&EPA
Off ice of Water
www.epa.gov
United States
Environmental Protection
Agency
EPA 821-R-14-011
September 2014
Method 1600: Enterococci in
Water by Membrane Filtration
Using membrane-Enterococcus
Indoxyl-p-D-Glucoside Agar (mEI)
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-14-011
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Method 1600
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
waste water 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, loannice 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, loannice 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
September 2014
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Method 1600
Disclaimer
The Engineering and Analysis Division, of the Office of Science and Technology, has reviewed and approved
this report for publication. The Office of Science and Technology directed, managed, and reviewed the work of
DynCorp in preparing this report. Neither the United States Government nor any of its employees, contractors,
or their employees make any warranty, expressed or implied, or assumes any legal liability or responsibility for
any third party's use of or the results of such use of any information, apparatus, product, or process discussed in
this report, or represents that its use by such party would not infringe on privately owned rights. Mention of
trade names or commercial products does not constitute endorsement or recommendation for use.
Questions concerning this method or its application should be addressed to:
Robin K. Oshiro
Engineering and Analysis Division (4303T)
U.S. EPA Office of Water, Office of Science and Technology
1200 Pennsylvania Avenue, NW
Washington, DC 20460
oshiro .robin@epa.gov
202-566-1053 (facsimile)
September 2014
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Method 1600
Table of Contents
Acknowledgments i
Disclaimer ii
1.0 Scope and Application 1
2.0 Summary of Method 1
3.0 Definitions 1
4.0 Interferences 2
5.0 Safety 2
6.0 Equipment and Supplies 2
7.0 Reagents and Standards 3
8.0 Sample Collection, Handling, and Storage 7
9.0 Quality Control 7
10.0 Calibration and Standardization 12
11.0 Procedure 12
12.0 Verification Procedure 13
13.0 Data Analysis and Calculations 14
14.0 Sample Spiking Procedure 14
15.0 Method Performance 18
16.0 Pollution Prevention 21
17.0 Waste Management 21
18.0 References 22
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Method 1600
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).
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Method 1600
Method 1600: Enterococci in Water by Membrane Filtration Using
membrane-Enterococcus Indoxyl-p-D-Glucoside Agar (mEI)
September 2014
1.0 Scope and Application
1.1 Method 1600 describes a membrane filter (MF) procedure for the detection and enumeration of the
enterococci bacteria in water. This is a single-step method that is a modification of EPA Method 1106.1
(mE-EIA). Unlike the mE-EIA method, it does not require the transfer of the membrane filter to
another medium. The modified medium has a reduced amount of triphenyltetrazolium chloride (TTC)
and includes indoxyl (3-D-glucoside, a chromogenic cellobiose analog used in place of esculin. In this
procedure, p-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 CFRPart 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.
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. gattinarium, and their variants. The genus Enterococcus includes the enterococci formerly
assigned to the Group D fecal streptococci.
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Method 1600
4.0 Interferences
4.1 Water samples containing colloidal or suspended particulate materials can clog the membrane filter and
prevent filtration, or cause spreading of bacterial colonies which could interfere with enumeration and
identification of target colonies.
5.0 Safety
5.1 The analyst/technician must know and observe the normal safety procedures required in a microbiology
laboratory while preparing, using, and disposing of cultures, reagents, and materials, and while operating
sterilization equipment.
5.2 The selective medium (mEI) and azide-dextrose broth used in this method contain sodium azide as well
as other potentially toxic components. Caution must be exercised during the preparation, use, and
disposal of these media to prevent inhalation or contact with the medium or reagents.
5.3 This method does not address all of the safety issues associated with its use. It is the responsibility of
the laboratory to establish appropriate safety and health practices prior to use of this method. A
reference file of material safety data sheets (MSDSs) should be available to all personnel involved in
Method 1600 analyses.
5.4 Mouth-pipetting is prohibited.
6.0 Equipment and Supplies
6.1 Glass lens with magnification of 2-5X or stereoscopic microscope
6.2 Lamp, with a cool, white fluorescent tube
6.3 Hand tally or electronic counting device
6.4 Pipet container, stainless steel, aluminum or borosilicate glass, for glass pipets
6.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, of appropriate volume
6.6 Sterile graduated cylinders, 100-1000 mL, covered with aluminum foil or kraft paper
6.7 Sterile membrane filtration units (filter base and funnel), glass, plastic or stainless steel, wrapped with
aluminum foil or kraft paper
6.8 Ultraviolet unit for sanitization of the filter funnel between filtrations (optional)
6.9 Line vacuum, electric vacuum pump, or aspirator for use as a vacuum source (In an emergency or in the
field, a hand pump or a syringe equipped with a check valve to prevent the return flow of air, can be
used)
6.10 Flask, filter, vacuum, usually 1 L, with appropriate tubing
September 2014 2
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Method 1600
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 Om 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 Water bath 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 Ib 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).
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Method 1600
7.4 Phosphate buffered saline (PBS)
7.4.1 Composition:
Sodium dihydrogen phosphate (NaFfcPO/i) 0.5 8 g
Disodium hydrogen phosphate (NfeHPC^) 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 (3-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 x 50 mm or 15 x 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
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Method 1600
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 water bath. Pour the medium
into each 15 x 60 mm culture dish to a 4-5 mm depth (approximately 4-6 mL), and allow to
solidify. Final pH should be 7.3 ± 0.2.
7.7 Brain heart infusion broth (BFflB)
7.7.1 Composition:
Calf brains, infusion from 200.0 g 7.7 g
Beef heart, infusion from 250.0 g 9.8g
Proteose peptone 10.0 g
Sodium chloride (NaCl) 5.0 g
Disodium hydrogen phosphate (N^FIPO/O 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 (BfflB) with 6.5% NaCl
7.8.1 Composition:
BfflB with 6.5%NaCl is the same as BfflB above (Section 7.7), but with additional NaCl.
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 BfflB 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 (BfflA)
7.9.1 Composition:
BfflA contains the same components as BfflB (Section 7.7), with the addition of 15.0 g agar per
liter of BfflB.
7.9.2 Add agar to formula for BfflB 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.
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Method 1600
7.10 Bile esculin agar (BEA)
7.10.1 Composition:
Beef Extract 3.0g
Pancreatic Digest of Gelatin 5.0 g
Oxgall 20.0 g
Esculin l.Og
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 water bath, and dispense into sterile petri dishes.
Final pH should be 6.8 ± 0.2. Store in a refrigerator.
7.11 Azide dextrose broth (ADB)
7.11.1 Composition:
Beef extract 4.5 g
Pancreatic digest of casein 7.5 g
Proteose peptone No. 3 7.5 g
Dextrose 7.5 g
Sodium chloride (NaCl) 7.5 g
Sodium azide 0.2 g
Reagent-grade water 1.0 L
7.11.2 Add reagents to 1 L of reagent-grade water and dispense in screw cap bottles. Autoclave at
121 °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 (bioMerieux, Durham NC)
7.12.2 Negative control organism (for each organism, either of the following are acceptable)
• Stock cultures of Escherichia coll (E. coll) ATCC 11775
• E. coli ATCC 11775 BioBalls (bioMerieux, Durham NC)
• Stock cultures of Enterobacter aerogenes (E. aerogenes) ATCC 13048
• E. aerogenes NCTC 10006 BioBalls (bioMerieux, Durham NC)
September 2014
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Method 1600
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.
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
September 2014
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Method 1600
(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
Initial precision and recovery (IPR)
Mean percent recovery
Precision (as maximum relative standard deviation)
Ongoing precision and recovery (OPR) as percent recovery
Lab-prepared spike
acceptance criteria
31% -127%
28%
27% -131%
BioBall™
acceptance criteria
85% -106%
14%
78% -113%
9.4 Ongoing precision and recovery (OPR)—To demonstrate ongoing control of the analytical system, the
laboratory should routinely process and analyze spiked PBS samples. The laboratory should analyze
one OPR sample after every 20 field and matrix spike samples or one per week that samples are
analyzed, whichever occurs more frequently. OPR samples must be accompanied by an acceptable
method blank (Section 9.9) and appropriate media sterility checks (Section 9.11). The OPR analysis is
performed as follows:
9.4.1 Spike a 100-mL PBS sample with E. faecalis ATCC 19433 according to the spiking procedure
in Section 14. Spiking with laboratory-prepared suspensions is described in Section 14.2 and
spiking with BioBalls is described in Section 14.3. Filter and process each OPR sample
September 2014
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Method 1600
according to the procedures in Section 11 and calculate the number of enterococci per 100 mL
according to Section 13.
9.4.2 Calculate the percent recovery (R) for the OPR sample using the appropriate equation in Section
14.2.2 or 14.3.4 for samples spiked with laboratory-prepared spiking suspensions or BioBalls,
respectively.
9.4.3 Compare the OPR result (percent recovery) with the corresponding OPR recovery criteria in
Table 1, above. If the OPR result meets the acceptance criteria for recovery, method
performance is acceptable and analysis of field samples may continue. If the OPR result falls
outside of the acceptance criteria, system performance is unacceptable. In this event, identify
the problem by evaluating each step of the analytical process, media, reagents, and controls,
correct the problem and repeat the OPR analysis.
9.4.4 As part of the laboratory QA program, results for OPR and IPR samples should be charted and
updated records maintained in order to monitor ongoing method performance. The laboratory
should also develop a statement of accuracy for Method 1600 by calculating the average percent
recovery (R) and the standard deviation of the percent recovery (sr). Express the accuracy as a
recovery interval from R - 2sr to R + 2sr.
9.5 Matrix spikes (MS)—MS analysis are performed to determine the effect of a particular matrix on
enterococci recoveries. The laboratory should analyze one MS sample when disinfected wastewater
samples are first received from a source from which the laboratory has not previously analyzed samples.
Subsequently, 5% of field samples (1 per 20) from a given disinfected wastewater source should include
a MS sample. MS samples must be accompanied by the analysis of an unspiked field sample
sequentially collected from the same sampling site, an acceptable method blank (Section 9.9), and
appropriate media sterility checks (Section 9.11). When possible, MS analyses should also be
accompanied by an OPR sample (Section 9.4), using the same spiking procedure (laboratory-prepared
spiking suspension or BioBalls). The MS analysis is performed as follows:
9.5.1 Prepare two, 100-mL field samples that were sequentially collected from the same site. One
sample will remain unspiked and will be analyzed to determine the background or ambient
concentration of enterococci for calculating MS recoveries (Section 9.5.3). The other sample
will serve as the MS sample and will be spiked with E.faecalis ATCC 19433 according to the
spiking procedure in Section 14.
9.5.2 Select sample volumes based on previous analytical results or anticipated levels of in the field
sample in order to achieve the recommended target range of enterococci (20-60 CPU, 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
September 2014
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Method 1600
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
Percent recovery for MS
Lab-prepared acceptance criteria
29% -122%
BioBall™ acceptance criteria
63% -110%
9.5.8 Laboratories should record and maintain a control chart comparing MS recoveries for all
matrices to batch-specific and cumulative OPR sample results analyzed using Method 1600.
These comparisons should help laboratories recognize matrix effects on method recovery and
may also help to recognize inconsistent or sporadic matrix effects from a particular source.
9.6 Culture Controls
9.6.1 Negative controls—The laboratory should analyze negative controls to ensure that the mEI
agar is performing properly. Negative controls should be analyzed whenever a new batch of
media or reagents is used. On an ongoing basis, the laboratory should perform a negative
control every day that samples are analyzed.
9.6.1.1 Negative controls are conducted by filtering a dilute suspension of viable E. coll
(e.g., ATCC 11775) orE! aerogenes (e.g., ATCC 13048) and analyzing as described
in Section 11. Viability of the negative controls should be demonstrated using a
non-selective media (e.g., nutrient agar or tryptic soy agar).
9.6.1.2 If the negative control fails to exhibit the appropriate response, check and/or replace
the associated media or reagents, and/or the negative control, and reanalyze the
appropriate negative control.
9.6.2 Positive controls—The laboratory should analyze positive controls to ensure that the mEI agar
is performing properly. Positive controls should be analyzed whenever a new batch of media
or reagents is used. On an ongoing basis, the laboratory should perform a positive control
September 2014 10
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Method 1600
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 K 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
Bileesculin agar(BEA)
Brain heart infusion broth (BHIB) with 6.5% NaCI
Brain heart infusion broth (BHIB) incubated at 45 °C
Positive Control
£ faecalis
E. faecalis
E. faecalis
Negative Control
£ co//
£ co//
£ 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 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
11 September 2014
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Method 1600
single analyst. If no positive field samples are available, an OPR sample may be substituted for these
determinations.
10.0 Calibration and Standardization
10.1 Check temperatures in incubators twice daily with a minimum of 4 hours between each reading to ensure
operation within stated limits.
10.2 Check thermometers at least annually against a NIST certified thermometer or one that meets the
requirements of NIST Monograph SP 250-23. Check mercury columns for breaks.
10.3 Refrigerators used to store media and reagents should be monitored daily to ensure proper temperature
control.
11.0 Procedure
11.1 Prepare the mEI agar as directed in Section 7.5.
11.2 Mark the petri dishes and report forms with sample identification and sample volumes.
11.3 Place a sterile membrane filter on the filter base, grid-side up and attach the funnel to the base so that the
membrane filter is now held between the funnel and the base.
11.4 Shake the sample bottle vigorously about 25 times to distribute the bacteria uniformly, and measure the
desired volume of sample or dilution into the funnel.
11.5 Select sample volumes based on previous knowledge of the enterococci level, to produce 20-60
enterococci colonies on membranes. It is recommended that a minimum of three dilutions be analyzed
to ensure that a countable plate (20-60 enterococci colonies) is obtained.
11.6 Smaller sample size or sample dilutions can be used to minimize the interference of turbidity or for high
bacterial densities. Multiple volumes of the same sample or sample dilutions may be filtered.
Note: When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS or
phosphate-buffered dilution water should be added to the funnel or an aliquot of sample should
be dispensed into a dilution blank prior to filtration. This will allow even distribution of the
sample on the membrane.
11.7 Filter the sample, and rinse the sides of the funnel at least twice with 20-30 mL of sterile buffered rinse
water. Turn off the vacuum and remove the funnel from the filter base.
11.8 Use sterile forceps to aseptically remove the membrane filter from the filter base, and roll it onto the mEI
Agar to avoid the formation of bubbles between the membrane and the agar surface. Reseat the
membrane if bubbles occur. Run the forceps around the edge of the filter outside the area of filtration,
close to the edge of the dish, to be sure that the filter is properly seated on the agar. Close the dish,
invert, and incubate at 41°C ± 0.5 °C for 24 ± 2 hours.
September 2014 12
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Method 1600
Note: If the medium is prepared in 15 x 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.
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 BfflB tube and onto a BfflA 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 BfflB tube to BEA, BHIB, and BfflB
with 6.5% NaCl.
12.3.1 Incubate BEA and BfflB with 6.5% NaCl at 35 °C ± 0.5 °C for 48 ± 3 hours.
12.3.2 Incubate BfflB 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 BfflA slant.
13
September 2014
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Method 1600
12.6 Gram-positive cocci that grow and hydrolyze esculin on BEA (i.e., produce a black or brown
precipitate), and grow in BfflB with 6.5% NaCl at 35 °C ± 0.5 °C and BfflB 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% Nad.
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:
Number of enterococci colonies
Enterococci/100mL= x 100
Volume of sample filtered (mL)
13.2 See general counting rules in Reference 18.7 (see Appendix B).
13.3 Report results as enterococci per 100 mL of sample.
14.0 Sample Spiking Procedure
14.1 Method 1600 QC requirements (Section 9.0) include the preparation and analysis of spiked reference
(PBS) and matrix samples in order to monitor initial and ongoing method performance. For the IPR
(Section 9.3), OPR (Section 9.4), and MS (Section 9.5) analyses it is necessary to spike samples with
either laboratory-prepared spiking suspensions (Section 14.2) or BioBalls (Section 14.3) as described
below.
14.2 Laboratory-Prepared Spiking Suspensions
14.2.1 Preparation
14.2.1.1 Stock Culture. Prepare a stock culture by inoculating a TSA slant (or other
non-selective media) with E.faecalis ATCC 19433 and incubating at 35 °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
September 2014 14
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Method 1600
approximately 1.0 * 106-1.0x 107 E.faecalis colony forming units (CPU) 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^
mL of the original undiluted spiking suspension.
14.2.1.3.3 Dilution "C"—Aseptically transfer 11.0 mL of dilution "B" to 99 mL of
sterile PBS and mix thoroughly by shaking the bottle a minimum of 25
times. This is spiking suspension dilution "C" and 1 mL contains 10"5
mL of the original undiluted spiking suspension.
14.2.1.3.4 Dilution "D"—Aseptically transfer 11.0 mL of dilution "C" to 99 mL
of sterile PBS and mix thoroughly by shaking the bottle a minimum of
25 times. This is spiking suspension dilution "D" and 1 mL contains
10"6 mL of the original undiluted spiking suspension.
14.2.2 Sample spiking
14.2.2.1 Add 3.OmL of the spiking suspension dilution "D" (Section 14.2.1.3.4)to lOOmLor
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 i oo 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.
15 September 2014
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Method 1600
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 CPU per plate.
14.2.4 Recovery calculations for spiked samples
14.2.4.1 Calculate the concentration of enterococci (CFU/mL) in the undiluted spiking
suspension (Section 14.2.1.2) according to the following equation. Example
calculations are provided in Table 4, below.
Enterococci mdiiuted splke = (CFUi + CFU2 + ...+ CFUn)/(Vi + V2 + ... + Vn)
Enterococci mdiiuted 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.
September 2014 16
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Method 1600
Table 4. Example Calculations of Laboratory-prepared Enterococci Spiking Concentration
Examples
Example 1
Example 2
CFU/plate (triplicate analyses) from
TSA plates
10"5 mL plates
94, 106,89
32, 55, 72
10-6 mL plates
9, 11,28
8,5,3
10'7 mL plates
1,0,4
0,0,0
Enterococci CFU/mL in undiluted
spiking suspension
(EnterOCOCCi undiluted spike)3
(94+106+89)/(10-5+10-5+10-5) =
289/(3.0x10-5) = 9,633,333 =
9.6 x 10s CFU/mL
(32+55+72)/(10-5+10-5+10-5) =
159/(3.0x10-5) = 5,300,000 =
5.3 x 10s CFU/mL
3 Enterococci undiluted spike is calculated using all plates yielding counts within the countable range of 30 to 300 CPU per plate
14.2.4.2 Calculate true concentration of spiked enterococci (CFU/100 mL) according to the
following equation. Example calculations are provided in Table 5, below.
1 spiked Enterococci
Where,
(EnterOCOCCi undiluted spike) X (V spiked per 100 mL sample)
Tspked Enterococci = Number of spiked Enterococci (CPU/100 mL)
Enterococci undiluted spike = Enterococci (CFU/mL) in undiluted spiking suspension
V spiked per i oo mL sample = mL of undiluted spiking suspension per 100 mL sample
Table 5. Example Calculations for Determination "True" Spiked Enterococci Concentration
EnterOCOCCi undiluted spike
9.6 x106 CFU/mL
5.3 x106 CFU/mL
V spiked per 100 mL sample
3.0X10-6mLper100mLof
sample
3.0X10-6mLper100mLof
sample
Tspiked Enterococci
(9.6 x 1 06 CFU/mL) x (3.0 x 1 0'6 mL/1 00 mL) =
28.8 CFU/100 mL
(2.8x1 06 CFU/mL) x (3.0 x 1 0'6 mL/1 00 mL) =
8.4CFU/100mL
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=100x
(Ns - Nu)
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)
17
September 2014
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Method 1600
Table 6. Example Percent Recovery Calculations for Lab-prepared Spiked Samples
Ns(CFU/100mL)
42
34
10
Nu (CFU/100 mL)
<1
10
<1
Tspiked Enterococci (CFU/100 mL)
28.8
28.8
8.4
Percent recovery (R)
1 00 x (42-1)728.8
= 142%
1 00 x (34-10)728.8
= 83%
100x(10-1)/8.4
= 107%
14.3 BioBall™ Spiking Procedure
14.3.1 Aseptically add 1 BioBall™ to 100 mL of PB S 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 = 100x
(Ns - Nu)
Where,
R
Ns =
Nu =
Percent recovery
Enterococci (CFU/100 mL) in the spiked sample (Section 13)
Enterococci (CFU/100 mL) in the unspiked sample (Section 13)
True spiked enterococci (CFU/100 mL) in spiked sample based on the lot mean
value provided by manufacturer
Table 7. Example BioBall™ Percent Recovery Calculations
Ns (CFU/100 mL)
24
36
Nu (CFU/100 mL)
<1
10
T (CFU/100 mL)
11.2
32
Percent recovery (R)
1 00 x (24 -1)732 = 72%
1 00 x (36 -10)732 = 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.
September 2014
18
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Method 1600
15.1.3 Specificity - The ability of a method to select and/or distinguish the target bacteria from other
bacteria in the same water sample. The specificity characteristic of a method is usually
reported as the percent of false positive and false negative results. The specificity for this
medium as reported for various environmental water samples was 6.0% false positive and 6.5%
false negative.
15.1.4 Multilaboratory variability - A collaborative study was conducted among fourteen collaborators
at twelve laboratories to examine the interlaboratory reproducibility of the method.
Reproducibility among laboratories (RSDR) for freshwater, marine water, chlorinated
secondary effluent, and non-chlorinated primary effluent ranged from 2.2% for marine water to
18.9% for freshwater with a low enterococcal density.
15.2 Interlaboratory Validation of Method 1600 in Disinfected Wastewater (Reference 18.3)
15.2.1 Twelve volunteer participant laboratories, two enterococci verification laboratories, and two
research laboratories participated in the U.S. Environmental Protection Agency's (EPA's)
interlaboratory validation study of EPA Method 1600. The purposes of the study were to
characterize method performance across multiple laboratories and disinfected wastewater
matrices and to develop quantitative quality control (QC) acceptance criteria. A detailed
description of the study and results are provided in the validation study report (Reference 18.3).
Results submitted by laboratories were validated using a standardized data review process to
confirm that results were generated in accordance with study-specific instructions and the
September 2002 version of EPA Method 1600.
15.2.2 Recovery - Method 1600 was characterized by mean laboratory-specific recoveries of
enterococci from disinfected wastewater samples spiked with BioBalls™ 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 BioBalls™ ranging from 0% to
69.5%, with an overall pooled, within-laboratory RSD of 22.6%. For PBS samples spiked
with BioBalls, laboratory-specific RSDs ranged from 3.1% to 13.7%, with an overall pooled,
within-laboratory RSD of 8.1%.
15.2.4 False positive confirmation rates - Method 1600 laboratory-specific false positive confirmation
rates for unspiked disinfected/secondary results combined, ranging from 0.0% to 10.0%. For
secondary wastewater (excluding disinfected results), only 2 of 123 typical colonies submitted
to verification were non-enterococci, resulting in a false positive confirmation rate of 1.6%.
For disinfected wastewater (excluding secondary results), none of the 66 typical colonies
submitted to verification were non-enterococci, resulting in a false positive confirmation rate of
0.0%. Since all 2184 typical colonies observed during the study could not be submitted to
confirmation, the percent of total colonies that would have resulted in a false positive result was
estimated (see Table 6, Reference 18.3). It is estimated that 0.0% and 1.2% of the total
colonies would have resulted in a false positive for disinfected wastewater and secondary
wastewater, respectively.
15.2.5 False negative rates - Method 1600 laboratory-specific false negative rates laboratory-specific
false negative confirmation rates for unspiked disinfected/secondary results combined, ranged
from 28.6% to 100.0%. For secondary wastewater (excluding disinfected results), 62 of 79
19 September 2014
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Method 1600
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
Disinfected
Secondary
Disinfected +
Secondary
Total colonies
Typical
391
1793
2184
Atypical
105
734
839
False positive (FP) assessment
Typical
colonies
submitted
66
123
189
No. FP
colonies
0
2
2
FP
confirmation
rate (%) a
0.0
1.6
1.1
Estimated
% of total
colonies
that would
have been
aFPb
0.0
1.2
0.8
False negative (FN) assessment
Atypical
colonies
submitted
8
79
87
No. FN
colonies
8
62
70
FN
confirmation
rate (%) c
100.0
78.5
80.5
Estimated %
of total
colonies that
would have
been a FN d
21.2
22.8
22.3
False positive confirmation rate = number of false positive colonies/number of typical colonies submitted
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 x(2/123))/(1793+734)] x 100 = 1.2%
False negative confirmation rate = number of false negative colonies/number of atypical colonies submitted
Percent of total colonies estimated to be false negatives = [(total atypical colonies* FN confirmation rate)/(total number of
typical and atypical colonies observed)] x 100; e.g., [(734 x(62/79))/(1793+734)] x 100 = 22.8%
15.2.6 During evaluation of the study results, it was noted that many of the false negatives (atypical
colonies submitted to verification which were identified as enterococci) were pink to red in
color but simply lacked a blue halo. The predecessor to EPA Method 1600 for enterococci is
EPA Method 1106.1 which uses mE and EIA media. For EPA Method 1106.1, pink to red
colonies on mE, which produce a brown precipitate after transfer to EIA are considered positive
for enterococci. Tetrazolium chloride (TTC), the reagent responsible for producing pink to red
enterococci colonies on mE, is also included as a reagent in mEI. A follow-on study was
conducted, for which pink to red colonies without halos from unspiked secondary wastewaters
were submitted to verification. For pink to red colonies without halos that were >0.5 mm
colony size, 54 of 90 colonies submitted were identified as enterococci, resulting in a 60.0%
verification rate.
Results of the verification analyses from the initial study were assessed with pink to red colonies
without halos being counted as enterococci. When pink to red colonies without halos are
September 2014
20
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Method 1600
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.)
Disinfected
(Samples 1-4)
Secondary
(Samples 5,
6)
Disinfected &
Secondary
(Samples 1-6)
Total colonies
Typical
477
2291
2768
Atypical
19
236
255
False positive (FP) assessment
Typical
colonies
submitted
69
166
235
No. FP
colonies
0
7
7
FP
confirmation
rate (%) a
0.0
4.2
3.0
Estimated
% of total
colonies
that would
have been
aFPb
0.0
3.8
2.7
False negative (FN) assessment
Atypical
colonies
submitted
4
32
36
No. FN
colonies
3
27
30
FN
confirmation
rate (%) c
75.0
84.4
83.3
Estimated %
of total
colonies that
would have
been a FN d
2.9
7.9
7.0
False positive confirmation rate = number of false positive colonies/number of typical colonies submitted
Percent of total colonies estimated to be false positives = [(total typical colonies * FP confirmation rate)/(total number of
typical and atypical colonies observed)] x 100; e.g., [(2291 x(7/166))/(2291+236)] x 100 = 3.8%
False negative confirmation rate = number of false negative colonies/number of atypical colonies submitted
Percent of total colonies estimated to be false negatives = [(total atypical colonies x FN confirmation rate)/(total number of
typical and atypical colonies observed)] x 100; e.g., [(236 x(27/32))/(2291+236)] x 100 = 7.9%
16.0 Pollution Prevention
16.1 The solutions and reagents used in this method pose little threat to the environment when recycled and
managed properly.
16.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize the
volume of expired materials to be disposed.
17.0 Waste Management
17.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations governing
waste management, particularly the biohazard and hazardous waste identification rules and land disposal
restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fume
21
September 2014
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Method 1600
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 HC1 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 APFIA. 1998. Standard Methods for the Examination of Water and Wastewater. 20th Edition. American
Public Health Association, Washington D.C.
18.7 Bordner, R, J.A. Winter, and P.V. Scarpino (eds.). Microbiological Methods for Monitoring the
Environment: Water and Wastes, EPA-600/8-78-017. Cincinnati, OH: U.S. Environmental Protection
Agency, 1978.
September 2014 22
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Appendix A
Part II (General Operations)
Section A (Sample Collection, Preservation, and Storage)
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Sample Collection1
1.0 Sample Containers
1.1
1.2
1.3
1.4
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.
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.
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.
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
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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.
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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
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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.
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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
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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.
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Appendix B
Part II (General Operations)
Sections C.3.5 (Counting Colonies) and C.3.6 (Calculation of Results)
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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.
1
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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)
= 30 CPU / 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:
— xlOO = 550 CFU/100 mL
10 '
and
30
— xlOO = 3000 CFU/100 mL
Calculate the arithmetic mean as in section 2.3.1 above:
(550 CFU/100 mL + 3000 CFU/100 mL)
1 = 1775 CPU / 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.
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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
— xlOO = 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:
— xlOO = 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:
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110
xlOO = 1,100,000 CFU/100 mL
0.01 '
Report this as estimated count 1.1 x 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
xlOO = 600,000 CFU/100 mL
0.01 '
Report this as > (greater than) 6 x 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
— xlOO = 180 CFU/100 mL
10 '
Report this as estimated count 180 CFU/100 mL.
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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.
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