should be available to all personnel involved in these analyses.

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 EPA logo

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 Filter flask, vacuum, usually 1 L, with appropriate tubing

6.11 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

2 September 2014
Method 1603

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 × 50 mm, with tight-fitting lids; and 15 × 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 Sterile disposable applicator sticks

6.23 Incubator maintained at 35°C ± 0.5°C, with approximately 90% humidity if loose-lidded petri dishes are 

used

6.24 Water bath maintained at 44.5°C ± 0.2°C

6.25 Water bath maintained at 50°C for tempering agar

6.26 Test tubes, 20 × 150 mm, borosilicate glass or plastic

6.27 Test tubes, 10 × 75 mm, borosilicate glass (durham tubes)

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

6.29 Test tubes screw-cap, borosilicate glass, 16 × 125 mm or other appropriate size

6.30 Whirl-Pak® bags or equivalent

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

minutes

6.32 Filter paper

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 18.3). The agar used in preparation of culture media must be of 

microbiological grade.

3 September 2014
Method 1603

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 18.4).

7.4 Phosphate buffered saline (PBS)

7.4.1 Composition:

Monosodium phosphate (NaH2PO4) 0.58 g

Disodium phosphate (Na2HPO4) 2.5 g

Sodium chloride 8.5 g

Reagent-grade water 1.0 L

7.4.2 Dissolve the ingredients 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 at 121°C (15 PSI) for 15 minutes. Final pH should be 7.4 ± 0.2.

Note: The initial and ongoing precision and recovery (IPR and OPR) performance criteria 

established for Method 1603 were determined using spiked PBS samples (Section 9.3, 

Table 1). Laboratories must use PBS when performing IPR and OPR sample 

analyses. However, phosphate-buffered dilution water (Section 7.5) may be 

substituted for PBS as a sample diluent and filtration rinse buffer. 

7.5 Phosphate buffered dilution water (Reference 18.5) 

7.5.1 Composition of stock phosphate buffer solution:

Monopotassium phosphate (KH2PO4) 34.0 g

Reagent-grade water 500.0 mL

Preparation: Dissolve KH2PO4 in 500 mL reagent-grade water. Adjust the pH of the solution 

to 7.2 with 1 N NaOH, and bring the volume to 1 L with reagent-grade water. Sterilize by 

filtration or autoclave at 121°C (15 PSI) for 15 minutes.

7.5.2 Preparation of stock magnesium chloride (MgCl2) solution: Add 38 g anhydrous MgCl2 or 81.1 

g magnesium chloride hexahydrate (MgCl2 • 6H2O) to 1 L reagent-grade water. Sterilize by 

filtration or autoclave at 121°C (15 PSI) for 15 minutes. 

7.5.3 After sterilization, store the stock solutions in the refrigerator until used. Handle aseptically. 

If evidence of mold or other contamination appears, the affected stock solution should be 

discarded and a fresh solution should be prepared.

7.5.4 Working phosphate buffered dilution water: Mix 1.25 mL of the stock phosphate buffer and 5 

mL of the MgCl2 stock per liter of reagent-grade water. Dispense in appropriate amounts for 

dilutions and/or for use as rinse buffer. Autoclave at 121°C (15 PSI) for 15 minutes. Final pH 

should be 7.0 ± 0.2.

4 September 2014
Method 1603

7.6 Modified mTEC agar

7.6.1 Composition:

Protease peptone #3 5.0 g

Yeast extract 3.0 g

Lactose 10.0 g

Sodium chloride 7.5 g

Dipotassium phosphate (K2HPO4) 3.3 g

Monopotassium phosphate (KH2PO4) 1.0 g

Sodium lauryl sulfate 0.2 g

Sodium desoxycholate 0.1 g 

Chromogen (5-bromo-6-chloro-3-indolyl-β-D-glucuronide) 0.5 g

Agar 15.0 g

Reagent-grade water 1.0 L

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

Autoclave at 121°C (15 PSI) for 15 minutes, and cool in a 50°C water bath; adjust pH to 7.3 ± 

0.2. with 1.0 N hydrochloric acid or 1.0 N sodium hydroxide. Pour the medium into each 9 × 

50 mm culture dish to a 4-5 mm depth (approximately 4-6 mL), and allow to solidify. Store in 

a refrigerator.

7.7 Nutrient agar

7.7.1 Composition:

Peptone 5.0 g

Beef extract 3.0 g

Agar 15.0 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 into screw-cap tubes, and autoclave at 121°C (15 PSI) for 15 minutes. Remove the 

tubes and slant. Final pH should be 6.8 ± 0.2.

5 September 2014
Method 1603

7.8 Tryptic/trypticase soy broth

7.8.1 Composition:

Pancreatic digest of casein 17.0 g

Enzymatic/papaic digest of soybean meal 3.0 g

Sodium chloride 5.0 g

Dextrose 2.5 g

Dipotassium phosphate (K2HPO4) 2.5 g

Reagent-grade water 1.0 L

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

Dispense into screw-cap tubes, and autoclave at 121°C (15 PSI) for 15 minutes. Final pH 

should be 7.3 ± 0.2.

7.9 Simmons citrate agar

7.9.1 Composition:

Magnesium sulfate (MgSO4) 0.2 g

Monoammonium phosphate (NH4H2PO4) 1.0 g

Dipotassium phosphate (K2HPO4) 1.0 g

Sodium citrate (Citric acid) 2.0 g

Sodium chloride 5.0 g

Bromthymol Blue 0.08 g

Agar 15.0 g

Reagent-grade water 1.0 L

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

Dispense into screw-cap tubes, and autoclave at 121°C (15 PSI) for 15 minutes. Cool the tubes 

and slant. Final pH should be 6.9 ± 0.2.

7.10 Tryptone water

7.10.1 Composition:

Tryptone 10.0 g

Sodium chloride 5.0 g

Reagent-grade water 1.0 L

7.10.2 Add reagents to 1 L of reagent grade water and mix thoroughly to dissolve. Dispense in 5-mL 

volumes into tubes, and autoclave at 121°C (15 PSI) for 15 minutes. Final pH should be 7.3 ± 

0.2.

6 September 2014
Method 1603

7.11 EC broth

7.11.1 Composition:

Tryptose or trypticase peptone 20.0 g

Lactose 5.0 g

Bile salts No.3 1.5 g

Dipotassium phosphate (K2HPO4) 4.0 g 

Monopotassium phosphate (KH2PO4) 1.5 g

Sodium chloride 5.0 g

Reagent-grade water 1.0 L

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

Dispense into fermentation tubes (20 × 150 mm tubes containing inverted 10 × 75 mm tubes). 

Autoclave at 121°C (15 PSI) for 15 minutes. Final pH should be 6.9 ± 0.2. 

Note: Do not use tubes if the inverted tubes (durham tubes) are not completely filled with 

medium after sterilization.

7.12 Oxidase reagent

7.12.1 Composition:

N,N,N’,N’-tetramethyl-ρ-phenylenediamine dihydrochloride, 1% aqueous solution (1 g per 100 

mL sterile reagent-grade water).

Note: Prepared oxidase test slides are commercially available and are recommended for 

colony verification (Section 12.0).

7.13 Kovacs indole reagent

7.13.1 Composition:

ρ-dimethylaminobenzaldehyde 10.0 g

Amyl or isoamyl alcohol 150.0 mL

Concentrated (12 M) hydrochloric acid 50.0 mL

7.13.2 Preparation: Dissolve ρ-dimethylaminobenzaldehyde in alcohol, slowly add hydrochloric acid, 

and mix.

7 September 2014
Method 1603

7.14 Tryptic soy agar (TSA)

7.14.1 Composition:

Pancreatic digest of casein 15.0 g

Enzymatic digest of soybean meal 5.0 g

Sodium chloride 5.0 g

Agar 15.0 g

Reagent- grade water 1.0 L

7.14.2 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. Pour the medium 

into each 15 × 100 mm culture dish to a 4-5 mm depth and allow to solidify. Final pH should 

be 7.3 ± 0.2.

7.15 Lauryl tryptose broth (LTB)

7.15.1 Composition:

Tryptose 20.0 g

Lactose 5.0 g

Dipotassium phosphate (K2HPO4) 2.75 g

Monopotassium phosphate (KH2PO4) 2.75 g

Sodium chloride 5.0 g

Sodium lauryl sulfate 0.1 g

7.15.2 Preparation: Add reagents (Section 7.15.1) to 1 L of reagent-grade water, heat with frequent 

mixing, and boil for one minute to dissolve completely. Autoclave at 121°C (15 PSI) for 15 

minutes. Final pH should be 6.8 ± 0.2.

7.16 Control cultures

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

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

• E. coli ATCC 11775 BioBalls (BTF Pty, Sydney, Australia) 

7.16.2 Negative control organism (either of the following are acceptable):

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

• E. faecalis ATCC 19433 BioBalls (BTF Pty, Sydney, Australia)

OR

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

8 September 2014
Method 1603

8.0 Sample Collection, Handling, and Storage

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

Microbiological Methods for Monitoring the Environment: Water and Wastes, Part II, Section A (see 

Appendix A). Adherence to sample handling procedures and holding time limits is critical to the 

production of valid data. Samples should not be analyzed if these conditions are not met. 

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 in 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 1603 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 18.5. 

9.2 The minimum analytical QC requirements for the analysis of samples using Method 1603 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 1603 are based on the results of the interlaboratory validation 

of Method 1603 in PBS and disinfected wastewater matrices. Although the matrix spike 

9 September 2014
Method 1603

recovery criteria (Section 9.5, Table 2) pertain only to disinfected wastewaters, 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. 

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. coli ATCC 11775 

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 E. coli 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 E. coli 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)

46% - 119%

36%

detect - 144%

61%

Ongoing precision and recovery (OPR) as percent recovery 38% - 127% detect - 144%

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. coli ATCC 11775 according to the spiking procedure in 

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

10 September 2014
Method 1603

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 E. coli 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 1603 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 E. 

coli 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 E. coli for calculating MS recoveries (Section 9.5.3). The other sample will 

serve as the MS sample and will be spiked with E. coli ATCC 11775 according to the spiking 

procedure in Section 14.

9.5.2 Select sample volumes based on previous analytical results or anticipated levels of E. coli in 

the field sample in order to achieve the recommended target range of E. coli (20-80 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 

11 September 2014
Method 1603

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 E. coli (CFU/100 mL) according to Section 13 and 

adjust the colony counts based on any background E. coli observed in the unspiked matrix 

sample.

9.5.5 Calculate the percent recovery (R) for the MS sample (adjusted based on ambient E. coli 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 

recreational waters). 

Table 2.Matrix Spike Precision and Recovery Acceptance Criteria 

Performance test Lab-prepared acceptance 

criteria

BioBallTM acceptance 

criteria

Percent recovery for MS 12% - 149% 17% - 117%

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 1603. 

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 

modified mTEC 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. faecalis

(e.g., ATCC 19433) 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 modified 

mTEC agar is performing properly. Positive controls should be analyzed whenever a new 

12 September 2014
Method 1603

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. coli (e.g., 

ATCC 11775) 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

Cytochrome oxidase reagent E. faecalis E. coli

Kovac’s indole reagent E. coli E. aerogenes

Simmons citrate agar E. aerogenes E. coli

EC broth (44.5°C ± 0.2°C) E. coli 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°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 or phosphate-buffered dilution water, place the 

filter on a modified mTEC agar plate and process according to Section 11. 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 or phosphate-buffered dilution water 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, modified mTEC, 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% 

13 September 2014
Method 1603

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, 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 modified mTEC agar as directed in Section 7.6.

11.2 Mark the petri dish and report form with the sample identification and volume.

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 held between the funnel and the base.

11.4 Shake the sample bottle vigorously at least 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 pollution level, to produce 20-80 E. coli

colonies on the membranes. It is recommended that a minimum of three dilutions be analyzed to ensure 

that a countable plate (20-80 E. coli colonies) is obtained. Sample volumes of 1-100 mL may be tested 

at half-log intervals (e.g., 100, 30, 10, 3 mL). 

11.6 Smaller sample sizes 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 

modified mTEC 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 

14 September 2014
Method 1603

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 35°C ± 0.5°C for 2 ± 0.5 hours. 

11.9 After a 2 ± 0.5 hour incubation at 35°C ± 0.5°C, transfer the plates to a Whirl-Pak® bag, seal the bag, 

and submerge in a 44.5°C ± 0.2°C water bath for 22 ± 2 hours.

Note: Do not overfill the Whirl-Pak® bag because this will prevent proper sealing allowing liquid to 

enter the bag and possibly contaminating the plates. 

11.10 After 22 ± 2 hours, remove the plates from the water bath, count and record the number of red or 

magenta colonies with the aid of an illuminated lens with a 2-5X magnification or a stereoscopic 

microscope (See Photo 1).

Photo 1. E. coli colonies on modified mTEC agar are red to magenta.

12.0 Verification Procedure

12.1 Red or magenta colonies are considered “typical” E. coli. Verification of typical and atypical colonies 

may be required in evidence gathering and is also recommended as a means of quality control. The 

verification procedure follows.

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

typical and 10 well-isolated atypical colonies to nutrient agar plates or slants and to tryptic/trypticase soy 

broth. Incubate the agar and broth cultures for 24 ± 2 hours at 35°C ± 0.5°C. 

12.3 After incubation, transfer growth from the nutrient agar slant and perform cytochrome oxidase test. If 

the area where the bacteria were applied turns deep purple within 15 seconds, the test is positive. 

Note: Use only platinum, plastic, or wooden applicators to perform the oxidase test. Do not use iron 

or other reactive wire because it may cause false positive reactions.

12.4 Transfer growth from the tryptic/trypticase soy broth tube to Simmons citrate agar, tryptone water, and 

an EC broth.

15 September 2014
Method 1603

12.4.1 Incubate the Simmons citrate agar for 4 days at 35°C ± 2°C in an aerobic atmosphere. A 

positive reaction is indicated by growth with an intense blue color on the slant. E. coli is citrate 

negative, and thus for this organism on this medium there should be either no growth or trace 

growth with no change in agar color (i.e., medium remains dark green).

12.4.2 Incubate the EC broth at 44.5°C ± 0.2°C in a water bath for 24 ± 2 hours. The water level must 

be above the level of the EC broth in the tube. A positive test is indicated by turbidity and 

production of gas as seen in the inner durham tube.

12.4.3 Incubate the tryptone broth for 18-24 hours at 35°C ± 2°C with loosened caps. After the 

incubation period, add 0.5 mL of Kovacs Indole Reagent and shake the tube gently. Allow the 

tubes to stand for 5-10 minutes at room temperature. A positive test for indole is indicated by a 

deep red color which develops in the alcohol layer on top of the broth. 

12.5 E. coli are oxidase- negative, citrate- negative, EC growth- and gas-positive, and indole-positive.

12.6 Alternately, commercially available multi-test identification systems may be used to verify colonies. 

Inoculate the colonies into an identification system for Enterobacteriaceae that includes lactose 

fermentation, σ-nitrophenyl-β-D-galactopyranoside (ONPG), and cytochrome oxidase test reactions.

13.0 Data Analysis and Calculations

Use the following general rules to calculate the E. coli count (CFU) per 100 mL of sample:

13.1 If possible, select a membrane filter with 20-80 magenta or red colonies, and calculate the number of E. 

coli per 100 mL according to the following general formula:

E. coli / 100 mL =

Number of E. coli colonies

× 100

Volume of sample filtered (mL)

13.2 See general counting rules in Microbiological Methods for Monitoring the Environment: Water and 

Wastes, Part II, Sections C.3.5 and C.3.6 (see Appendix B).

13.3 Report results as E. coli CFU per 100 mL of sample. 

14.0 Sample Spiking Procedure

14.1 Method 1603 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) tests 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 of laboratory-prepared spikes

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

non-selective media) with Escherichia coli ATCC 11775 and incubating at 35°C ± 

16 September 2014
Method 1603

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 lauryl tryptose broth 

(LTB) by combining 99 mL of sterile PBS and 1 mL of sterile single-strength LTB 

in a sterile screw cap bottle or re-sealable dilution water container. Inoculate the 

1% LTB using a small amount of growth from the stock culture. 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 × 107

 - 1.0 × 108 E. coli 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 using laboratory-prepared suspensions

14.2.2.1 Add 0.3 mL of the spiking suspension dilution "D" to 100 mL of 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 10-7 mL. Filter the spiked sample and analyze the filter 

according to the procedures in Section 11. 

14.2.3 Enumeration of laboratory-prepared spiking suspension

14.2.3.1 Prepare trypticase soy agar (TSA) spread plates, in triplicate, for spiking

suspension dilutions "B", "C", and "D".

17 September 2014
Method 1603

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. Refer to Section 14.2.4 for

calculation of E. coli concentration in the undiluted spiking suspension. The 

number of E. coli (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 samples spiked with laboratory-prepared spiking suspensions

14.2.4.1 Calculate the concentration of E. coli (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.

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

 E. coli undiluted spike = E. coli (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

18 September 2014
Method 1603

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 E. coli Spiking Suspension Concentration

Examples

CFU / plate (triplicate analyses) from 

TSA plates in Section 2.2.5

E. coli CFU / mL in undiluted 

spiking suspension

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

Example 1 TNTC, TNTC, 

TNTC 94, 106, 89 10, 0, 4

(94+106+89) / (10-6+10-6+10-6) = 

289 / (3.0 x 10-6) = 96,333,333 = 

9.6 x 107 CFU / mL

Example 2 269, 289, 304 24, 30, 28 0, 2, 0

(269+289+30) / (10-5+10-5+10-6) = 

588 / (2.1 x 10-5) =28,000,000 = 

2.8 x 107 CFU / mL

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

14.2.4.2 Calculate true concentration (CFU / 100 mL) of spiked E. coli (T spiked E. coli) 

according to the following equation. Example calculations are provided in Table 

5, below. 

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

Where,

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

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

Vspiked per 100 mL sample = mL of undiluted spiking suspension per 100 mL sample

Table 5. Example Calculations of Spiked E. coli

EC undiluted spike V spiked per 100 mL sample T Spiked E. coli

9.6 x 107 CFU / mL 3.0 X 10-7 mL per 100 mL of sample (9.6 x 107 CFU/mL) x (3.0 x 10-7 mL/100 mL) =

28.8 CFU / 100 mL

2.8 x 107 CFU / mL 3.0 X 10-7 mL per 100 mL of sample (2.8 x 107 CFU/mL) x (3.0 x 10-7 mL/100 mL) = 

8.4 CFU / 100 mL

14.2.4.3 Calculate percent recovery (R) of spiked E. coli (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 = E. coli (CFU/100 mL) in the spiked sample (Section 13)

Nu = E. coli (CFU/100 mL) in the unspiked sample (Section 13)

19 September 2014
Method 1603

T = True spiked E. coli (CFU/100 mL) in spiked sample (Section 14.2.4.2)

Table 6 Example Percent Recovery Calculations

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

16 <1 8.4

100 x (16 - 1) / 8.4

= 179%

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 (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 E. coli (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 = E. coli (CFU/100 mL) in the spiked sample (Section 13)

Nu = E. coli (CFU/100 mL) in the unspiked sample (Section 13)

T = True spiked E. coli (CFU/100 mL) in spiked sample based on the lot mean value 

provided by manufacturer

Table 7. Example 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%

20 September 2014
Method 1603

15.0 Method Performance

15.1 Performance Characteristics (Reference 18.6)

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 modified mTEC 

method precision was found to be fairly representative of what would be expected from counts 

with a Poisson distribution.

15.1.2 Bias – The persistent positive or negative deviation of the average value of the method from the 

assumed or accepted true value. The bias of the modified mTEC method has been reported to 

be -2% of the true value.

15.1.3 Specificity – The ability of a method to select and or distinguish the target bacteria under test 

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 false positive 

rate reported for modified mTEC medium averaged 6% for marine and fresh water samples. 

Five percent of the E. coli colonies observed gave a false negative reaction.

15.1.4 Upper Counting Limit (UCL) – That colony count above which there is an unacceptable 

counting error. The error may be due to overcrowding or antibiosis. The UCL for E. coli on 

modified mTEC medium has been reported as 80 colonies per filter.

15.2 Interlaboratory validation of Method 1603 in disinfected wastewater (Reference 18.2)

15.2.1 Eight volunteer laboratories, an E. coli verification laboratory, and a research laboratory 

participated in the U.S. Environmental Protection Agency's (EPA's) interlaboratory validation 

study of EPA Method 1603. 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 18.2). 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 1603.

15.2.2 Recovery - Method 1603 was characterized by mean laboratory-specific recoveries of E. coli

from disinfected wastewater samples spiked with laboratory-prepared spikes ranging from 

47.8% to 106%, with an overall mean recovery of 80.7%. For PBS samples spiked with 

laboratory spiking suspensions, mean laboratory-specific recoveries ranged from 70.7% to 

109.7%, with an overall mean recovery of 82.9%.

15.2.3 Precision - Method 1603 was characterized by laboratory-specific relative standard deviations 

(RSDs) from disinfected wastewater samples spiked with laboratory-prepared spikes ranging 

from 6.1% to 51.4%, with an overall pooled, within-laboratory RSD of 25.9%. For PBS 

samples spiked with laboratory-prepared spiking suspensions, laboratory-specific RSDs ranged 

from 7.7% to 29.6%, with an overall pooled, within-laboratory RSD of 19.6%.

15.2.4 False positive rates - Method 1603 laboratory-specific false positive rates determined from all 

unspiked disinfected and secondary results combined, ranged from 0% - 6.7%. For secondary 

wastewater (excluding disinfected results), only one of 41 typical colonies submitted to 

21 September 2014
Method 1603

verification was non-E. coli, resulting in a false positive rate of 2.4%. For disinfected 

wastewater (excluding secondary results), only one of 67 typical colonies submitted to 

verification was non-E. coli, resulting in a false positive rate of 1.5%. Since all 785 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 

18.2). It is estimated that 0.6% and 1.4% 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 1603 laboratory-specific false negative rates determined from all 

unspiked disinfected and secondary results combined, also ranged from 0% - 6.7%. For 

secondary wastewater (excluding disinfected results), two of 33 atypical colonies submitted to 

verification were identified as E. coli, resulting in a false negative rate of 6.1%. For disinfected 

wastewater (excluding secondary results), three of 75 atypical colonies submitted to verification 

were identified as E. coli, resulting in a false negative rate of 4.0%. Since all 732 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 

2.5% and 2.6% 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 163 263 67 1 1.5% 0.6% 75 3 4.0% 2.5%

Secondary 622 469 41 1 2.4% 1.4% 33 2 6.1% 2.6%

Disinfected & 

Secondary 785 732 108 2 1.9% 1.0% 108 5 4.6% 2.2%

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., [(622 ×(1/41))/(622+469)] × 100 = 1.4% 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., [(469 ×(2/33))/(622+469)] × 100 = 2.6%

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.

22 September 2014
Method 1603

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 required.

17.2 Samples, reference materials, and equipment known or suspected to have viable E. coli 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 Dufour, A.P. Health Effects Criteria for Fresh Recreational Waters, EPA-600/1-84-004. Research 

Triangle Park, NC: U.S. Environmental Protection Agency, 1984.

18.2 USEPA. 2004. Results of the Interlaboratory Validation of EPA Method 1603 (modified mTEC) for E. 

coli in Wastewater Effluent. EPA-821-R-04-009. December 2004.

18.3 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.

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

Public Health Association, Washington D.C.

18.5 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.

18.6 Smith, B. G. and A. P. Dufour. 1997. A Modified mTEC Medium for Monitoring Recreational Waters. 

American Society for Microbiology General Meeting, Miami Beach, FL, May 1997.

23 September 2014
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 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.

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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. 

A-2 
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 

A-3 
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.

A-4 
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 

A-5 
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.

A-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 

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.

B-1 

 
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. 

B-2 
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:

B-3 
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.

B-4
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.

B-5