PROTOCOL FOR ALTERNATE TEST PROCEDURES FOR COLIFORM BACTERIA
IN COMPLIANCE WITH DRINKING WATER REGULATIONS
PRESENCE/ABSENCE LIQUID CULTURE METHODS FOR FINISHED WATERS
Version 1.2
December, 1995
U. S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL EXPOSURE RESEARCH LABORATORY
CINCINNATI, OHIO 45268
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PROTOCOL FOR ALTERNATE TEST PROCEDURES FOR COLIFORM BACTERIA
IN COMPLIANCE WITH DRINKING WATER REGULATIONS
PRESENCE/ABSENCE LIQUID CULTURE METHODS FOR FINISHED WATERS
I. INTRODUCTION
1.1 Regulatory Background
1.1.1 The Administrator, U.S. Environmental Protection Agency (EPA), approves
analytical methods for all contaminants regulated under the Safe Drinking Water
Act (SDWA). When EPA publishes a regulation under the SDWA, the regulation
generally provides for at least one method for detection and/or quantification
of that contaminant. After any regulation 1s published, the Administrator may
approve additional methods or modifications to approved methods, based on a staff
recommendation, after satisfactory comparability tests by the proposer, following
the Alternate Test Procedure (ATP) protocol.
1.1.2 The June 29, 1989 and January 8, 1991 regulations on the microbiological
characterization of finished drinking water samples require the determination of
the presence or absence of total conforms rather than their quantitative
enumeration.1''- These regulations also require that when-total conforms are
present, the presence or absence of either fecal coliforms or £. coli must also
be determined rather than enumerated. Consequently, the protocol for ATPs was
modified to evaluate this different type of method. The methods selected by EPA
for evaluation of proposed methods, are designated as Reference Methods in this
protocol.
1.1.3 If the data evaluation demonstrates that the applicant's method performs
at least as well as the currently approved method, the Ecological Exposure
Research Division of the National Exposure Research Laboratory (NERL) in
Cincinnati (formerly the Environmental Monitoring Systems Laboratory at
Cincinnati) will recommend approval to the Office of Ground Water and Drinking
Water, which begins the regulation development process. Regulation development
includes a Federal Register notice proposing to approve an ATP, public comment
on the proposed method, and (depending on public comment) a final rule published
in the Federal Register that approves the method. The regulation development
process may take about one year or more.
1.2 Comparability Determination
\ ^^—»«a^.
1.2.1 This protocol describes the method description and Comparability Study
data which EPA requires to evaluate an ATP for presence/absence liquid culture
(LC) methods in microbiology. The ATP program is intended to be flexible in that
EPA's NERL-Cincinnati may tailor the criteria to a particular method. For this
reason, before beginning the comparability testing, the applicant is required to
contact the ATP Coordinator, Ecological Exposure Research Division, NERL-
Cincinnati, U.S. Environmental Protection Agency, 26 West Martin Luther King
Drive, Cincinnati, Ohio 45268, to assure agreement on the test design.
1.2.2 Generally the reference method selected by EPA for use in the Compara-
bility Study will be the same type of test as the proposed method. Consequently,
if an LC method were proposed for the target organism, the corresponding LC
method for the target organism would be used in the Comparability Study.
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1.2.3 If a proposed method consists of more than one configuration of tubes o"
bottles, the ATP Program of NERL-Cincinnati will require the testing of only one
configuration, providing that the proposer submits sufficient data showing tne
equivalence of other configurations.
2. APPLICATION FOR ATP
2.1 General Requirements
2.1.1 The general requirements for an application for nationwide approval of a
new or revised method for total coliforms, fecal coliforms, and/or E. coli
currently include the name and address of the applicant and/or authorized
representative; the microbiological analyte for which the new procedure is
proposed; justification for the proposed new method; the title, company
identification number, the date of submission, and a complete write-up that is
a stand-alone description of the proposed method in the required format (see
Section 2.3 below).
2.1.2 If the applicant believes the proposed method is very similar to a
promulgated method, and/or represents a minor optional change of a reference
method, the applicant should also prepare a two-column side-by-side description
of the sections of the refsrence and proposed methods and highlight differences
between the methods. If the method is a proposed commercial version of a
previously approved method, differences in reagents, interferences, test
conditions, etc. should be presented with available performance data from the
proposed and reference methods.
2.1.3 NERL-Cincinnati will judge the proposed method to be: 1) an acceptable
version of or an optional minor modification of a previously promulgated method,
which does not require approval as an ATP or 2) a significantly different method
which requires an application for an ATP approval.
2.2 'Every 'application for approval of a method must"b'e'made in triplicate
(original + 2 copies) and forwarded to the Director, Ecological Exposure Research
Division, NERL-Cincinnati, USEPA, Cincinnati, OH 45268. Upon receipt of the
Application, the ATP Coordinator will assign it an identification number, which
should be used in all future communications. NERL-Cincinnati staff will initiate
its technical reviews. The initial review will concentrate on the clarity and
completeness of the description of the proposed method, the applicability of the
proposed microbiological principles and reactions and the performance
characteristics described for the method. The ATP staff will evaluate the
submitted information and advise the applicant whether or not a comparability
study is required.
2.3 Method Description
2.3.1 Each method description must include the following topics, listed in the
EMMC method format , in the order given. The purpose of the description is to:
1) permit a fair comparison of the proposed and reference methods and 2) provide
a clean, clear description of the method that can be easily used by laboratories.
The method should read like a scientific paper.
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2.3.1.1 Scope and Application
Include target organisms, type of test, e.g., membrane filter, chromogemc
test, flubrogenic test, etc. and the sample types to which it is applicable.
2.3.1.2 Summary of Method
Include a brief outline of the method that describes its essential features
without extraneous details.
2.3.1.3 Definitions
Include special terms or unique usage of terms. Do not include common
microbiological terms.
2.3.1.4 Interferences
Include Information and data generated by applicant during method
development using typical samples containing a specific quantity of an
interference such toxic materials, particulates, non-target organisms, etc.
2.3.1.5 Safety
Refer to good laboratory practices and use of a hood, goggles, and/or
protective clothing, if appropriate. Emphasize any special procedure or
precaution.
2.3.1.6 Instrumentation, Equipment and Supplies
Describe the necessary instrumentation, equipment and supplies and reference
applicable manuals.
2.3.1.7 Reagents, Standards and Media
v Describe reagent, standard and media formulations and preparation. Indicate
shelf life of packaged materials and special storage requirements.
2.3.1.8 Sample Collection, Dechlorination, Preservation, Shipment, and Storage
Detail sample collection and handling requirements. Consider the sample
collector, sample containers, dechlorination, sample holding times and
temperature conditions as specified In Standard Methods4.
2.3.1.9 .Quality Control (QC)
Indicate the specific QC procedures and the frequency of performance
required for the proposed method. They should include sterility checks, positive
and negative controls, verification/confirmation of the target organism, media
performance checks, duplicate analyses, etc. Document that a general QA/QC
program is in operation and that routine QC checks are recorded and action taken
if a problem is indicated.5'6
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2.3.1.10 Calibration and Standardization
If applicable, include the calibration steps that are performed on pH meter,
analytical balance, thermometer, autoclave, etc.
2.3.1.11 Procedure
Detail the sample preparation and analytical steps in the proposed method
write-up. Exceptions are those, routine microbiological procedures, such as
membrane filtration of samples, that are known to professionals and that may be
incorporated by reference.
2.3.1.12 Data Analyses and Calculations
Describe the procedures for data analyses, calculations, interpretation.and
reporting of results.
2.3.1.13 Method Performance Characteristics (sensitivity, specificity, recovery,
and precision)
Provide available information on the performance characteristics of the
proposed method and the procedures by which they were determined. Specificity
data should demonstrate the ability of the proposed method to recover and
distinguish the target organism from other organisms in the sample. The proposer
should have data on method sensitivity, the variability of replicate analyses and
data on recoveries of known numbers of target and non-target organisms by the
proposed method. Summaries of these evaluative data should be included.
, (However, in addition to the data provided above in the method description,
a separate formal Specificity Study will be required as part of the ATP process.
The method for determining the specificity of a method is presented in the
Appendix.)
2.3.1.14 Pollution Prevention
Pollution prevention is any technique that reduces or eliminates the
quantity or toxicity of waste at the point of generation. It is the
environmental management tool preferred over waste disposal or recycling. Uhen
feasible, laboratory staff should use a pollution prevention technique such as
preparation of the smallest practical volumes of reagents, standards and media
or downsizing of the test units in a method.
For information about pollution prevention that may be applicable to
laboratories and research institutions, consult "Less is Better: Laboratory
Chemical Management for Waste Reduction," available from the American Chemical
Society's Department of Government Regulations and Science Policy, 1155 16th
Street N.W., Washington, DC 20036, (202) 872-4477.
If the proposed method prevents or reduces exposure to toxicity, pollution
of the laboratory or the general environment including reduced generation of
wastes, cite here. Also indicate non-applicability.
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2.3.1.15 Waste Management
The Environmental Protection Agency requires that laboratory waste
management practices be conducted consistent with all applicable rules and
regulations. Excess reagents and samples and method process wastes should be
characterized and disposed of in an acceptable manner. The Agency urges
laboratories to protect the air, water, and land by minimizing and controlling
all releases from hoods and bench operations, complying with the letter and
spirit of any waste discharge permit and regulations, and by complying with all
solid and hazardous waste regulations, particularly the hazardous waste
identification rules and land disposal restrictions. For further information on
waste management consult "The Waste Management Manual for Laboratory Personnel,"
available from the American Chemical Society.
Describe the proper disposal methods for waste reagents, materials, supplies
and samples.
2.3.1.16 References
.Cite those source documents and publications which-are.necessary sources of
information to properly perform the method.
2.3.1.17 Tables, Diagrams, Flowcharts, and Validation Data
Provide as needed.
2.3.1.18 Proprietary Information
Mark proprietary Information in the proposed method description as
"Confidential". EPA staff will treat proprietary information according to the
regulations outlined in Subparts A and B in Part 2 of Title 40 of the CFR.
2.4 Study Approval
2.4.1 Method comparability data and quality control data will be required for
each application of a new or significantly modified method. The applicant is
urged not to initiate the Comparability Study until EPA has completed an
evaluation of the method description and the preliminary performance
characteristic information and has approved the Comparability Study design.
3. COMPARABILITY STUDY DESIGN
3.1 Summary of Design
3.1.1 Typically, the comparability data are generated from a single finished
water which has been spiked with different (e.g., 10) wastewaters or polluted
surface waters containing the target organisms (total coliforms and fecal
coliforms or E. coli). The spiked samples are analyzed then diluted to a 10s/
100 ml level of target organisms. In a preliminary study, spiked samples are
exposed to a chlorinating agent to stress and reduce the numbers of target
organisms. After an appropriate exposure period, sodium thiosulfate is added to
deactivate the chlorine in the samples and the target organisms are enumerated
using a membrane filter procedure to determine the dilution that will yield a
density of 1-10 organisms per 100 mL of sample. Based on these data, serial
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dilutions of each of the chlorinated samples are prepared using one of two
options. In the Comparability Study, presence/absence analyses are performed on
each of the serial dilutions by both the reference and proposed methods so as to
bracket the 1-10 target organisms/100 ml and assure a reasonable split in
positive and negative results. The identities, preparation, dilutions, and
analytical observations on the samples are recorded and submitted to the NERL-
Cincinnati staff for evaluation. See the schema on the following page.
3.1.2 A minimum of twenty (20) replicate analyses are performed by each method
on the dilutions selected for each of the 10 spiked samples: Replicate analyses
on the three or more dilutions of each spiked sample must be performed on the
same day for both the reference and proposed methods.
3.1.3 See the following example of a Comparability Study Design.
Example of ATP Study Design
Natural Sources No. Samples/
of Organisms Source
Reference Method
and 10 1
Proposed Method
Replicate Tests Minimum Comparability
of 100 ml Volumes Results Required
For Each Sample per Method
,Reference Method 20 200
Proposed Method 20 200
3.2 Laboratory Participation
3.2.1 Since the purpose of the study is to compare the proposed method to the
reference method with minimal variability due to individual analyst error, the
number of laboratories participating in the study should be minimized. It is
strongly recommended that only one lab perform the analyses. This laboratory
must be certified under the drinking water laboratory certification program. An
applicant having a vested Interest in the method, instrumentation, apparatus,
reagents, media, or associated kits, may not perform the comparability study
analyses in the applicant's laboratory.
3.3 Quality Control Data
3.3.1 QC checks must be performed on each day of analyses with known positive
and negative cultures, sterility controls, and verification of colonies.
Maintain records of QC checks performed. See the EPA Manual for the
Certification of Laboratories Analyzing Drinking Water5 and the Intralaboratory
Quality Control Guidelines in Standard Methodl^.
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SCHFMA OF COMPARABILITY STUDY
Preparation
Collect Spike (Effluent or Polluted Water)
Determine Density of Target Organisms
Spike Drinking Water to 10s Level
Conduct Preliminary Chiorination Study
Determine Target Organism Densities
Comparability Study
Chlorinate Spiked Sample to Stress Target Organisms
Dechlorinate Sample Over Increasing Exposure Times
Select Dilutions to Bracket 1-10 Organisms/100 ml
Determine Target Organism Densities (Option A Only)
Conduct Comparability Study Analyses
If No Acceptable Split in P/A Responses, Repeat Study
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3.4 Sample Collection and Handling
3.4.1 Each sample should be collected and held in a single sterile, wide-mouth
bottle of heat-resistant plastic or borosilicate glass with a leak-proof screw
cap or ground-glass stopper. The container must be resistant to the solvent
action of water and survive sterilization without any deformity or production of
toxic materials. Screw caps must not produce any bacteriostatic, toxic or
nutritive products during sterilization. Each new lot of bottles and closures
should be checked for these effects before use in the study
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2 Samples should be maintained at 1-4 C during transit and holding time.
4. PREPARATION FOR COMPARABILITY STUDY
y
4.1 Preparation of Spiked Samples
4.1.1 Since it is unlikely that finished drinking water samples can be collected
which contain total coliforms and fecal coliforms or E. coli for the
comparability study, a single drinking water sample can be spiked with these
target organisms from different sources. Recommended spiking sources are non-
chlorinated secondary sewage effluents or polluted surface waters. Both contain
large numbers of total and fecal conforms and non-coliforms; however, effluent
has the advantage of providing a wide range of £. coli strains, whereas polluted
surface waters may be more variable in quality. These sources must come from ten
or more geographically dispersed sites.
To prepare spiked samples:
,4.1.1.1 Collect at least five liters of each surface water or effluent to be
used to spike the drinking water.
4.1.1.2 Obtain a sufficient volume of an oxidant-free and reductant-free
(chlorine-free and sodium thiosulfate-free) drinking water sample to make
dilutions of the spike water to obtain 10s target organisms/100 ml. A single
drinking water must be used for all dilutions and testing of a spiked test
sample. Refrigerate all waters at 1-4 C until tests are' completed.
4.1.1.3 Perform MF analyses of the spike source waters/effluents to determine
the original densities of the target organisms.
4.1.1.4 Using the density information, add sufficient oxidant-free and
reductant-free drinking water to provide 10s each total coliforms, fecal
coliforms or E. coli/lQQ ml.
4.2 Chlorination of Spiked Samples
4.2.1 The microorganisms in the samples must be stressed by exposure to
chlorination at ambient temperatures under conditions similar to those in
drinking water treatment facilities. No two samples are expected to produce the
same levels of injured target organisms after chlorination. After exposure, and
prior to comparability testing, analyses must be conducted to confirm that each
sample contains the desired low numbers of injured total coliforms, fecal
coliforms, or E. coli.
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4.2.2 -The disinfection process Is dependent upon a number- of physical and
biological factors including the type of sample to be disinfected (e.g., sewage
effluent or source water), the initial titer of the target organism(s) present
in the sample, the chlorine demand of the sample, the type and concentration of
chlorinating agent, the exposure time, s'ample mixing, pH, and temperature8'9.
These physical and biological factors should be carefully monitored and recorded
for each sample or experiment.
4.3 Preliminary Chlorination Study
4.3.1 Conduct a preliminary chlorination study to establish the exposure time
needed to reduce the 10 colony-forming units (CFUJ/100 ml level in each spiked
sample to a 1-10 CPU/100 mi level, using the following procedure. Determine the
total residual and free residual chlorine concentrations initially, at mid-point,
and at the end of the exposure time just prior to dechlorination. Make these
measurements using the approved N,N-diethyl-p-phenylened1am1ne (DPD) colorimetric
method10. Record the measurements.
4.3.2 Place 2 liters of the spiked water sample in a 3-liter glass container.
If a sample has an appreciable chlorine demand (e.g., a primary effluent or a
sewage sample), add dilute sodium hypochlorite solution until a total residual
chlorine level between 2.0 and 2.5 mg/L 1s maintained in the absence of free
chlorine.
4.3.3 If a sample has a low chlorine demand, avoid over-stressing or killing the
organisms by prolonged exposure to free residual chlorine. The free residual
chlorine concentration should not exceed 0.5-1.0 mg/L.
•4.3.4 The exposure time is directly dependent upon the initial titer of the
target organism present, the sample demand, and the form of chlorine present.
For example, expose a sewage sample to 2.0-2.5 mg total chlorine/L for over a
range of times such as 10, 20 and 30 minutes to reduce the E. coli density from
10 CPU to a 1-10 CPU/100 ml sample. However, the required period of exposure
of a sample with a low chlorine demand of 0.5-1.0 mg free residual chlorine/L,
may be significantly shorter than 20-30 minutes.
4.3.5 Add an appropriate volume of a diluted solution of reagent grade sodium
hypochlorite e.g., a 1:20 dilution of 5% (w/v) stock solution, to achieve the
desired level of chlorinating agent. Stir the sample continuously during the
Chlorination.
4.3.6 Stop the action of the chlorine at the end of the exposure period by
adding 0.8 mL of a 10% (w/v) sodium thiosulfate solution/L sample.
4.4 Determination of Bacterial Density
4.4.1 After deactivating the chlorine in the exposed samples, analyze the
samples to determine the exposure times which achieve residual levels of 1-10
CPU/100 mL, for total coliforms and fecal coliforms/f.' coTi using the MP method
and medium designated for each target organism cited in Table 1.
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Target Organism
Total conforms
Fecal coliforms
E. coli
TABLE 1
Method
SM 9222B11
SM 9222D12
SM 9222B"
EPA 110513*
Medium
M-Endo LES Agar
M-FC
M-Endo LES Agar/
Nutrient Agar MUG
Count the colonies of target organisms after 24 h to determine the densities and
record, the exposure.time producing the desired counts
4.5 Chiorination of Samples for Comparability Study
4.5.1 Proceed with the chlorination of samples for the formal Comparability
Study using the exposure time and necessary dilution to produce the desired
survival count of 1-10 CFU/100 mL for total coliforms and fecal coliforms/
E. coli.
4.5.2 The proposer may modify the guidance in this protocol for the spiking,
chlorination, dechlorination steps as seen fit with the provisos that:
4.5.2.1 The chlorination reduces the 10s CFU/100 ml in the spiked water samples
by <32£ZT6i)s to assure the presence of stressed organisms. The final coli form,
fecal coli form or f. coli target range for the Comparability Study is 1-10
CFU/100smL and 100 mL sample volumes are tested.
4.5.3 Place 5 liters of each test water in a glass container. Add an
appropriate volume of a diluted solution of reagent grade sodium hypochlorite
(e.g., a 1:20 dilution of 5% (w/v) stock solution) to achieve the desired level
of chlorinating agent.
4.5.4 Determine the total residual and free residual chlorine concentrations
initially, at midpoint and at end of the exposure time. Make these measurements
using the approved N,N-diethyl-p-phenylenediamine (DPD)- colorimetric method.
Record the measurements and submit with the study results.
4.5.5 Expose each sample to the disinfectant for the selected time, based upon
preliminary data, to achieve an inactivation of 3-4 orders of magnitude (99.9-
99.99%) of the initial 10s population. Stir the sample continuously during the
chlorination.
4.5.6 Stop the action of the chlorine at the end of the exposure period by
adding 0.8 mL of 10% (w/v) sodium thiosulfate solution/L sample and mixing.
*Equivalent to Reference Method, NA + MUG .
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4.6 Dilution of Chlorinated Sample for Comparability Study
4.6..1. Since .the study is intended to compare the performance of the proposed
method to the performance of the reference method, the data used for comparison
should be from the dilution(s) which produces results closest to an equal number
of positive and negative MF results for the reference method. The results for
the proposed method must come from that same dilution.
4.6.2 The sample dilutions are prepared according to one of two options:
Option A utilizes.preliminary analyses to establish the density of target
organisms and hence requires a 24 h holding period for samples.
Option B does not use preliminary analyses and, therefore, does not require
a 24 h holding period. However, an expanded series of dilutions is needed to
bracket the density of the target organisms.
4.6.3 Option A
4.6.3.1 After chlorination, and deactivation of the chlorine and prior to
comparability testing, conduct MF analyses to confirm that each sample contains
the desired low numbers of 1-10 total coliforms, fecal coliforms, or E. coli/\OQ
ml using the media designated in Table 1.
4.6.3.2 After initiating the assay of the target organism density of each of the
dechlorinated samples in 4.6, Immediately refrigerate the samples at 4-10 C until
density.data are obtained (about 24 h). This holding period .also simulates the
period of drinking water transit in a distribution system. Unpublished data
indicate that this holding period does not significantly affect the recovery of
target organisms, cause die-off, or allow repair from chlorine injury. Read the
plates after 24 h to determine the approximate density of the target organisms.
4.6.3.3 Use the density assay results to estimate the dilution of each sample
with oxidant-free and reductant-free drinking water that is necessary to reach
the target organism density of 1-10 cells per unit test volume. Make that
dilution and at least two others that bracket, for exarole, halve and double that
dilution. One -of these dilutions should contain the desired 1-10 target
organisms per test volume. Immediately conduct the comparability analyses with
each sample using these three dilutions. •
4.6.4 Option B
4.6.4.1 Use oxidant-free and reductant free drinking water to prepare equal
volumes of five serial five-fold (1:5) dilutions of each of the dechlorinated
samples immediately after initiation of the MF density assays. Comparability
analyses will be performed on each of the dilutions in each set without delay by
both the approved and proposed methods.
4.6.5 If one of the dilutions does not produce an acceptable split in positive
and negative results for the reference method, the proposer must return to the
original spike sample and begin again to prepare samples1for the Comparability
Study. See Section 4 and the Schema. Although the intention of the ATP design
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is to achieve a 50%/50% split in positive and negative results, the split in
responses may be as much as 25%/75% in either direction and still be acceptable.
5. Comparability Study
5.1 Test Methods
5.1.1 The description of the reference methods and guidelines for use of the
proposed method follow. For valid comparisons, the samples, sample volumes and
dilutions must be the same for both methods. Each test unit must be 100 ml, but
may be configured as a ten x 10 ml, five x 20 ml or one x 100 ml volume. The
spiked samples must be stirred continuously while portions are withdrawn for
testing. Include the testing of pure cultures of organisms of known positive and
negative responses for total coliform, fecal coliform and £. coli cultures to
insure a proper interpretation of the results. It is particularly important to
include a non-f. coli thermotolerant fecal coliform culture such as Klebsiella
pneumonias (negative control) along with a NUG-positive f. co 7/culture to insure
a proper interpretation of the fluorescent responses.
5.2 Reference Methods
5.2.1 The approved reference methods used in the Comparability Study are usually
the same type of test as the proposed method. The reference methods to be used
are given in Table 2.
Table 2
Target Organism Reference Method Med i urn
Total coliforms (TC) SM 9221B15 LTB & BGLB
TC Completed Test SM 9221B15 M Endo LES or
MacConkey agar/LTB
Fecal coliforms
f. COli
SM 9221B15/9221E.l16
SM 9221B15/EPA 110413*
LTB/EC
LTB/EC MUG
5.2.2 Reference Method for Total Coliforms
5.2.2.1 Inoculate LTB with the volumes of sample or dilution which will bracket
a total coliform level of 1-10 organisms/100 ml. Use the LTB tube or bottle
configurations approved by NERL-Cincinnati.
5.2.2.2 Incubate the LTB tubes for 22-24 h at 35 C and examine for a positive
test of growth, growth and gas or growth and acid. Record results and inoculate
positive tubes into BGLB for confirmation.
5.2.2.3 Incubate negative tubes for another 24 h and examine for growth and gas.
Record results and inoculate positive tubes into BGLB for confirmation.
'Equivalent to Reference Method, EC MUG .
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5.2.2.4 Incubate BGLB tubes for 24 h at 35 C. Examine for a positive test of
growth and gas at 24 h. Incubate negative tubes for an additional 24 h and
examine for growth and gas. Record results.
5.2.2.5 Perform the completed test on one of the 20 replicate test units of the
dilution selected for one of 10 samples included in the'Comparability Study.
Streak each BGLB positive tube on M-Endo agar for isolation and incubate for 24 h
at 35 C . Transfer a portion of a total coliform (sheen) colony from each M-Endo
plate into LTB. Incubate the LTB tubes for 24 h at 35 C. Examine for growth and
gas or growth and acid. Record results. If no growth and gas, incubate for an
additional 24 h and examine as above. Record results.
5.2.3 Reference Methods for Fecal Coliforms and E. coli
5.2.3.1 Inoculate tubes of EC and/or EC MUG medium with growth from positive LTB
tubes from 5.2.2.2-5.2.2.3 using the dilutions that bracket 1-10 fecal coliforms
or £. C07//100 mL. These dilutions may differ from those used to obtain 1-10
total coliforms/100 mL because fecal coliforms and E. coTi are only a part of the
total coliform group.
5.2.3.2 Incubate the EC medium at 44.5 C for 24 h and examine for growth and
gas, which constitutes a positive P/A test for fecal coliform bacteria.
5.2.3.3 Incubate the EC MUG medium at 44.5 C for 24 h and examine for blue-white
fluorescence under long wave light, which constitutes a positive P/A test for
E. co7f.
5.3 Proposed Presence/Absence Method
5.3.1 Analyze the same volumes of spiked finished waters' 'and/or dilutions as
tested for the reference method, following the instructions for the proposer's
method.
5.3.2 Incubate the cultures as directed in the method and examine for positive
and negative results. Record all results.
5.3.3 Complete verification steps according to method instructions.
5.4 Recording Results
5.4.1 Record the individual positive and negative results (raw data) for the
reference and proposed methods on a bench form similar to Attachment 1. Include
information on the analyst, laboratory, date of analyses, supervisory approval,
etc.
6. DATA REPORTING
6.1 All the data from the Comparability Study, i.e., the replicate observations
of the samples by both the EPA-approved reference method and the applicant's
proposed method and the quality control observations should be forwarded to the
Director, Ecological Exposure Research Division, NERL-Cincinnati (address on page
1). The final test unit results should be recorded in the formats suggested in
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the Attachments 2, 3, and 4 and forwarded to NERL-Cincinnati, see 5.4.1. Include
information on the analyst, laboratory, date of analyses, supervisory approval.
etc. The individual test results (raw data) should be available for review by
EPA, if requested. The evaluation of the application can be accomplished more
quickly by the NERL-Cincinnati program and statistical staff if the information
is also forwarded on disks compatible with an IBM-PC computer. The text on the
disc should be presented in the latest version of WordPerfect (currently 5.1) and
the data presented in WordPerfect or in ASCII.
7. DATA REVIEW
7.1 Upon receipt of an applicant's data sets, NERL-Cincinnati staff will
initiate Us technical and statistical reviews. Appropriate criteria will be
used to determine the acceptability of the reference method data as a basis in
the evaluation of the analyses by the proposed method.
7.2 For presence/absence methods, the Chi-square test will be used to compare
the number of positive test units in the proposed method analyses with the number
of positive test units in the reference method analyses. This statistical test
will also afford a comparison of the results of the reference and proposed
methods for each of the samples. The Breslow-Day test will be used to
investigate the interaction between method and sample.
8. METHOD RECOMMENDATION AND APPROVAL
8.1 After completion of the technical and statistical reviews, NERL-Cincinnati
will prepare its recommendation for approval/disapproval of the new or
significantly revised method, notify the applicant of its recommendation, and
forward the recommendation to the Office of Ground Water and Drinking Water
(OGWDW), which has the responsibility for proposing the method in the Federal
Register. Following a three-month public comment period, OGWDW will review
submitted comments and prepare the final nationwide approval/disapproval decision.
and promulgation notice in the Federal Register.
8.2 Upon approval of the method, the applicant will be responsible for the
publication and distribution of the approved method to anyone requesting a copy.
9. REFERENCES
1. U.S. Environmental Protection Agency. Drinking Water; National Primary
Drinking Water Regulations; Total Coliforms (Including Fecal Coliforms and
f. co/j); Final Rule. Federal Register 54(124):27562-27568, June 29,
1989.
2. U.S. Environmental Protection Agency. National Primary Drinking Water
Regulations; Analytical Techniques; Coliform Bacteria, Federal Register
56(5):636-643, January 8, 1991.
3. Villa, 0. and L. Reed, Co-Chairs, EMMC Methods Integration Panel. Final
Version of Approved EMMC Format (Memorandum to Members of EMMC Steering
Committee, Methods Integration Panel, and Work Group, Tri-Chairs). U.S.
Environmental Protection Agency, February 14, 1992. pp. 1-2.
14
-------
4. Standard Methods for the Examination of Water and Wastewater. 18th Ed..
APHA, Washington, DC, 1992, Section 9060A and B, p. 9-18 thru 9-20.
5. U.S. Environmental Protection Agency. Manual for the Certification of
Laboratories Analyzing Drinking Water: Criteria and Procedures, Quality
Assurance, 3rd ed. EPA/570-9-90-008A. Chapter 5, Microbiology (revised)
Office of Drinking Water, Washington, D.C., October 1991, pp. 37-48.
6. Standard Methods Section 9020B.
7. Standard Methods.... Section 9020B, pp. 9-5 and 9-6.
8. White, G. C. Chemistry of Chlorine. In: Handbook of Chlorination for
Potable Water. Wastewater. Coolino Water. Industrial Processes, and
Swimming Pools. Van Nostrand Reinhold Co., New York, 1972. pp. 182-227.
9. Jolley, R. L. and J. H. Carpenter. A Review of the Chemistry and
Environmental Fate of Reactive Oxidant Species of Chlorinated Water. In:
Water Chlorination: Environmental Impact and Health Effects. Volume 4. Book
1, Chapter 1. Ann Arbor Science, Ann Arbor, 1983. pp. 3-47.
10. Standard Methods.... Method 4500-C1 G. pp.4-45 and 4-46.
NOTE: To obtain total chlorine in one reading, add the full amount of
potassium iodide at the start with the specified amounts of buffer reagent
and DPD indicator.
11. Standard Methods.... Section 9222B, pp. 9-54 thru 9-58.
12. Standard Methods.... Section 9222D, pp. 9-60 thru 9-61.
13. U.S.. Environmental Protection Agency. Tests Methods.,for.Escherichia co1i
in Drinking Water: EC Medium with MUG Tube Procedure and Nutrient Agar
with Mug Membrane Filter Procedure. EPA/600/4-91/016. Environmental
Monitoring Systems Laboratory, Cincinnati, Ohio. July 1991.
14. U.S. Environmental Protection Agency. National Primary Drinking Water
Regulations; Analytical Techniques; Coliform Bacteria, Federal Register. 40
CFR Part 141, 56 (5) 636-643, January 8, 1991.
15. Standard Methods.... Section 9221B, pp. 9-46 thru 9-49.
16. Standard Methods.... Section 9221E.1, pp. 9-52 and 9-53.
17. Fleiss, J. F., Statistical Methods for Rates and Proportions. 2nd ed., John
Wiley & Sons, New York, NY, 1973.2
15
-------
ATTACHMENT I. EXAMPLE BENCH FORM tor PRESENCE/ABSENCE COLIFORM MFT ANALYSIS
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16
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ATTACHMENT 2. EXAMPLE FORMAT
RESULTS OF ATP COMPARABILITY STUDY OF METHOD FOR TOTAL COUFORM BACTERIA
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-------
ATTACHMENT 3. EXAMPLE FORMAT
RESULTS OF ATP COMPARABILITY STUDY OF METHOD FOR FECAL COUFORM BACTERIA
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-------
ATTACHMENT 4. EXAMPLE FORMAT
RESULTS OF ATP COMPARABILITY STUDY OF METHOD FOR ESCHERICHIA COU
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-------
APPENDIX
DETERMINING THE SPECIFICITY OF A PROPOSED PRESENCE/ABSENCE
LIQUID CULTURE METHOD FOR TOTAL
COLIFORNS. FECAL COLIFORMS, AND/OR E. coli
-------
APPENDIX
DETERMINING THE SPECIFICITY OF A PROPOSED PRESENCE/ABSENCE LIQUID CULTURE
METHOD FOR TOTAL COLIFORMS, FECAL COLIFORMS, AND/OR E. coli
1. INTRODUCTION
1.1 This appendix provides guidance for determining the specificity of
presence-absence (P/A) methods for total coliforms, fecal coliforms, or f. coli.
Specificity is determined by evaluating isolates from aquatic environments, which
are representative of target and nontarget organisms that may impact on drinking
water, and using them in the determination of the false positive error and the
undetected target error of the proposed test. A false positive error occurs when
a non-target organism produces the reaction expected from the target organism.
A false -negative error occurs when the target organism fails to produce the
expected positive reaction 1n the test procedure. The false positive and false
negative errors may be calculated according to the American Society of Testing
Materials (ASTM) Standard Practice 0 3870-79.'
1.2 The tests for specificity may be conducted separately from the formal ATP
Comparability Study or for economy may utilize positive and negative test units
from the Comparability Study Itself. In either case, the results of the
specificity tests will be evaluated independently of the Comparability Study and
the specificity results will not be used to adjust or modify the Comparability
Study results.
2. SPECIFICITY STUDY DESIGN
2.1 One water sample is collected from each of ten different, geographically
dispersed wastewater or ambient water sources that are known to contain the
target organisms (total coliforms, fecal coliforms, or f. coli) for use as spikes
into a finished drinking water. Preliminary quantitative analyses are conducted
to establish the density of the target organism in each source water.
2.2 Based on the results of the density analyses, the source water volume or
dilution is determined for spiking into the finished drinking water to yield 1-10
target organism CFU/100 ml. This concentration of organisms should generate an
approximately even split between positive and negative responses wherein a
positive response consists of one or more positive results-in a test unit and a
negative response consists of all negative results in a test unit.
2.3 The proposer must perform sufficient replicate analyses on the selected
volume or dilution for each sample to generate 100 positive and 100 negative
responses. Ideally, 10 positive and 10 negative responses would be obtained from
each of the 10 spiked drinking water samples.
2.4 If the proposed method can analyze for more than one target organism,
different sample or dilution volumes may be needed to achieve the desired even
split in positive and negative responses for the different organisms.
2.5 Each specificity analysis must be performed on a drinking water sample
volume of 100 ml, consisting of ten-10 mL, five-20 mL, one-100 mL or other
configuration equalling 100 mL approved by the ATP Program for the study.
21
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2.6 The testing of pure cultures of organisms of known positive and negative
responses for total coliform, fecal coliform and E. coli Is included as quality
control checks. It is particularly important to include a non-f. coli
thermotolerant fecal coliform culture such as Klebsiella -pneumoniae (negative
control) along with a MUG-positive E. coli culture to insure a proper
interpretation of the fluorescent response in the EC MUG test.
3. DETERMINATION OF FALSE POSITIVE ERROR (See Figure 1)
3.1 Total Conforms2
3.1.1 For each of the ten spiked water samples, examine the sample volume or
dilution selected in 2.2 and 2.3, for units positive for total coliforms. For
each sample, transfer a loopful of growth from the selected dilution of each of
the ten replicate positive 100 ml test units into tubes of LTB, for a total of
100 transfers. Incubate the 100 LTB tubes for 24 h at 35 C. Examine for visible
growth with gas or acid production (a positive test).
3.1.2 If no visible growth after 24 h, incubate for an additional 24 h and again
examine for visible growth. If none, record as a true negative response and
interpret as a false positive test for total coliform bacteria by the proposed
method.
3.1.3 If growth is visible in LTB after 24-48 h, record results and transfer a
loopful of culture into a tube of Brilliant Green Lactose Bile Broth (BGLB}2.
Incubate BGLB tubes at 35 C for 24-48 h. Examine tubes for growth and gas
production.
3.1.4 If no visible growth and gas in BGLB after 24-48 h, record as a true
negative response and interpret as a false positive response for total coliform
bacteria by the proposed method.
3.1.5 Growth and gas in BGLB after 24-48 h is a true positive response for total
coliform bacteria.
3.1.6 Record results.
3.2 Fecal Coll forms3
3.2.1 For each of the ten spiked water samples, examine the sample volume or
dilution selected in 2.2 and 2.3 for units positive for fecal coliforms. For
each sample, transfer a loopful of growth from the selected dilution of each of
10 replicate positive test units into tubes of LTB, for a total of 100 transfers.
Incubate tubes at 35 C for 24 h and examine for growth and gas or growth and acid
(a positive test).
3.2.2 If no visible growth after 24 h, incubate for an additional 24 h and again
examine for visible growth. If none, record as a true' negative response and
interpret as a false positive response for fecal coliform bacteria by the
proposed method.
3.2.3 If growth is visible after 24-48 h, record results and transfer a loopful
of culture into EC. medium and incubate EC tubes at 44.5 C for 24 h and examine.
22
-------
3.2.4 Growth and gas in EC medium after 24 h is a positive test for feca"
coliform bacteria.
3.2.5 No growth and gas in EC medium after 24 h is interpreted as a false
positive response for fecal coliform bacteria by the proposed method.
3.2.6 Record results.
3.3 f. co7/*
3.3.1 For each of the ten spiked water samples, examine the sample volume or
dilution selected in 2.2 and 2.3 for units positive for E. coli. For each
sample, transfer a loopful of growth from the selected dilution of each of 10
replicate positive test units into tubes of LIB, for a total of 100 transfers.
Incubate LIB tubes at 35 C for 24 h and examine for growth and gas or growth and
acid (a positive test).
3.3.2 If no visible growth in LIB after 24 h, incubate for an additional 24 h
and examine for growth. If none, record as a negative response and interpret as
a false positive E. coli by the proposed method.
3.3.3 If growth is visible after 24-48 h, transfer one loopful of each culture
to EC MUG medium4 in a tube and incubate at 44.5 C for 24 h.
3.3.4 Examine under long wave (366nm) UV light. If no fluorescence with or
without growth, response is interpreted as a false positive test for E. co7i by
the proposed method.
3.3.5 If fluorescence occurs with growth, response is a true positive response.
If fluorescence occurs without growth, it is judged autofluorescence and a
negative response.
3.3.6 Record results.
3.4 Report all results from the Specificity Study of the 100 test units positive
for the target organism, in a form similar to the attached Example Forms 5-7.
Note that the forms have only one entry space for LIB or BGLB verification
results for each transfer for the proposed liquid culture method. The values
entered should be the sums of the 24 and 48 h responses.
3.5 The. false positive error will be calculated as follows:
False Positive Error = A/B
Where A = False positives which are the number of initial positive
response by the proposed method, which were not verified by the
reference methods.
and B - Total number of initial positive responses produced with the
proposed method (> 100).
23
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4. DETERMINATION OF FALSE NEGATIVE ERROR (See Figure 2)
4.1 Total Coliforms
4.1.1 For each of the ten spiked water samples, examine the sample volume or
dilution selected in 2.2 or 2.3 for units negative for total col.iforms. For each
sample, -transfer one ml of culture from the selected dilution of each of 10
replicate negative test units into tubes of LIB, for a total of 100 transfers.
Incubate at 35 C for 24 h. Examine for visible growth with or without gas or
acid production (a positive response).
4.1.2 If no growth, incubate for another 24 h at 35 C and again examine for
growth. If no growth, record as a true negative response.
4.1.3 If growth is visible in LTB after 24-48 h, transfer a loopful into a tube
of BGLB. Incubate BGLB tube at 35 C ± 0.5 C for 24-48 h.
4.1.4 Examine BGLB tubes. Growth and gas after 24-48 h is a positive response
and is interpreted as a false negative response for total coliform bacteria by
the proposed method.
4.1.5 No growth and no gas in BGLB after 24-48 h for total coliforms is a true
negative test.
4.1.6 Record results.
4.2 Fecal Coliforms
4.2.1 For each of the ten spiked water samples, if the test is for total
coliforms and fecal coliforms, examine the sample volume or dilution selected in
2.2 and 2.3 for units positive for total coliforms and-negative for fecal
coliforms. For each sample, transfer one mL of culture from the selected
dilution of each of 10 replicate negative 100 mL test units into tubes of LTB,
for a total of 100 transfers. Incubate at 35 C for 24 h and examine for growth
and gas or growth with acid production (a positive response).
4.2.2 If the test is for fecal coliforms only, transfer a loopful of culture
into LTB from each of 100 test units that are negative for fecal coliforms.
Incubate at 35 C for 24 h and examine for growth.
4.2.3 If no growth, incubate for another 24 h at 35 C and again examine for
growth. If no growth, record as a true negative response.
4.2.4 If growth is visible in LTB after 24-48 h, record results and transfer a
loopful into a tube of EC medium. This transfer may be done at the same time as
for the total coliforms in 4.1.2. Incubate at 44.5 C for 24 ± 2 h. Examine for
growth and gas.
4.2.5 Growth and gas in EC after 24 H is a positive response and is interpreted
as a false negative test for fecal coliform bacteria by the proposed method.
4.2.6 No visible growth or gas in EC after 24 h for fecal coliforms is a true
negative response.
4.2.7 Record'results. ""' '
24
-------
4.3 f. co7?
4.3.1 For each of the ten spiked water samples, if test is for total coliforms
and E. coli, examine the sample volume or dilution selected in 2.2 and 2.3 for
units positive for total coliforms and negative for E. coli. For each sample.
transfer one ml of culture from the selected dilution of each of 10 replicate
negative 100 ml test units into tubes of LTB, for a total of 100 transfers.
Incubate at 35 C for 24 h and examine for growth and gas.
4.3.2 If the test is for E. co7; only, transfer a loopful of culture into LTB
from each of 100 test units that are negative for E. coli. Incubate at 35 C for
24 h.
4.3.3 Examine LTB tubes for visible growth with or without gas or acid
production. If none, reincubate for another 24 h and examine for growth, if
none, record as a true negative response and interpret as a false positive
response by the proposed method.
4.3.4 If growth is visible after 24-48 h, transfer one loopful of each culture
to EC MUG medium in a tube and incubate at 44.5 C + 0.2 C for 24 h.
4.3.5 Examine EC MUG tubes for fluorescence under long wave (366nm) UV light.
Fluorescence with growth is a positive response and is interpreted as a false
negative response for f. coli by the proposed method.
4.3.6 No fluorescence is a true negative result response 'for E. coli.
Fluorescence without growth is judged autofluorescence and a negative response.
4.3.7 Record results.
4.4 Report all results from the Specificity Study of the 100 test units negative
for the target organisms, in a form similar to the attached Example Forms 8-10.
Note that the forms have only one entry space for LTB or BGLB verification
results for each transfer from the proposed liquid culture method. The values
entered should be the sums of the 24 and 48 h responses.
4.5 The false positive error will be calculated as follows:
False Negative Error = C/(B - A + C)
Where A - False positives which are the number of initial positive
responses by the proposed method which were not verified by the
reference methods.
B » Total number of initial positive responses produced with the
proposed method (> 100)
and C = False negatives which are the number of initial negative
responses by the proposed method which were positive by the
reference methods.
25
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REFERENCES
1. American Society of -Testing Materials. D 3870-79: Practice for
Establishing Performance Characteristics for Colony Counting Methods In
Bacteriology. In: 1991 Annual Book of ASTM Standards. Vol. 11.02, Water
Philadelphia, PA.
2. Standard Methods for the Examination of Water and Wastewater. 18th ed
Section 9221 B, APHA, Washington, DC 1992. pp. 9-46-to 9-49.
3. Standard Methods Section 9221 E, pp. 9-52 to 9-53.
4. United States Environmental Protection Agency (USEPA). Test Methods for
f. coli in Drinking Water. EPA 600/4-91/016, ORD, NERL-Cincinnati, July.
1991.
26
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FIGURE 1. SPECIFICITY TESTS FOB TOTAL Cm
AND E. COLI IJETIiODS FOR
FECAL CO
'
PROPOSED METHOD
FOR EACH POSITIVE RESPONSE:
ONE LOOPFUL OF LIQUID CULTURE INTO LTB
INCUBATE
LTB
GROOTH-
FALSE POSITIVE
LTB
GROWTH* w/wo GAS OR ACID
GAS-
FALSE
POSITIVE
TOTAL
COLIFORM
GAS+
TRUE POSITIVE
TOTAL
COLIFORM
FLUORESCENCE
FALSE
POSITIVE
£•
FLUORESCENCE
TRUE POSITIVE
£. £QLX
GA
FALSE POSITIVE
FECAL COLIFORM
AS-*-
TRUE POSITIVE
FECAL COLIFORM
-------
FIGURE 2,
SPECIFICITY TESTS FOR TOTAL CoLIFORM, FECAL CoLIFORM
AND E. COLI METHODS FOR FALSE NEGATIVE ERROR
PROPOSED METHOD
FOR EACH NEGATIVE RESPONSE:
ONE ML OF LIQUID CULTURE INTO LTB
INCUBATE
LTB
GROWTH-
TRUE NEGATIVE
LTB
GROWTH* w/wo GAS OR ACID
EC HUG
GAS-
TRUE
NEGATIVE
TOTAL
COLIFORM
GAS*
FALSE
NEGATIVE
TOTAL
COLIFORM
No
FLUORESCENCE
TRUE NEGATIVE
£.
FLUORESCENCE
FALSE
NEGATIVE
£•
TRUE NEGATIVE
FECAL COLIFORM
GAS+
FALSE NEGATIVE
FECAL COLIFORM
-------
ATTACHMENT 5. EXAMPLE FORMAT
SPECIFICITY STUDY FOR FALSE POSITIVE RESPONSES BY THE PROPOSED METHOD FOR TOTAL COUFORM BACTERIA
Analyst:
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Indicate: + « presence - = absence
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-------
ATTACHMENT 6. EXAMPLE FORMAT
SPECIFICITY STUDY FOR FALSE POSITIVE RESPONSES BY THE PROPOSED METHOD FOR FECAL COUFORM BACTERIA
Lob Nam* A Location:
A
tOljflYS
Swporvbor'* Signature
Indicate: 4- = presence - a absence
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ATTACHMENT 7. EXAMPLE FORMAT
SPECIFICITY STUDY FOR FALSE POSITIVE RESPONSES BY THE PROPOSED METHOD FOR ESCHERICHIA COU
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ATTACHMENT 8. EXAMPLE FORMAT
SPECIFICITY STUDY FOR FALSE NEGATIVE RESPONSES BY THE PROPOSED METHOD FOR TOTAL COUFORM BACTERIA
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ATTACHMENT?. EXAMPLE FORMAT
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ATTACHMENT 10. EXAMPLE FORMAT
SPECIFICITY STUDY FOR FALSE NEGATIVE RESPONSES BY THE PROPOSED METHOD FOR ESCHERICHIA COU
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