PROTOCOL  FOR ALTERNATE TEST PROCEDURES FOR COLIFORM  BACTERIA
                      IN COMPLIANCE WITH WATER AND WASTEWATER REGULATIONS

                             QUANTITATIVE MEMBRANE FILTER METHODS
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 C~                                        Version 1.0

 ^                                      December,  1995

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IV

                             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 WATER AND WASTEWATER REGULATIONS FOR QUANTITATIVE MEMBRANE FILTER METHODS

I.   INTRODUCTION

1.1  Regulatory Background

1.1.1  The Administrator, U.S. Environmental Protection Agency (EPA), approves
analytical methods for contaminants regulated under the Clean Water Act (CWA) and
the Safe Drinking Water  Act  (SDWA).  When EPA publishes  a regulation  under these
Acts, which sets permit  or maximum contaminant  levels,  it generally  approves at
least one method for detection and/or quantification of that contaminant.  After
a regulation is published, the Administrator may approve additional methods or
modifications  of   approved  methods  that  have  satisfactorily  completed  a
comparability study  under the Alternate Test Procedure (ATP) Program.

1.1.2   Although  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  coliforms  rather  than  their
quantitative enumeration.1'2   The quantitation of these organisms  is still needed
for source water, other ambient waters and wastewaters under the Surface Water
Treatment  Rule  of  the  SDWA3 and  the 304(h)  regulations  of  the  CWA4.   The
quantitative  MF   methods   for   the   evaluation  of  proposed   quantitative
microbiological  methods for  total  and  fecal  coliform bacteria  are  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 National Exposure Research
Laboratory at Cincinnati (NERL-Cincinnati) will recommend  approval  to the Office
of  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  can take one year or more.

1.2  Comparability Determination

1.2.1  This  protocol  describes the method  description  and Comparability Study
data which EPA needs to evaluate  an ATP  for quantitative  membrane filter (MF)
methods  in  microbiology.    The  ATP  program  is  intended to  be  flexible,
consequently, EPA's NERL-Cincinnati  may modify the study design for a particular
proposed 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, for  approval  of  the  test
design.

1.2.2    Generally  the   reference  method  selected  by  EPA for  use   in  the
Comparability Study will be the  same type of  test  as  the proposed method.
Consequently,  if an  MF method  were proposed  for the  target organism,  the
corresponding MF reference method for the target organism would be used in the
Comparability Study.  However, for ATP applications in which a new technology is

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proposed and for which there  is no reference method counterpart,  the ATP Program
will use professional  judgment  in  determining  which  other method would be the
most appropriate as the reference method for the ATP Comparison Study.

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 and EPA regulation 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, 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 an Agency
method,  and/or represents a  minor  optional  change  to  an Agency method,  the
applicant  should  also  prepare  a  two-column side-by-side description  of  the
sections of the Agency reference method  and  the  proposed method 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  be 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 format5,  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, chromogenic
test, fluorogenic 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

      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

      Detafl sample collection and  handling requirements.  Consider the sample
collector,  sample  containers,  dechlorination (if applicable),  sample holding
times and temperature as specified in Standard Methods*.

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, that routine QC checks are recorded and that actions are
taken if a problem is indicated7'*.

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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.  When
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.

      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.

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

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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 Study  Design

3.1.1   Typically,  the  Comparability  Study data are  generated  from replicate
analyses of ten or more ambient waters and/or wastewaters from geographically-
dispersed areas,  which contain  the target  organism  or   organisms  in  varying
numbers and different water or wastewater compositions.  To simplify  the tests,
preliminary screening analyses are  recommended to establish approximate numbers
and reduce the number of dilutions and analyses in the formal study.  The volumes
or dilutions of water and wastewater samples tested must generate data  within the
acceptable range of the Agency reference method.

3.1.2  A minimum of twenty  (20)  replicate  analyses are  performed  by each method
on the 3 or more dilutions selected for each of the 10 samples.  The replicate
filtrations on the three or more dilutions of each sample must be performed on
the same day for both  the reference and proposed methods.

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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 MF               Minimum Comparability
                     Filtrations per Organism*          Results Required
	and Sample Dilution	per Method
Reference Method               20                              200

Proposed Method                20                              200

*Total Coliform, Fecal Coliform, etc.

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  laboratory 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   Conduct QC  checks on each  day  of  analyses  including  temperature of
incubators, analyses of known positive and negative  response  cultures, sterility
controls and verification of colonies.  Maintain records of QC checks performed.
See the Manual  for the Certification of  Laboratories  Analyzing  Drinking Water7
and the  Intralaboratory Quality  ControlGuidelinesin  Standard Methods8 for
general guidance.

3.4  Sample Collection and Handling

3.4.1  Each sample should be  collected and held  in a single  sterile, wide-mouth
bottle of non-toxic  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 deformity or production
of  toxic  materials.   Screw caps  must  not  produce  bacteriostatic,  toxic or
nutritive products during sterilization.  Bottles and closures  should be checked
for these effects before use in the study*.

3.4.2  Samples should be  maintained  at  1-4 C  during  transit  and  holding time.

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4.   PREPARATION FOR COMPARABILITY STUDY

4.1  Selection and Collection of Water and Uastewater Samples

4.1.1   The  reason  for requiring ten different  water/wastewater samples is to
obtain, as roach as is practical,  a  good  presentation of  the wide range of water
types with their even  wider  range  of  target  organisms,  to which a method must
respond appropriately.  The ten samples  can be collected over time  as needed to
complete the Comparability Study, with the understanding that all  testing of a
water or wastewater must  be completed in a single day for both the reference and
proposed methods.

4.1.2  Collect each water or  wastewater  sample in sufficient volume to complete
all replicate analyses of sample or dilution volumes by both the reference and
proposed methods.

4.2  Sample Volumes and Dilutions for the Comparability Study

4.2.1   Since the study is  intended  to compare the  performance  of  the proposed
method to the reference method, the data  for the comparison must  be from the same
sample  volumes or dilutions  for  both methods based on  those  sample volumes or
dilutions  that produce  plate counts  within the  appropriate  range for  the
reference method.

4.2.2  The sample volumes or dilutions are prepared by one of two  options:

     Option  A  utilizes  preliminary  analyses  to   establish  target  organism
densities before beginning the Comparability Study.  This approach reduces the
number of dilutions  needed to assure a bracketing of the range count  but requires
the additional effort  of these analyses and a 24 h holding period  for samples.

     Option B proceeds directly with the Comparability Study.   The samples are
not  held  for  24  h  and  this  option  does not  require  preliminary  analyses.
Instead, the analyst  gambles on her/his ability to "guess-timate" the  sample
volumes or dilutions necessary to bracket the  reference method's counting range.
This usually requires  an expanded series of dilutions.

4.3  Option A.  Determination of Bacterial Density

4.3.1  After collecting a water or wastewater sample(s), determine the density
of the target organism  using the MF procedure and media  designated for the target
organism:

Target Organism
Total col i forms
Fecal col i forms
TABLE 1
Method
SM 9222B10
SM 92220"

Medium
M-Endo LES Agar
M-FC
4.3.2   Count  the colonies of  target  organisms  after 24 h  to determine their
approximate density  in each water  or  wastewater  sample.   Use these results to

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establish the sample volumes and/or dilution series needed to bracket the desired
counting range  for  the  reference  method(s)  in  the  Comparative Study.  For the
total coliform  method using M-Endo LES agar, the range is 20-80 colonies/plate
and for the fecal coliform method  using  M-FC agar, the range  is 20-60 colonies/
plate.  Sample volumes of less than 1  ml^njust be added as a  dilution.  Filtration
volumes  of  tess than  10  ml must  be proceeded  in  the filtration  process by
approximately 10 ml  of sterile dilution water.

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 and be based  on the  appropriate
dilutions  for  the   reference  method.    The  spiked samples  must  be  stirred
continuously  while  portions are  withdrawn  for filtration  and  preparation of
dilutions.

5.1.2  Include  the testing of pure cultures of organisms of known positive and
negative responses  for  total coliform and  fecal  coliform  cultures to insure a
proper interpretation of the results.

5.2  Reference  Methods

5.2.1  The current reference methods  that will be  used in the comparison studies
are listed in Table  2.

                                   Table 2

     Target Organism         Reference Method              Medium

     Total coliforms          SM 9222B10/9221B12        M-Endo LES/LTB/BGLB

     Fecal coliforms          SM 9222B10/9221E.11J      M-FC/LTB/EC


5;3  Reference  Method forTotal Coliform Bacteria

5.3.1  Filter the appropriate volumes of water or  wastewater and/or dilutions to
yield 20-80 total coliform bacteria on M-Endo LES agar.

5.3.2  Incubate at 35 C for 22-24 h.   Golden green metallic sheen colonies are
coliforms.  Count and record sheen colonies.

5.3.3  For each  sample, randomly pick  10  sheen colonies from each of 20 replicate
plates,  into  LTB tubes.  Incubate for  24-48 h at  35 C and examine.   Growth,
growth and gas, or growth and acid are positive presumptive tests.

5.3.4  Transfer growth  from positive  tubes into BGLB tubes and incubate at  35 C
for 24-48 h.  Growth and gas  verify  total  coliform bacteria.  Adjust original
count based on  verified count, and record.

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  5.4   Reference Method for Fecal  Coliform Bacteria

  5.4.1  Filter the appropriate volumes of  water or wastewater and/or dilutions to
  yield  20-60 fecal  coliform bacteria  on M-FC  agar  plates.

  5.4.2   Incubate M-FC  agar plates  for 24 h  at 44.5  C.   Count and  record  blue
  colonies  as fecal  coliform bacteria.

  5.4.3   From each  sample  randomly pick  10  blue  colonies  on each of the  20
  replicate plates  into LIB  tubes.   Incubate for 24-48 h at 35 C.  Growth,  growth
  with gas,  or growth and acid are presumptive positive tests.

  5.4.4  Transfer growth from the positive  LIB  tubes  into EC tubes and incubate at
  44~5  C for 24 h.   Growth  and gas  in EC  medium verify  fecal  coliform bacteria.
 (Mjustj)rifljina 1 count based on  pps1t1_ye_ver1f1cat1on and  record  results^.

  5.5   Proposed Method

  5.5.1  Filter the appropriate amounts of water or wastewater and/or dilutions and
  follow developer's directions for  analyses.

  5.5.2   Incubate tests units as directed.  Follow  developer's  instructions for
  identifying and enumerating target colonies.

  5.5.3   Complete verification steps according to the  developer's  directions.

  5.6   Recording Results

       Record the  colony counts  for  all   volumes  tested by  the  reference  and
  proposed  methods.

  6.   DATA REPORTING

  6.1  All the data from the comparability  study, i.e.,  the replicate observations
  of  the samples by the EPA  reference  method and  the  proposed method and  the
  quality  control  observations should  be  forwarded to the Director, Ecological
  Exposure  Research Division,  National  Exposure  Research Laboratory - Cincinnati
  (address  on page  1).

  6.2  The  results  from the  analyses of the ten samples should be recorded  in the
  formats  suggested in Attachments  1  and  2. "Note  that the forms  have  only one
-entry  space for LTB  and BGLB verification results for each pick from M-Endo LES
j  of  M-FC  agar.    The values entered  should  be the  sums of  the  24  and  48  h
< _resppjis.e_s_.j The evaluation of the  application  can be accomplished more quickly
  by the EERD, NERL-Cincinnati ATP program  if the information is also forwarded on
  disks  compatible with  an  IBM-PC  computer.   The  text  on  the disks  should  be
  presented in the  latest  version of WordPerfect  (currently  5.1)  and  the  data
  presented in WordPerfect  or  in ASCII.    Confirm  the current  formats  with EPA
  before generating study data.

  7.   DATA REVIEW

  7.1    Upon receipt  of an  applicant's  data  sets,  NERL-Cincinnati  staff  will
  initiate  its technical and statistical   reviews.   Appropriate  criteria will  be

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 used  to  determine the acceptability  of  the  reference method data as a basis in
 the evaluation of the analyses by the proposed method.   If this evaluation is
 favorable,  the proposed  method will  be  recommended  for approval  by the program
 office  and  the applicant so notified.   If  the results of  the analyses  by  the
 approved method are not  satisfactory, the applicant will be  notified that  the
 application -has not been approved.

 7.2   The statistical review will consist of two parts:   an examination  of  the
 data  including descriptive statistics and a comparability analysis of the data.
 First, the  data will be screened  for  outliers and unusual patterns.  Descriptive
 statistics, such as the  mean,  standard  deviation  and coefficient of variation,
 will  then  be. calculated  for  each set ofjreplicates._'The  "second  part  of  the
 statistical review will compare the precision and-recovery of the proposed method
 with  that of the reference method using appropriate statistical techniques.  The
^rec4sit>rrTTf~^enP^^                                 that of the reference
 method  by  means of a  nonparameteric  statistical   procedure attributable  to
 Scheffe14.  An analysis of variance (ANOVA) will be used to compare the recovery
 of the proposed method to that of the approved method.

 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.      It   will   notify  the  applicant of  its
 recommendation,  and  forward the recommendation  to the Office  of Water  (OW), which
 has   the  responsibility   for  proposing  the  method  in  the Federal  Register.
 Following a three-month public comment period,  OW 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.
                                       10

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9.   REFERENCES

1.   U.S.  Environmental  Protection Agency.  Drinking  Water;  National Primary
     Drinking Water Regulations;  Total  Coliforms  (Including  Fecal  Coliforms and
     f.  co7/);  Final  Rule.    Federal  Register 54(124):27562-27568,  June 29,
     1989.  -

2.   U.S.  Environmental  Protection Agency.  National  Primary  Drinking   Water
     Regulations;  Analytical  Techniques;  Colifonn  Bacteria,  Federal Register
     56(5):636-643, January 8, 1991.

3.   40 CFR Part 141 and 142, Drinking Water:  National Primary Drinking Water
     Regulations; Filtration,  Disinfection, Turbidity, Giardia lamblia, Viruses,
     LegioneTla and Heterotrophic Bacteria; Final Rule June 29, 1989,  pp. 27530-
     27531.

4.   40 CFR Part 136, Guidelines Establishing Test  Procedures  for the Analysis
     of Pollutants Under the Clean Water Act; Final  Rule and Interim  Final Rule
     and Proposed  Rule, October 26, 1984,  p. 19.

5.   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.

6.   Standard Methods  for  the Examination of Water and Wastewater.  18th Ed.,
     APHA, Washington, DC, 1992, Section 9060A and  9060B, pp.  9-18 thru 9-20.

7.   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.

8.   Standard Methods	 Section 9020B, pp.  9-3 thru 9-12.

9.   Standard Methods	Section 9020B, p. 9-5.

10.  Standard Methods	Section 9222B, pp.  9-54 thru  9-58.

11.  Standard Methods	 Section 9222D, pp. 9-60 thru  9-61.

12.  Standard Methods.... Section 9221B, pp. 9-46 to 9-50.

13.  Standard Methods	Section 9221E.1, pp. 9-52 and 9-53.

14.  Scheffe, Henry.  The Analysis of Variance. John Wiley and Sons, New York,
     NY, 1959.
                                      11

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                                ATTACHMENT 1.  EXAMPLE FORMAT
   RESULTS OF ATP COMPARABILITY STUDY OF QUANTITATIVE MF METHOD FOR TOTAL COUFORM BACTERIA
Title of Proposed Method:	   Lab Nome & Location:  	
Analyst:  	   Supervisor's Signature:   	
        RECORD COLONY COUNTS and VERIFIED COUNTS by REFERENCE and PROPOSED METHODS

                            Sample # and Description:	
                                   Date of Analysis:—.L	L—
       M-ENDO LES
               REFERENCE METHODS
PROPOSED METHOD
PROPOSED METHOD
    VERIFICATION

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1
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11
11
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11
17
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11
Sum*
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                                                                             USEM. NEIUdndnnaM, »/»3

-------
                                ATTACHMENT 2.  EXAMPLE FORMAT
   RESULTS OF ATP COMPARABILITY STUDY OF QUANTITATIVE MF METHOD FOR FECAL COUFORM BACTERIA
THI« of Proposed Method: 	   Lob Nome * Location:  	
Analyst: 	   Supervisor's Signature:  	
         RECORD COLONY COUNTS and VERIFIED COUNTS by REFERENCE and PROPOSED METHODS

                           Samplo # and Description:	
                                  Date of Anarpte—L	I—
t»tiHmt»
1








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11
11
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REFERENCE METHODS
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11
11
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ROPOSED METHOD
1 II II































































PROPOSED METHOD
VEHRCATIOK
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1
1
1
4
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7
1
f
11
11
11
11
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17
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Sum*
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nrva;
                                                                           USIM, NUtL-dndnnatl, 9/95

-------