I
United States Office of EPA 570/9-90-008A
Environmental Protection Drinking Water (WH-550D) October 1991
Agency Washington DC 20460
&EPA Manual for the
Certification of
Laboratories Analyzing
Drinking Water
Criteria and Procedures
Quality Assurance
Third Edition
Prepared by
The Laboratory Certification Program Revision Committee
Change 1 - October 1991
Supersedes EPA/570/9-82/002, October 1982, entitled Manual for the Certification of
Laboratorie: Analyzing Drinking Water
Printed on Recycled Paper
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List of Effective Pages
REMOVE SUPERSEDED PAGES AND INSERT REVISED PAGES
^
I
Dates of issue for original and changed pages are as follows:
Original 0 April 1990
Change 1 October 1991
Page/Chapter Change No.*
No.
i (Title Page) 1
ii (Effective Pages) (Added) 1
iii (Notice) 1
iv (Foreword) (Added) 1
v-vi (Preface) 1
vii-ix (Acknowledgments) 1
x (blank) 1
xi-xii (Contents) 1
1-36 (Chapters I-IV) 0
37-58.8 (Chapter V) 1
59-74 (Chapter VI) 0
75-86 (Appendices A-D) 0
87-91 (Appendices E-G) . 1
93-95 (Appendix H) (Added) 1
'Zero in this column indicates an original page.
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Notice
This manual has been reviewed by the Office of Drinking Water and the Office of
Research and Development and approved for publication. The mention of
commercial products does not constitute endorsement by the U.S. Environmental
Protection Agency.
iii Change 1
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Foreword
Each of the approximately 200,000 public water systems in
the United States must routinely monitor its drinking water to
determine if it is adequately protected from regulated
microbiological, chemical, and radiochemical contaminants.
Because of the need to safeguard public health, it is imperative
that every laboratory analyzing drinking water generate accurate
data on a continuing basis. This laboratory certification manual
will help the laboratory accomplish this task and also provide a
means for the U.S. Environmental Protection Agency (EPA) to
evaluate laboratory quality.
Specifically, this manual describes the operational and
technical criteria and procedures that EPA will use to evaluate a
laboratory for its ability to properly analyze regulated drinking
water contaminants. The certification program described in this
manual extends to the EPA Regional laboratories, principal State
laboratories in States which have primary enforcement
responsibility (primacy), and to all laboratories that perform
analyses under the Safe Drinking Water Act in the few States
without primacy. The vast majority of primacy States have their
own laboratory certification programs. Although many of them use
the EPA's program as presented in this manual, individual State
programs may vary. Therefore, any laboratory outside the EPA's
program that wants to become certified should contact the State
program.
This manual is the result of sustained work by many
individuals, representing EPA, States, and water systems.
I hope the manual will be used as a practical tool for upgrading
and maintaining laboratory quality. Your comments or suggestions
will be considered in developing subsequent revisions of this
manual.
James R. Elder, Director
Office of Ground Water
and Drinking Water
IV
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Preface
Since 1978, the U.S. Environmental Protection Agency (EPA) has had a
program for certifying Regional laboratories, principal State laboratories in
primacy States, and local laboratories in non-primacy States performing
drinking water analyses required by regulations issued pursuant to the Safe
Drinking Water Act. This document is the third edition of the manual
describing the program's implementation procedures and technical criteria. It
supersedes the Manual for the Certification of Laboratories Analyzing Drinking
Water, EPA-570/9-82-002 (October 1982).
This revision was necessary to address the increased complexity of the
revised drinking water regulations, clarify Regional responsibilities concerning
State laboratory certification programs, reduce the time a laboratory can be
"provisionally certified," and improve feedback to EPA on how laboratories
perform on a routine basis. This edition is based on an ongoing review of the
laboratory certification program to improve implementation and technical
criteria in light of newly approved methodology and six additional years of
experience with the program.
The document was prepared by a committee chaired by the EPA's Office of
Drinking Water (ODW). Comments from the Regions and States were
solicited and considered at several points in the preparation of this revision.
These included recommendations from a workshop held in April 1987, at
which all Regions and States were invited to share their views about both the
implementation strategy and the technical criteria. Regions and States were
represented on the revision steering committee and its various subcommittees
and subgroups.
The EPA quality assurance program covers all activities relating to data
collection, processing, and reporting. This is managed by the Office of
Research and Development, Quality Assurance Management Staff (QAMS).
This manual represents ODW's implementation of the QAMS program
applicable to laboratories conducting drinking water analyses.
Like the previous edition, this program is not regulatory in nature (except for
analytical methodology and requirements in the primary drinking water
regulations), but rather offers guidance describing the recommended
procedures and criteria for assuring data validity. Laboratories may use
equivalent criteria, if these criteria are approved by the certifying authority.
EPA is currently developing new regulations for laboratory certification and
certain pre-laboratory and post-laboratory activities. The Agency is
undertaking this effort to ensure that all primacy States include in their
certification programs those few basic elements that the Agency regards as
critical to assuring data validity (e.g., certification downgrading procedures,
training of on-site evaluators). EPA does not expect that the recommended
procedures and criteria in this manual will conflict with these forthcoming
regulations.
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Unlike previous editions, this edition is in a loose-leaf format which will allow
EPA to more easily update it from time to time. EPA will furnish revised pages
to each State drinking water administrator and State laboratory director.
Holders of this manual should check with the EPA Region or the State
occasionally to make sure their manual is current.
In conclusion, EPA will use the certification criteria in this manual for
evaluating all laboratories that it certifies (Regional laboratories, principal State
laboratories, and local laboratories in non-primacy States). The Agency will
also use this manual as guidance in determining the adequacy of State
certification programs for local laboratories.
vi Change 1
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TO USERS OF THE MANUAL -FOR THE CERTIFICATION OF LABORATORIES
ANALYZING DRINKING WATER (1990 EDITION).
Enclosed is Change 1 to the -manual, which revises Chapter V.
This revision includes the certification criteria for the revised
Total Coliform Rule and Surface Water Treatment Requirements.
The change is to be inserted into the manual, and the previous
edition of Chapter V completely removed, along with the other
pages indicated on page ii of the update.
Enclosure
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Acknowledgments
This manual was prepared through the efforts of many individuals, including
representatives from U.S. Environmental Protection Agency program offices and
laboratories, Regional offices, States, and utility laboratories. The principal
contributors are listed below.
Executive Committee, Laboratory Certification Program Revision
J. Cotruvo (ODW) A. Perler (Advisor)
T. Clark (EMSL-CI) P. Berger (Advisor)
R. Booth (EMSL-CI)
Steering Committee. Laboratory Certification Program Revision
P. Berger, Program Manager (ODW)
B. Bathija, Deputy Program Manager (ODW)
H. Brass (ODS)
T. Clark (EMSL-CI)
D. Easterly (EMSL-LV)
G. Englund (Minnesota)
G. Foree (Region VII)
E. Geldreich (RREL)
W. Knight (Region IV)
J. Lichtenberg (EMSL-CI)
C-K. Liu (EMSL-LV)
H. Nash (RREL)
A. Perler (ODW)
D. Pickering (Washington Aqueduct)
I. Pomerantz (ODW)
M. Silver (OGC)
A. Tiedemann (Virginia)
J. Trax (ODW)
N. Wentworth (QAMS)
J. Cole (ODW)
vii Change 1
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Implementation Subcommittee
T. Clark, Chairman (EMSL-CI)
B. Bathija (ODW)
P. Berger (ODW)
D. Easterly (EMSL-LV)
B. Hamill (New Jersey)
W. Hausler (Iowa)
G. Hicks (Cincinnati Water Works)
C. Jones (Region III)
C-K. Liu (EMSL-LV)
C. Ryan (Region I)
P. Ryker (Kentucky)
R. Thomas (EMSL-CI)
J. Winter (EMSL-CI)
A. Wolfgang (Colorado)
Quality Assurance Manaqement Subcommittee
I. Pomerantz, Chairman (ODW)
B. Bathija (ODW)
P. Berger (ODW)
J. Cole (ODW)
R. Graves (EMSL-CI)
D. Pickering (Washington Aqueduct)
C. Ritchey (Region VI)
N. Roberts (Louisiana)
P. Stamp (Region IV)
A. Tiedemann (Virginia)
J. Trax (ODW)
N. Wentworth (QAMS)
J. Westrick (ODW)
Technical and Quality Control Subcommittee
J. Lichtenberg, Chairman (EMSL-CI)
Chemistry Subgroup
G. McKee, Chairman (EMSL-CI)
J. Barren (ODW)
B. Bathija (ODW)
D. Beesley.(North Carolina)
J. Blosser (Nebraska)
P. Britton (EMSL-CI)
B. Fleck (Illinois)
M. Gomez-Taylor (ODW)
J. Longbottom (EMSL-CI)
J. Pfaff (EMSL-CI)
R. Thomas (EMSL-CI)
VIII
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Microbiology Subgroup
H. Nash, Chairman (RREL)
P. Berger (ODW)
R. Bordner (EMSL-CI)
T. Covert (EMSL-CI)
A. Dufour (EMSL-CI)
E. Geldreich (RREL)
R. Gentry (Region IV)
M. Long (Region V)
A. McDaniels (EMSL-CI)
D. Reasoner (RREL)
G. Rice (RREL)
P. Ryker (Kentucky)
L. Shadix (ODW)
J. Standridge (Wisconsin)
J. Vasconcelos (Region X)
Radiochemistry Subgroup
C-K. Liu, Chairman (EMSL-LV)
F. Novielli (EMSL-LV)
D. Easterly (EMSL-LV)
R. Cothern (EPA Science Advisory Board)
S. Gold (EMSL-CI)
R. Holloway (EMSL-LV)
D. McCurdy (Massachusetts)
R. Tauer (Region VIII)
G. Uyesugi (California)
ix Change 1
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Contents
Page
CHAPTER I: INTRODUCTION , -,
CHAPTER II: RESPONSIBILITIES 3
Office of Drinking Water ...•'. 3
Office of Research and Development 3
Regional Offices 3
Drinking Water Laboratory Certification7 Work Group . . . . . . . . . . . . . 4
CHAPTER III: IMPLEMENTATION 5
Regional Laboratories and Programs 5
Principal State Laboratories ............ . . . 5
Local Laboratories ' g
Other Considerations for Certification 7
Quality Assurance Plan ....'... 7
Performance on Routine Water Samples .................. s
Chain-of-Custody Procedures '..'.'.'.'.'.' 8
Requirements for Maintaining Certification Status.....'.'.'.'.'.'.'.'.'.'.'. Q
Criteria and Procedures for Downgrading/Revoking
Certification Status ............ o
Reciprocity 10
Training T. ........... 1 o
Technical Services '.'.'.'.'.'.'.'. n
Reference Samples . '-...'.....'.'.'.'.'.'.'.'.'.'.''.'.'.' 11
Early Warning System for Problems with Test
Supplies and Equipment 11
Alternate Analytical Techniques '.'.'.'.'.'.'.'.'.'' 14
CHAPTER IV: CHEMISTRY 15
1. Personnel 15
2. Laboratory Facilities '.'.'.'.'.'.'.'.'.''"' 16
3. Laboratory Equipment and Instrumentation '.'.'.'.'. 16
4. General Laboratory Practices 16
5. Analytical Methodology '.'.'.'.'.'.'.'.'.'"' 16
6. Sample Collection, Handling, and Preservation ........ . . . . . 17
7. Quality Assurance 17
8. Records and Data Reporting 18
9. Action Response to Laboratory '.'.'.'.'.'.'.'. 19
Contents (continued)
xi Change 1
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Page
CHAPTER V: MICROBIOLOGY 37
1. Personnel • 37
2. Laboratory Facilities 37
3. Laboratory Equipment and Supplies 37
4. General Laboratory Practices 40
5. Analytical Methodology 44
6. Sample Collection, Handling, and Preservation 47
7. Quality Assurance 48
8. Records and Data Reporting 48
9. Action Response to Laboratory 48
CHAPTER VI: RADIOCHEMISTRY 59
1. Personnel 59
2. Laboratory Facilities 59
3. Laboratory Equipment and Supplies 60
4. General Laboratory Practices 61
5. Analytical Methodology 61
6. Sample Collection, Handling, and Preservation 61
7. Quality Assurance 61
8. Records and Data Reporting . 62
9. Action Response to Laboratory 62
APPENDICES
Appendix A: Chain-of-Custody 75
Appendix B: Recommended Protocol for Regions Conducting
On-site Laboratory Evaluations 81
Appendix C: • Abbreviations 83
Appendix D: EPA Policy on Third Party Certification 85
Appendix E: Required Analytical Capability for
Principal State Laboratory Systems 87
Appendix F: Additional Contaminants Scheduled for
Rules in 1992-1993 89
Appendix G: §1445 Unregulated Chemicals to be
Monitored 91
Appendix H Analytical Methods for Microbiology 93
XII
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Chapter V
Microbiology
Note: quality control items are designated as "QC"
and necessitate written records which are to be
retained for five years.
1. Personnel
1.1 Supervisor/Consultant
The supervisor or consultant is ,a professional
scientist experienced in water microbiology. If a
supervisor is not available, a consultant having the
same qualifications may be substituted. State
laboratory personnel would be a primary source for
consultants.
1.1.1 Academic Training: Minimum of a
bachelor's degree in science.
1.1.2 Job Training: Minimum of two weeks
training from a Federal agency, State agency, or
academic institution in microbiological analysis
of drinking water.
1.2 Analyst (or equivalent job title)
The analyst performs microbiological tests with
minimal supervision.
1.2.1 Academic training: Minimum of high
school education.
1.2.2 Job training: Training in microbiological
analysis of drinking water, acceptable to the
State (or EPA for nonprimacy States), plus a
minimum of 30 days on-the-job training.
Personnel should take advantage of workshops
and training programs available from Federal
and State regulatory agencies and professional
societies.
1.2.3 Experience: At least six months of
bench experience in sanitary, water, milk, or
food microbiology.
2. Laboratory Facilities
Laboratory facilities are clean and temperature and
humidity controlled, and have adequate lighting at
bench tops. The laboratory has provisions for disposal
of microbiological waste. It is recommended that the
laboratory contain 150-200 square feet and 5 to 6
linear feet of usable bench space per analyst.
Laboratory facilities should include sufficient bench-
top area for processing samples; storage space for
media, glassware, and portable equipment; floor
space for stationary equipment (incubators,
waterbaths, refrigerators, etc.); and associated area(s)
for cleaning glassware and sterilizing materials.
While safety criteria are not an aspect of laboratory
certification, laboratory personnel should be aware of
general and customary safety practices for
laboratories. Each laboratory is encouraged to have a
safety plan available.
3. Laboratory Equipment and Supplies
3.1 pH Meter
3.1.1 Accuracy and scale graduations within
±0.1 units.
3.1.2 Use pH buffer aliquot only once.
3.1.3 Maintain electrodes according to
manufacturer's recommendations.
QC 3.1.4 Standardize pH meter each use period
with pH 7.0 and pH 4.0 standard buffers.
QC 3.1.5 Date commercial buffer solution container
upon receipt, and when opened. Discard before
expiration date.
3.2 Balance (top loader or pan)
3.2.1 Balance detects 100 mg at a 150 gram
load.
QC 3.2.2 Calibrate balance monthly using Class S
or S-1 reference weights (minimum of three
traceable weights which bracket laboratory
weighing needs) or weights traceable to Class S
or S-1 weights. Calibrate non-reference weights
annually with Class S or S-1 reference weights.
37
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Correction data necessary with S or S-1
reference weights.
QC 3.2.3 Maintain service contract or internal
maintenance protocol and maintenance records.
Maintenance conducted annually at a minimum.
3.3 Temperature Monitoring Device
3.3.1 Use glass/mercury or dial thermometers
graduated in 0.5°C increments or less in
incubator units. Mercury column in glass
thermometers is not separated.
QC 3.3.2 Check calibration of in-use glass/mercury
thermometers annually and in-use dial
thermometer quarterly, at the temperature used,
against a reference National Institute of
Standards and Technology (formerly National
Bureau of Standards) (NBS) thermometer or
one that meets the requirements of NBS
Monograph SP 250-23.
QC 3.3.3 Recalibrate continuous recording devices
annually which are used to monitor incubator
temperature. Use same reference thermometer
described in QC 3.3.2.
3.4 Incubator Unit
3.4.1 Incubator unit has an internal
temperature monitoring device and maintains a
temperature of 35°±0.5°C and, if used,
44.5° ±0.2°C. For nonportable incubators, place
thermometers on the top and bottom shelves of
the use area with the thermometer bulb
immersed in liquid. If an aluminum block is
used, culture dishes and tubes fit snugly.
QC 3.4.2 Record temperature for days in use at
least twice per day with readings separated by
at least 4 hours.
3.4.3 An incubation temperature of
44.5°±0.2°C can best be maintained with a
water bath equipped with a gable cover.
3.5 Autoclave
3.5.1 Autoclave has a temperature gauge with
a sensor on the exhaust, a pressure gauge, and
an operational safety valve. Autoclave maintains
sterilization temperature during the sterilizing
cycle and completes an entire cycle within 45
minutes when a 12-15 minute sterilization period
is used. Autoclave depressurizes slowly to
ensure media do not boil over and bubbles do
not form in inverted tubes.
3.5.2 Because of safety concerns and
difficulties with operational control, pressure
cookers and vertical autoclaves are not
acceptable.
QC 3.5.3 Record date, contents, sterilization time,
and temperature for each cycle. Establish
service contract or internal maintenance
protocol, and maintain records.
QC 3.5.4 Use maximum-tempeirature-registering
thermometer, heat-sensitive tape, or spore
strips or ampoules during each autoclave cycle
and record temperature. Avoid overcrowding.
QC 3.5.5 Check automatic timing mechanism with
stopwatch quarterly.
3.6 Hot Air Oven
3.6.1 The oven maintains a stable sterilization
temperature of 170°-180°C for at least two
hours. Sterilize only dry items and avoid
overcrowding. The oven thermometer is
graduated in 10°C increments or less, with the
bulb placed in sand during use.
QC 3.6.2 Record date, contents, and sterilization
time and temperature of each cycle.
3.7 Colony Counter
Use colony counter, dark field model, to count
Heterotrophic Plate Count colonies.
3.8 Conductivity Meter
Suitable for checking laboratory pure water. Readable
in ohms or mhos, with a range from at least 2 ohms
to 2 megohms or equivalent micromhos ± 2%. Unit
may be in-line/bench or portable/battery operated.
QC 3.8.1 Conductivity meter is calibrated monthly
with a 0.01 M KCI solution, or lower
concentration if desired (see Method 120.1 in
Methods for Chemical Analyses of Water and
Wastes, 1979, EPA/600/4-79/020 (revised
1983); or Section 205, "Conductivity," pp. 76-
80, in Standard Methods for the Examination of
Water and Wastewater (16th ed.), 1985). An
inline conductivity meter need not be calibrated.
3.9 Refrigerator
3.9.1 Refrigerator maintains a temperature of
1° to 5°C. Thermometer graduated in at least
1°C increments with the thermometer bulb
immersed in liquid.
QC 3.9.2 Record temperatures for days in use at
least once per day.
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3.10 Inoculating Equipment
Metal or plastic loops, or wood applicator sticks
sterilized by dry heat. The metal inoculating loops
and/or needles are made of nickel alloy or platinum.
3.11 Membrane Filtration Equipment (if MF
procedure is used)
3.11.1 MF units are stainless steel, glass, or
autoclavable plastic, not scratched or corroded,
and do not leak.
3.11.2 10X to 15X magnification device with
fluorescent light source used to count sheen
colonies.
3.11.3 Membrane filters approved by the
manufacturer for total coliform water analysis.
Approval based on data from tests for toxicity,
recovery, retention, and absence of growth-
promoting substances. Filters are cellulose
ester, white, gridmarked, 47 mm diameter, and
0.45 pm pore size, or alternate pore sizes if
manufacturer provides performance data equal
to or better than the 0.45 um pore size.
Membrane filters are purchased presterilized or
autoclaved before use.
QC 3.11.4 Record the lot number and date
received for membrane filters. If the quality and
performance of membrane filters are
questionable, new lot(s) of membrane filters can
be checked by comparing recovery of coliform
organisms against membrane filters from a
previously acceptable lot. (Suggested
procedure: Obtain a natural coliform-positive
water sample or prepare a laboratory water
sample using a pure coliform culture. New lots
of membrane filters are evaluated by passing a
sufficient volume of water sample through a
membrane filter from a new lot and a membrane
filter known to be acceptable so that 30 to 60
coliform colonies are observed on the
acceptable membrane filter after 24 hours
incubation at 35 °C. The colony counts on the
membranes are evaluated using the formula:
Critical value* =
A-B- 1
, where
If the critical value is not less than 1.96, the new
membranes should be considered unacceptable.)
Unacceptable membrane filters are returned to the
vendor with a request to replace them with membrane
filters from a different lot number. Replacement
membranes are submitted to the same comparative
procedure. (This comparative procedure will
demonstrate gross differences between the
membranes; other, more stringent comparative
procedures are acceptable).
QC 3.11.5 Record the lot number and date
received for membrane filters.
QC 3.11.6 Check sterility of each lot number of
membranes by placing one membrane in 50 ml_
volume of non-selective broth medium (e.g.,
tryptic soy broth) and check for growth after 24
hours incubation at 35° ± 0.5°C.
3.72 Culture Dishes (loose or tight lid)
3.12.1 Use presterilized plastic or sterilizable
glass culture dishes. To maintain sterility of
glass culture dishes, use stainless steel or
aluminum canisters, or wrap dishes in a heavy
aluminum foil or char-resistant paper.
3.12.2 Incubate loose-lid dishes in a tight-
fitting container, e.g., plastic vegetable crisper,
to prevent dehydration of membrane filter and
medium.
3.12.3 Reseal opened packs of disposable
culture dishes between major use periods.
3.73 Pipets
3.13.1 To sterilize and maintain sterility of
glass pipets, use stainless steel or aluminum
canisters, or wrap individual pipets in char-
resistant paper.
3.13.2 Pipets have legible markings and are
not chipped or etched.
3.13.3 Opened packs of disposable sterile
pipets are resealed between major use periods.
A is the count on the acceptable membrane filter, and
B is the count on the membrane filter from a new lot.
3.14 Culture Tubes, Containers, and Closures
3.14.1 Tubes and containers are made of
borosilicate glass or other corrosion-resistant
glass, or plastic.
*Hald, Statistical Theory with Engineering Applications. John
Wiley and Sons, Inc., New York, NY, 1960, p. 725.
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3.14.2 Culture tubes and containers are of a
sufficient size to contain medium plus sample
without being more than three quarters full.
3.14.3 Tube and container closures are
stainless steel, plastic, aluminum, or screw caps
with non-toxic liners. Cotton plugs are not
acceptable.
3.15 Sample Containers
3.15.1 Sample bottles are wide mouth plastic
or non-corrosive glass with a non-leaking
ground glass stopper or a cap with a non-toxic
liner which will withstand repeated sterilization,
or other appropriate sample containers.
Capacity of sample containers is at least 120
mL (4 02.).
3.15.2 Glass stoppered bottle closures are
covered with aluminum foil or char-resistant
paper for sterilization.
3.15.3 Glass and plastic bottles are sterilized
by autoclaving; glass bottles may also be
sterilized by dry heat. Moisten empty containers
with several drops of distilled water before
autoclaving to prevent an "air lock" sterilization
failure.
3.16 Glassware and Plasticware
3.16.1 Glassware is borosilicate glass or other
corrosion-resistant glass and free of chips and
cracks. Markings on graduated cylinders and
pipets are legible. Plastic items are clear and
non-toxic.
3.16.2 Graduated cylinders for measurement
of sample volumes have a tolerance of 2.5% or
less. In lieu of graduated cylinders, use pre-
califarated containers that have clearly marked
volumes of 2.5% tolerance. Spot check the
calibration of each lot of pre-calibrated
containers.
3.16.3 Pipets delivering volumes of 10 mL or
less are accurate within a 2.5% tolerance or
less.
3.17 Ultraviolet Lamp (if used)
3.17.1 Disconnect unit monthly and clean
lamps by wiping with soft cloth moistened with
ethanol.
QC 3.17.2 If UV lamp is used for sanitization, test
lamp quarterly with UV light meter and replace if
it emits less than 70% of initial output or if agar
spread plates containing 200 to 1000
microorganisms, exposed to the UV light for 2
minutes, do not show a count reduction of 99%.
Other methods may be used to test a lamp if
they are as effective as the two suggested
methods.
4. General Laboratory Practices
4.1 Sterilization Procedures
4.1.1 The times for autoclaving materials at
1219C are listed below. Except for membrane
filters and pads and carbohydrate-containing
media, indicated times are minimal times which
may necessitate adjustment depending upon
volumes, containers, and loads.
Item
Time
(minutes)
Membrane filters & pads
Carbohydrate containing media
Contaminated test materjals
Membrane filter assemblies
Sample collection bottles
Individual glassware
Djlution water blank
Rinse water
10
12-15
30
15
15
15
15
15
4.1.2 Remove autoclaved membrane filters and
pads and all media immediately after completion
of sterilization cycle.
4.1.3 Membrane filter equipment is autoclaved
at the beginning of each filtration series. A
filtration series ends when 30 minutes or longer
elapses after a sample is last filtered.
4.1.4 Ultraviolet light (germicidal lamp, 2537
angstroms) may be used alternatively to sanitize
equipment, if all supplies are presterilized and
QC checks are conducted as indicated in
paragraph 3.17. Ultraviolet light or boiling v/ater
may also be used to control bacterial carry-over
between samples during a filtration series. If
boiling water is used, membrane filter equipment
should be submerged for about two minutes and
then cooled to room temperature before filtering
the next sample.
4.2 Sample Containers
4.2.1 Add sodium thiosulfate (Na2S2O3;
anhydrous, 100 mg/L) to sample containers
before sterilization (0.1 mL of 10% Na2S2O3
solution per 120 mL capacity).
QC 4.2.2 Select at least one sample container at
random from each batch of sterile sample
bottles, or other appropriate containers, and
confirm sterility by adding approximately a 25
mL volume of a sterile non-selective broth (e.g.,
tryptic soy, trypticase soy, or tryptone broth).
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Incubate at 35° ±0.5°C for 24 hours and check
for growth.
4.3 Reagent Water
4.3.1 Use only satisfactorily tested reagent
water from stills or deionization units to prepare
media, reagents, and dilution/rinse water for
performing bacteriological analyses.
QC 4.3.2 Test the quality of the reagent water or
have it tested by a certified laboratory to assure
it meets the following:
Parameter
Limits
Frequency
Conductivity
Pb, Cd, Cr, Cu,
Ni, Zn
Total Chlorine
ResiduaH
Heterotrophic
Plate Count2
Bacteriolog-
ical Quality of
Reagent Water3
> 0.5 megohms Monthly
resistance or < 2
micromhos/cm at
25°C
Not greater than Annually
0.05 mg/L per
contaminant.
Collectively, no
greater than 0.1
mg/L
Nondetectable Monthly
< 500/mL Monthly
Ratio of growth rate Annually
0.8-3.0
1 DPD Method not required if source water is not chlorinated,
2 Pour Plate Method.
3 Test for bacteriological quality of reagent water (Standard
Methods for the Examination of Water and Wastewater, 16th
Edition p. 835; also Microbiological Methods for Monitoring the
Environment, EPA-600/8-78-017, p.200). Control water for test is
defined as double distilled water using a glass still.
4.4 Dilution/Rinse Water
4.4.1 Prepare stock buffer solution or peptone
water using reagent water according to
Standard Methods for the Examination of Water
and Wastewater, 16th edition, p 855.
4.4.2 Stock buffer is autoclaved or filter-
sterilized. Label and date containers. Ensure
stored stock buffer is free of turbidity.
4.4.3 Dilution/rinse water is prepared by adding
1.25 mL volume of stock buffer solution and 5
mL volume of magnesium chloride (MgCIa)
solution (81.1 g MgCI2 • 6 H2O/L) per liter of
reagent water.
QC 4.4.4 Check each batch of dilution/rinse water
for sterility by adding 50 mL of water to a 50 mL
of a double strength non-selective broth (e.g.,
tryptic soy, trypticase soy or tryptose broth).
Incubate at 35° ± 0.5 °C for 24 hours and check
for growth.
4.5 Glassware Washing
4.5.1 Use distilled or deionized water for final
rinse.
QC 4.5.2 Perform the Inhibitory Residue Test
(Standard Methods for the Examination of Water
and Wastewater, 16th edition, p. 834, and
Microbiological Methods for Monitoring the
Environment, U.S. EPA-600/8-78-017 p. 199) on
the initial use of a washing compound and
whenever a different formulation of washing
compound, or washing procedure, is used to
ensure that glassware is free of toxic residue.
4.6 Analytical Media
4.6.1 General
4.6.1.1 Use of dehydrated or prepared
media manufactured commercially is
strongly recommended due to concern
about quality control. Store dehydrated
media in a cool, dry location and discard
caked or discolored media.
4.6.1.2 Date bottles of dehydrated media
upon receipt and also when initially
opened. Discard dehydrated media 6
months after opening; if stored in a
desiccator, storage is extended to 12
months. Discard any dehydrated media
that has passed the manufacturer's
expiration date.
QC 4.6.1.3 For media prepared in the
laboratory, record the date of preparation,
type of medium, lot number, sterilization
time and temperature, final pH,
technician's initials.
QC 4.6.1.4 For liquid media prepared
commercially, record date received, type
of medium, lot number, and pH verifica-
tion. Discard medium by manufacturer's
expiration date.
QC 4.6.1.5 Check each lot of commercial
and each batch of laboratory-prepared
medium before use for performance with
positive and negative culture controls.
These control organisms can be stock
cultures (periodically checked for purity)
41
Change 1
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or commercially available disks im-
pregnated with the organism.
4.6.1.6 Refrigerate prepared plates in
sealed plastic bags or containers to
minimize evaporation.
4.6.2 Membrane Filter (MF) Media (for total
coliforms)
4.6.2.1 Use m-Endo broth or agar or m-
Endo LES agar in the single step or
enrichment techniques. Ensure that
ethanol used in rehydration procedure is
not denatured. Prepare medium in a
sterile flask and use a boiling water bath
or, if constantly attended, a hot plate with
a stir bar to bring medium just to the
boiling point. Do not boil medium. Final
pH 7.2 ± 0.2.
4.6.2.2 Refrigerate MF broth no longer
than 96 hours, poured MF agar plates no
longer than 2 weeks, and ampouled m-
Endo broth in accordance with
manufacturer's expiration date. Discard
earlier if growth or surface sheen is
observed.
4.6.3 Multiple Tube Fermentation (MTF or
MPN) Media (for total coliforms)
4.6.3.1 Use lauryl tryptose broth or
lactose broth in the presumptive test and
single strength brilliant green lactose bile
(BGLB) broth in the confirmed test. The
appropriate presumptive test medium
concentration should be adjusted to
compensate for sample volume so that
the resulting medium after sample
addition is single strength. Autoclave
media at 121 °C for 12-15 minutes (see
Standard Methods for more specific
guidance). Final pH: presumptive test
media, 6.8 ± 0.2; BGLB broth, 7.2 ±
0.2).
4.6.3.2 Examine tubes following steriliza-
tion to ensure that the inverted vials are
one-third to one-half covered by the
medium and free of air bubbles.
4.6.3.3 If MPN media are refrigerated
after sterilization, incubate overnight at
35 °C before use. Discard tubes/bottles
showing growth and/or bubbles. Use
MPN media prepared in tubes/bottles
with loose-fitting closures within one
week. If prepared broth media are stored,
maintain in the dark at 4°C in screw-cap
tubes/bottles no longer than 3 months.
Discard media if evaporation exceeds
10% of original volume.
4.6.3.4 Use m-Endo agar LES for the
completed test. Use refrigerated medium
on plates within two weeks, and examine
before use. If growth is observed, discard
plates. Protect medium from light.
4.6.4 Presence-Absence (P-A) Coliform Test
Medium
4.6.4.1 Autoclave for 12 minutes at
121°C. Allow space between bottles.
4.6.4.2 If prepared medium is stored,
maintain in a culture bottle at 4°C in the
dark for no longer than 3 months.
Discard earlier if evaporation exceeds
10% of original volume.
4.6.4,3 Final pH: 6.8 ± 0.2.
4.6.5 EC Medium (for fecal coliforms)
4.6.5.1 Autoclave for 12-15 minutes at
121 °C.
4.6.5.2 Examine tubes following steriliza-
tion to ensure that the inverted vials are
one-third to one-half covered by the
medium and free of air bubbles.
4.6.5.3 Use prepared medium in tubes
with loose-fitting closures within one
week. If prepared medium is stored,
maintain in tightly closed screw-cap
tubes at 4°C no longer than 3 months.
Incubate refrigerated sterilized medium
overnight at 35 °C; discard tubes that
show growth and/or bubbles.
4.6.5.4 Final pH: 6.9 ± 0.2.
4.6.6 MMO-MUG Test Medium (for total coli-
forms)
4.6.6.1 Do not prepare this medium from
basic ingredients.
4.6.6.2 Protect medium from light.
4.6.6.3 Ingredients and tubes supplied by
manufacturer(s) are sterile. Do not
autoclave.
4.6.7 EC Medium + MUG (for E. coli)
QC 4.6.7.1 Incubate control cultures at 35° ±
0.5 °C for 24 hours in lauryl tryptose
Change 1
42
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broth. Transfer a loopful to EC Medium
+ MUG and incubate at 44.5° ± 0.2°C
for 24 hours. Read and record the
results.
4.6.7.2 Check test tubes and autoclaved
medium before use with a 366-nm
ultraviolet light to ensure they do not
ftuoresce. If the tubes or medium exhibits
faint fluorescence, either use non-
fluorescing tubes or another lot of
medium that does not fluoresce, or
include a MUG-positive (E. coli) and
MUG-negative (e.g., uninoculated)
control for each analysis.
4,6.7.3 Do not use an inverted vial; gas
production is not relevant to test, and the
use of an inverted vial may cause
confusion on test interpretation.
4.6.7.4 MUG may be added to EC
Medium before autoclaving. EC Medium
+ MUG is also available commercially.
Final MUG concentration is 50 jug/mL.
4.6.7.5 Final pH: 6.9 ± 0.2.
4.6.7.6 Use prepared medium in tubes
with loose-fitting closures within one
week. If prepared medium is stored,
maintain in tightly closed screw-cap
tubes at 4°C no longer than 3 months.
Incubate refrigerated sterile medium
overnight at 44.5° C; discard tubes with
growth.
4.6.8 Nutrient Agar Medium + MUG (for £.
coli)
QC 4.6.8.1 In accordance with paragraph
4.6.1.5, spot-inoculate control cultures
onto a membrane filter on m-Endo LES
agar and incubate at 35° ± 0.5°C for 18-
24 hours. Then transfer the membrane
.filter to Nutrient Agar + MUG and
incubate at 35°C for four hours. Read
and record the results.
4.6.8.2 Sterilize medium in 100-mL
volumes at 121°C for 15 minutes. MUG
may be added to Nutrient Agar before
autoclaving. Nutrient Agar + MUG is
also available commercially. Final MUG
concentration is 100 pg/mL.
4.6.8.3 Final pH: 6.8 ± 0.2.
4.6.8.4 If sterile medium is stored,
refrigerate the medium in petri dishes, in
a plastic bag or tightly closed container,
and use within two weeks. Incubate
stored medium overnight at 35 °C before
use; discard plates with growth.
4.6.9 Heterotrophic Plate Count (HPC) Media
(includes tryptone glucose extract agar, plate
count agar, and R2A agar)
4.6.9.1 Autoclave agar medium at 121°C
for 15 minutes. Final pH: Plate Count
Agar 7.0 '± 0.2; TGE Agar 6.8 - 7.0; R2A
Agar 7.2.
4.6.9.2 Temper melted agar at 44-46 °C
before pouring. Hold melted agar no
longer than 3 hours. Do not melt sterile
agar medium more than once.
4.6.9.3 Store sterile refrigerated medium
in bottles and petri dishes for up to 2
weeks. Store prepared petri dishes with
R2A medium for up to one week,
inverted and refrigerated, in a plastic bag
or tight container. Incubate stored
medium overnight at 35 °C before use;
discard plates with growth.
4.6.10 A-1 Medium (for fecal coliform enumera-
tion in source water only)
4.6.10.1 Sterilize by autoclaving at 121 °C
for 10 minutes.
4.6.10.2 Final pH: 6.9 ± 0.1.
4.6.10.3 Examine tubes following
sterilization to ensure that the inverted
vials are one-third to one-half covered by
the medium and free of air bubbles.
4.6.10.4 Store in dark at room
temperature not more than one week.
4.6.11 Fecal Coliform Membrane Filter (M-FC)
Broth/Agar (for fecal coliform enumeration in
source water only)
4.6.11.1 Sterilize M-FC broth (with or
without agar) by bringing it to boiling
point. Do not autoclave.
4.6.11.2 Final pH: 7.4.
4.6.11.3 Refrigerate prepared medium.
Discard broth medium after 96 hours,
43
Change 1
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and agar medium after two weeks.
Discard earlier if growth is observed.
5. Analytical Methodology
5.1 General
5.1.1 Use only the analytical methodology
specified in the Total Coliform Rule (40 CFR
141.21 (f)) and the Surface Water Treatment
Requirements (40 CFR 141.74(a)) (see
Appendix H).
5.1.2 A laboratory should be certified for all
analytical methods indicated below that it uses
for compliance purposes. At a minimum, the
laboratory must be certified for one total
coliform method 'and one fecal coliform or E.
coli method. In addition, for principal State
laboratories and other laboratories that may
enumerate heterotrophic bacteria for compliance
purposes, the laboratory should be certified for
the Pour Plate Method.
5.1.3 When impregnating an absorbent pad
with a liquid medium, ensure that pad is
saturated.
5.1.4 Shake water sample vigorously before
analyzing.
QC 5.1.5 Each month, perform 'the coliform
procedure normally used on a known coliform-
positive, and fecal- or £ co//-positive sample.
5.2 Membrane Filter Technique (for total
conforms in drinking water)
5.2.1 Sample volumes analyzed by the MF
procedure must be 100 mL ± 2.5 mL
QC 5.2.2 Conduct sterility check at the beginning
and end of each filtration series by filtering 20-
30 mL of dilution water through the membrane
filter. If control indicates contamination, reject all
data from affected samples and request
immediate resampling.
5.2.3 Incubate at 35° ± 0.5°C for 22-24 hours.
5.2.4 Invalidate all samples resulting in
confluent or TNTC (too numerous to count)
growth. Record as "confluent growth" or
"TNTC" and request an additional sample from
the same sampling site. Confluent growth is
defined as a continuous bacterial growth,
without evidence of sheen colonies (total
coliforms), covering the entire membrane filter.
TNTC is defined as greater than 200 colonies
on the membrane filter in the absence of
detectable coliforms. Do not invalidate sample
when the membrane filter contains at least one
total coliform colony.
5.2.5 Verify all sheen colonies (up to at least
ten colonies) using either single strength lactose
or LTB and then single strength BGLB broth
(same media used in MPM procedure), or EPA-
approved cytochrome oxidase and 0-
galactosidase rapid test procedure. Colonies
can be transferred with a sterile needle or
applicator stick.
5.2.6 Test total coliform colonies for either
fecal coliforms or £ coli. When EC Medium or
EC Medium + MUG is used, transfer the
colonies by using one of the options specified
by §141.21 (f) (5) (see Appendix H). If a swab is
used to transfer a total coliform-positive culture,
a single swab can be used to inoculate up to
three different media (e.g., EC Medium, lauryl
tryptose broth, and BGLB broth).
5.3 Multiple Tube Fermentation Technique (for
total coliforms in drinking water)
5.3.1 Various testing configurations can be
used (see 40 CFR 141.21 (f) (3) (i) and (4)), as
long as a total sample volume of 100 mil is
examined for each test.
5.3.2 Incubate at 35° ± 0.5°C for 24 ± 2
hours. If no gas is detected, incubate for
another 24 hours.
5.3.3 Invalidate all samples which produce a
turbid culture in the absence of gas production,
in lauryl tryptose broth or lactose broth. Collect,
or request the system collect, another sample
from the same location as the original
invalidated sample. (The laboratory may streak
the turbid, total coliform-negative culture onto
m-Endo agar, incubate, and examine for total
coliforms, and perform HPC. Although not
required, this information may help the system
assess its problem.)
5.3.4 Confirm 24 and 48 hour gas-positive
tubes using BGLB broth.
QC 5.3.5 Perform completed lest on not less than
10% of all coliform-positive samples per quarter.
If no coliform-positive samples have been
observed during a quarter, obtain one and
perform the method through the completed test.
5.4 Presence-Absence (P-A) Coliform Test (for
total coliforms in drinking water)
5.4.1 Inoculate 100-mL sample into P-A culture
bottle.
Change 1
44
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5.4.2 Incubate at 35° ± 0.5 °C, and observe for
yellow color after 24 and 48 hours.
5.4.3 Confirm yellow cultures in BGLB broth.
5.4.4 Invalidate all samples which produce a
non-yellow turbid culture in P-A medium.
Collect, or request the system collect, another
sample from the same location as the original
invalidated sample. (The laboratory may streak
the non-yellow, turbid, total coliform-negative
culture onto m-Endo agar, incubate, and
examine for total coliforms, and perform HPC.
Although not required, this information may help
the system assess its problem.)
5.5 Fecal Coliform Test (using EC Medium for
fecal coliforms in drinking water or source
water, or A-1 Medium for fecal coliforms in
source water only)
5.5.1 Use EC medium for determining whether
a total coliform-positive culture taken from the
distribution system contains fecal coliforms, in
accordance with the Total Coliform Rule.
Transfer a total coliform-positive culture from a
presumptive tube/bottle or colony to a tube
containing EC Medium and an inverted vial, as
specified by §141.21 (f) (5) (see Appendix H).
5.5.2 Use EC Medium to enumerate fecal
coliforms in source water, in accordance with
the Surface Water Treatment Requirements.
Initially, conduct MTF test, presumptive phase.
Use three sample volumes of source water (10
mL, 1 ml_, 0.1 mL), 5 or 10 tubes/sample
volume. Transfer culture from each total
coliform-positive tube to a tube containing EC
Medium and an inverted vial.
5.5.3 Use A-1 Medium as an alternative to EC
Medium to enumerate fecal coliforms in source
water, in accordance with the Surface Water
Treatment Requirements. Do not use A-1
Medium for drinking water samples. Use three
sample volumes of source/water (10 mL, 1 mL,
0.1 mL), 5 or 10 tubes/sample volume. Unlike
EC Medium, A-1 Medium can be directly
inoculated with a water sample.
5.5.4 Ensure water level of water bath is above
upper level of medium in the culture tubes.
5.5.5 Incubate EC Medium at 44.5° ± 0.2°C
for 24 ± 2 hours. Incubate A-1 Medium at
35° ± 0.5°C for 3 hours, then at 44.5° ± 0.2°C
for 21 ± 2 hours.
5.5.6 Any amount of gas detected in the
inverted vial of a tube that has turbid growth is
considered fecal coliform-positive.
5.6 MMO-MUG Test (for total coliforms in source
water or drinking water)
5.6.1 When using bulk medium, prepare and
incubate a sterility control for each analysis.
Control should consist of a test tube with the
MMO-MUG medium to which sterile water has
been added.
5.6.2 (for enumerating total coliforms in source
water) Use 5 or 10 tubes for each sample
volume tested. Dilution water (for the MMO-
MUG test only), if used, is sterile dechlorinated
tap water, deionized water, or distilled water.
5.6.3 (for determining presence of total
coliforms in drinking water) Use 10 tubes, each
containing 10-mL water sample, or a single
vessel containing 100-mL water sample.
5.6.4 Incubate sample at 35° ± 0.5 °C for 24
hours. A yellow color in the medium indicates
the presence of total coliforms.
5.6.5 If sample color is indeterminate after
incubation for 24 hours, incubate another 4
hours (do not incubate more than 28 hours
total). If the color is still indeterminate with a
reference comparator after 28 hours, invalidate
sample and request another.
5.6.6 Do not use the MMO-MUG test to verify
total coliforms on membrane filters. The filtration
step not only concentrates coliforms, but also
non-coliforms and turbidity, which, at high levels,
can suppress coliforms or cause false-positive
results in the MMO-MUG test.
5.6.7 Do not use the MMO-MUG test to
confirm total coliforms in the MTF or Presence-
Absence (P-A) Coliform Test. High densities of
non-coliforms in the inoculum may overload the
MMO-MUG suppressant reagent system and
cause false-positive results.
5.6.8 Avoid prolonged exposure of inoculated
test to direct sunlight. Sunlight may hydrolyze
indicator compounds, causing a false-positive
result.
5.6.9 Laboratories are encouraged to perform
parallel testing between the MMO-MUG test and
another EPA-approved procedure for
45
Change 1
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enumerating total coliforms for at least several
months and/or over several seasons to assess
the effectiveness of the MMO-MUG test for the
wide variety of water types submitted for
analysis.
5.7 EC Medium + MUG Test (for E. coli)*
5.7.1 Transfer a total coliform-positive cultures
from a presumptive tube/bottle or colony to EC
Medium + MUG, as specified by §141.21 (f) (5)
(see Appendix H).
5.7.2 Ensure water level of water bath is above
upper level of medium.
5.7.3 Incubate at 44.5° ± 0.2°C for 24 ± 2
hours.
5.7.4 Detect fluorescence using an ultraviolet
lamp (366-nm), preferably with a 6-watt bulb.
Ensure that weak autofluorescence of medium, if
present, is not misinterpreted as positive for £.
coli. A MUG-positive (£ coli) and MUG-negative
(e.g., uninoculated) control may be necessary for
each analysis where the medium autofluoresces.
QC 5.7.5 Verify at least 5% of both MUG-positive
results and turbid MUG-negative results for £
co//. Verification of a pure culture may be
conducted, for example, by the use of a multi-
test system (API 20E or equivalent); standard!
biochemical tests (e.g., citrate, indole, and
urease tests); serotyping after biochemical
identification, if desired; or the indole test at
44.5°C and growth in citrate.
5.8 Nutrient Agar + MUG Test (for E. coli)*
5.8.1 Transfer membrane filter containing
coliform colony(ies) from total coliform medium
to surface of Nutrient Agar + MUG medium.
Mark each sheen colony. A portion of the colony
may be transferred with a needle to total coliform
verification test before transfer to Nutrient Agar
+ MUG, or after the 4-hour incubation time.
Another method is to swab the entire membrane
filter surface on the Nutrient Agar + MUG med-
ium after the 4-hour incubation time, with a
sterile cotton swab, and transfer to the total
coliform verification test.
5.8.2 Incubate at 35° ± 0.5°C for 4 hours.
"Detailed procedure can be found in "Test Methods for
Eschoochia coli In Drinking Water," EPA/600/4-91/016. July 7997.
To receive a copy, write Resource Center (WH-550A), USEPA,
40) M Street SW, Washington, DC 20460.
5.8.3 Detect fluorescence using ah ultraviolet
lamp (366-nm), preferably with a 6-watt bulb.
Any amount of fluorescence in a halo around a
sheen colony is considered positive for £. coli.
QC 5.8.4 Verify at least 5% of both MUG-positive
results and MUG-negative, total coliform-positive
results for E. coli. Also verify any non-sheen
colonies that fluoresce. Verification may be
conducted with any of the tests in 5.7.5.
5.9 Pour Plate Method (for enumerating
heterotrophic bacteria; see §141.74 (a) (3) in
Appendix H. Also for use of R2A medium, see
variance criteria in preamble of Federal Register
notice 56:7556-7557, January 15, 1991)
5.9.1 For most potable water samples,
countable plates can be obtained by plating 1.0
mL or 0.1 mL volume of the undiluted sample.
Use at least two replicate plates.
5.9.2 Aseptically pipet sample into bottom of
100 mm x 15 mm petri dish. Add 12-15 mL of
tempered melted (44° - 46°C) agar to each petri
dish. Mix the sample and melted agar carefully to
avoid spillage. After agar plates have solidified
on a level surface, invert plates and incubate at
35° ± 0.5°C for 48 ± 3 hours (except for R2A
Medium). Stack plates in incubator to allow
proper air circulation to maintain uniform
incubation temperature. Do not stack plates
more than four high. Remelt sterile agar medium
only once.
5.9.3 Count colonies manually using a counting
aid such as a Quebec colony counter. Consider
only plates having 30 to 300 colonies in
determining plate count, except for plates
inoculated with 1.0 mL volume of undiluted
sample. Counts less than 30 for such plates are
acceptable. (Fully automatic colony counters are
not suitable because of the size and small
number of colonies observed when potable water
is analyzed for heterotrophic bacteria.)
5.9.4 Check each batch or flask of agar for
sterility by pouring final control plate. Reject data
if control is contaminated.
5.10 Spread Plate Method (for enumerating
heterotrophic bacteria as part of variance
criteria)
5.10.1 Pour 15 mL of R2A agar medium into a
petri dish (100 x 15 mm or 90 x 15 mm) and let
agar solidify. Inoculate agar and incubate as
described by 16th edition of Standard Methods,
Method 907B. Use at least two replicate plates.
5.10.2 Same as paragraph 5.9.3.
Change 1
46
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5.10.3 Same as paragraph 5.9.4.
5.11 Membrane Filter Method (for enumerating
heterotrophic bacteria as part of variance
criteria
5.11,1 Filter a volume that will yield between 20-
>:* 200 colonies. Transfer filter to a 50 x 9 mm petri
dish containing 5 mL of solidified R2A medium.
Incubate at 35 °C or lower for 5-7 days in a close
fitting box containing moistened paper towels.
Use at least two replicate plates.
5.11.2 Count colonies using a stereoscopic
microscope at 10 to 15 magnification.
5.11.3 Check each batch or flask of agar for
sterility by pouring final control plate. Reject data
if control is contaminated.
5.12 MF Procedure (for enumeration of total
coliforms in source water)
5.12.1 Same as paragraphs 5.2.2 - 5.2.5, except
that in paragraph 5.2.4, laboratories should
invalidate any sample which results in confluent
growth or TNTC, even when total coliform
colonies are present, since coliform density is to
be determined.
5.12.2 Use appropriate sample dilutions which
will yield no more than 80, and preferably at
least 20, total coliform colonies per membrane.
5.12.3 Adjust initial counts based upon
verification data.
QC 5.12.4 If two or more analysts are available,
each analyst should count monthly the total
coliform colonies on the same membrane.
Colony counts should agree within 10%.
5.13 Multiple Tube Fermentation Technique (for
enumeration of total coliforms in source
water)
5.13.1 Use 3 sample volumes of source water
(10 mL, 1 mL, 0.1 mL), and 5 or 10 tubes/sam-
ple volume.
5.13.2 Incubate at 35° ± 0.5 °C for 24 ± 2 hours.
5.13.3 Invalidate all samples which produce a
turbid culture in the absence of gas production,
in lauryl tryptose broth or lactose broth. Collect,
or request the system collect, another sample.
The laboratory may use another method to test
the second sample.
Alternatively, if a sample produces a turbid
culture in the absence of gas production,
perform a confirmed test. If the confirmed test is
total coliform-positive, report the most probable
number. If a confirmed test is total coliform-
negative, invalidate the sample and request
another one.
QC 5.13.4 Perform the completed test quarterly on a
coliform-positive tube(s)/bottle.
5.74 Fecal Coliform Membrane Filter Procedure
(for enumerating fecal coliforms in source
water)
5.14.1 Use appropriate sample volumes which
will yield 20 - 60 fecal coliform colonies per
membrane.
QC 5.14.2 Conduct sterility check at the beginning
and end of each filtration series by filtering 20-30
mL of dilution water through the membrane filter.
If control indicates contamination, reject all data
from affected samples and request immediate
resampling.
5.14.3 Incubate
hours.
at 44.5° ± 0.2°C for 24 ± 2
QC 5.14.4 If two or more analysts are available,
each analyst should count monthly the fecal
coliform colonies on the same membrane.
Colony counts should agree within 10%.
6. Sample Collection, Handling, and
Preservation
(Applicable to those laboratories that collect samples;
all laboratories are responsible for paragraphs 6.4 and
6.5.)
6.7 Sample Collector
Collector is trained in sampling procedures and, if
required, approved by the appropriate regulatory
authority or its designated representative.
6.2 Sampling
Samples must be representative of the potable water
distribution system. Water taps used for sampling are
free of aerators, strainers, hose attachments, mixing
type faucets, and purification devices. Maintain a
steady water flow for at least 2 minutes to clear the
service line before sampling. Collect at least a 100
mL sample volume, allow at least 1/2-inch air space
to facilitate mixing of sample by shaking.
47
Change 1
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6.3 Sample Icing
Sample collectors who deliver samples directly to the
laboratory should ice samples immediately after
sample collection.
6.4 Sample Holding/Travel Time
Holding/travel time between sampling and analysis is
not to exceed 30 hours. If laboratory is required by
State regulation to analyze samples after 30 hours
and up to 48 hours, the laboratory is to indicate that
the data may be invalid because of excessive delay
before sample processing. No samples received after
48 hours are to be analyzed for compliance. All
samples received in the laboratory are to be analyzed
on the day of receipt.
6.5 Report Form
Immediately after collection, enter on the sample
report form the sample site location, sample type
(e.g., routine, repeat), date and time of collection,
free chlorine residual, collector's initials, and any
remarks. Also include the date and time of sample
arrival at the laboratory and the date and time
analysis begins. Record additional information as
required by the National Primary Drinking Water
Regulations.
6.6 Cha!n*of-Custody
Follow applicable State regulations pertaining to
chain-of-custody.
7. Quality Assurance
The laboratory prepares and follows a written QA plan
(see Chapter Ill's discussion of QA plans) which is to
be available for inspection by the certification officer.
8. Records and Data Reporting
Records of microbiological analyses are kept by the
laboratory or are accessible to the laboratory for at
least five years. Actual laboratory reports may be
kept, or data may be transferred to tabular
summaries, provided that the following information is
included:
• Date, place, and time of sampling, name of
persons who collected the sample.
» Identification of sample as to whether it is a
routine distribution system sample, repeat
sample, raw or process water sample, or other
special purpose sample.
» Date and time of sample receipt and analysis.
• Laboratory and persons responsible for
performing analysis.
• Analytical technique/method used.
» Results of analysis.
Total coliform MPN data based on confirmed or
completed test (for broth media) and verified
counts (for MF media)
9. Action Response to Laboratory
Results
9.1 Testing Total Colitorm-Positive Cultures
(for Total Coliform Rule) Laboratory must test all total
coliform-positive cultures for presence of either fecal
conforms or E. coli.
9.2 Notification of Positive Results
(for Total Coliform Rule) Laboratory must notify
proper authority promptly of a positive total coliform,
fecal coliform, or E. coli result, so that appropriate
follow-up actions (e.g., collection of repeat samples)
can be conducted (see 40 CFR 141.21(b) and (e), 40
CFR 141.31, etc.). Total coliform-positive result is
based on confirmed phase for the Multiple Tube
Fermentation Technique and Presence-Absence (P-A)
Coliform Test, or verified test for Membrane Filter
Technique. No requirement exists for confirmation of
positive MMO-MUG tests, fecal coliform test, or E.
coli tests.
9.3 Invalidation of Total Coliform-Negative
Sample
(for Total Coliform Rule) Laboratory must notify
proper authority when results indicate that noncoli-
forms may have interfered with the total coliform
analysis, as described by 40 CFR 141.21(c) (2).
Change 1
48
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Sample Forms for On-Site Evaluation of Laboratories Analyzing Public Water Supplies—Microbiology
Laboratory,
Street
City.
Telephone Number.
Survey by_
Affiliation
State
Date
S - Satisfactory
Codes for Marking On-Site Evaluation Forms
X - Unsatisfactory U - Undetermined NA - Not Applicable
1. Personnel
Position/Title
Laboratory
Director
Supervisor/
Consultant
Professional
(note discipline)
Technician/
Analyst
Name
/
Time in Present
Position
Academic
Training and/or
Degree
Present
Specialty
Experience
(years/area)
49
Change 1
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2. Laboratory Facilities
Laboratory facilities clean, temperature and humidity controlled
Adequate lighting at bench top
Laboratory has provision for disposal of microbiological wastes
3. Laboratory Equipment. Supplies, and Materials
3.1 pH Meter
Manufacturer Model
Accuracy ± 0.1 units
Scale graduation, 0.1 units
Maintains electrodes according to manufacturer's
recommendations
pH buffer solution aliquots used only once _
QC Commercial buffer solutions dated when received and
discarded before expiration date
QC Standardize pH meter each use period with
pH 7.0 and 4.0 standard buffers
3.2 Balances (Top Loader or Pan)
Manufacturer Model.
Detects 100 mg at a 150 gram load
QC Calibrate balance monthly using Class S or S-1 reference
weights or weights traceable to Class S or S-1 weights.
If non-reference weights are used, calibrate non-reference
weights with Class S or S-1 reference weights
QC Correction data available with S or S-1 weights
QC Annual service contract or internal maintenance
protocol and record maintained
3.3 Temperature Monitoring Device
Use glass/mercury or dial thermometer in incubator.
Units graduated in no more than 0.5°C increments
No separation in mercury column
QC Check calibration of glass/mercury thermometers annually
and dial thermometers quarterly at the temperature used
against a reference NBS thermometer or one meeting the
requirements of NBS Monograph SP 250-23
Change 1 so
-------
QC Recalibrate continuous recording devices used to monitor
incubator temperature annually against a NBS thermometer or
one meeting the requirements of NBS Monograph SP 250-23
3.4 Incubator Unit
Manufacturer
Maintains internal temperature of 35° ± 0.5 °C,
and if used, 44.5° ± 0.2°C
Place thermometers on top and bottom shelves in
use area of non-portable incubators
Immerse thermometer bulb in liquid
Culture dishes and tubes fit snugly in aluminum
block incubator
QC
Record temperature twice daily for days in use, with
readings separated by at least four hours
3.5 Autoclave
Manufacturer
QC
QC Establish service contract or internal maintenance protocol
QC Heat-sensitive tape, spore strips or ampoules, or maximum
temperature registering thermometer used during each
autoclave cycle
QC Check automatic timing mechanism accuracy with
stop-watch quarterly
3.6 Hot Air Oven
Manufacturer
Model
Temperature gauge with sensor on exhaust
Operational safety valve
Maintains sterilization temperature during cycle
Completes entire cycle within 45 minutes when a
12-15 minute sterilization period is used
Depressurizes slowly to ensure media do not boil
over and bubbles do not form in fermentation tubes
Record date, contents, sterilization time, and
temperature for each cycle
Model
Hot air oven maintains a temperature of 170°-180° C
Thermometer graduated in no more than 10°C increments
Place thermometer bulb in sand
Model
51
Change 1
-------
QC Records include date, sterilization time, and
temperature of each cycle
3.7 Colony Counter
Manufacturer
A dark field colony counter available to count
Heterotrophic Plate Count colonies
Model
3.8 Conductivity Meter
Manufacturer
QC
Suitable for checking laboratory pure water. Readable in
ohms or mhos, has a range of 2 ohms to 2 megohms or
equivalent micromhos ± 2%
Conductivity meter is calibrated monthly with a
0.01 M KCI solution or lower concentration
Model
3.9 Refrigerator(s)
Manufacturer
Model
Maintains temperatures of 1 ° to 5°C
Thermometer(s) graduated in 1°C increments or less
Thermometer bulb(s) immersed in liquid
QC Temperature recorded for days in use
3.10 Inoculating Equipment
Metal or plastic loops, or applicator sticks sterilized by dry heat
Metal loops and/or needles are made of nickel alloy or platinum
3.11 Membrane Filtration Equipment, Membrane Filters and Pads
Manufacturer Mociel_
MF units of stainless steel, glass, or autoclavable plastic
Units do not leak, not scratched or corroded
10 to 15X magnification device with fluorescent light source
Forcep tips without corrugations
Membrane filters from cellulose ester material, white,
gridmarked, 47 mm diameter, 0.45 pm pore size
Alternate pore size used
Membrane filters recommended by manufacturer for
total coliform analysis
Membrane filters and pads are purchased presterilized
or autoclaved before use
Change 1
52
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QC Record lot numbers of membrane filters and date received
QC Determine sterility of each lot of membrane filters by placing
one membrane filter in non-selective broth medium
3.12 Culture dishes : , . :
Use presterilized plastic or sterilized glass dishes
Incubate loose-lid dishes in a tight fitting container
Sterilize glass culture dishes in stainless steel or aluminum
canisters or in heavy aluminum foil or char-resistant paper
Reseal open packs of disposable culture dishes between uses
3.13 Pipets
Sterilize glass pipets in stainless steel or aluminum canisters
or individual pipets wrapped in char-resistant paper
Reseal packs of disposable sterile pipets between
major use periods
Pipets not etched, mouthpiece and tip are not
chipped, graduation markings legible
3.14 Culture Tubes, Containers and Closures
Tubes and containers are borosilicate glass or other corrosion-
resistant glass or plastic
Tubes and containers are of sufficient size that medium
plus sample does not exceed 3/4 full
Tube and container closures are stainless steel, plastic,
aluminum, or screw caps with non-toxic liner
3.15 Sample Containers
Capacity at least 120 mL (4 oz)
Sample bottles are wide mouth plastic with a non-toxic
cap liner, or borosilicate glass with a ground glass stopper,
or other appropriate sample containers such as
single-service sterilized plastic sampling bags with
sodium thiosulfate
Cover glass-stoppered bottle top with aluminum
foil or char-resistant paper prior to sterilization
Glass bottles sterilized by autpclaving or dry heat. Plastic
bottles sterilized by autoclavirig. Empty containers moistened
before autoclaving
3.16 Glassware and Plastic ware
Glass made of borosilicate or other corrosion-resistant glass
Free of chips and cracks
53 Change!
-------
;. 12^15 rnih '
Graduation marks are legible . :
Plastic items are clear and non-toxic. , ''
Graduated cylinders and other pre-calibrated containers used to
measure sample volume have clearly marked volumes with
a 2.5% tolerance or less
Calibration of pre-calibrated containers spot checked
Pipets used to measure sample volumes have a
2.5% tolerance or less • )! ;'"'w:> •"'••';'"'*•;;:'"*''
3.17 Ultraviolet lamp (if used) , , ,, , (
. Lamps cleaned monthly with a soft cloth moistened with ethanol
QC Lamp used for sanitization tested every quarter
4. General Laboratory Practices • • . s • • i ,'•:-••
|tem •'•.'•• Autoclave time 121° C
Membrane filter and pads ,
Carbohydrate media
Contaminated test materials
Membrane filter assemblies
'* ' ' '••,•< , • • •
Sample collection bottles • ;-
Individual glassware T :
Dilution water blanks
Rinse water '
Remove autoclaved MF filters and pads and
all media immediately after sterilization cycle , >;;
Membrane filter assemblies are autoclaved at
start of each filtration series • . '
' If ultraviolet light is used to sanitize equipment, all supplies
are presterilized and QC checks conducted
Ultraviolet light or boiling water used to control bacterial carry-
over between samples during filtration series (optional) .
If boiling water is used to control bacterial carry-over, membrane
filter equipment is submerged for two minutes and then cooled
to room temperature before filtering next sample
4.2 Sample Containers ' " • • • ;' • '•'*
Stock 10% sodium thiosulfate solution free of turbidity
- 30;mih:'';':;";-—-'
15 min '
' ; T5:min' ""••-
: 15 mini
-,•,"''»; "•<'
15 miri_
15 min
Change 1
54
-------
Add sodium thiosulfate to sample containers prior
to sterilization
QC Determine sterility of each lot of sample bottles or presterilized
sample bags by adding non-selective broth, incubating at 35°C
for 24 hours and checking for growth
4.3 Reagent Water
Use reagent water to prepare media, reagents, and
dilution/rinse water
QC
Reagent water is tested to assure the following
minimum criteria are met:
Parameter
Conductivity
Metals-Pb , Cd, Cr,
Cu, Ni, Zn
Total chlorine
residual
Heterotrophic
Plate Count
Bacteriological
quality of
reagent water
4.4 Dilution/Rinse Water
Limits
> 0.5 megohms or
< 2 micromhos at
25°C
Not greater than
0.05 mg/L per con-
taminant. Collec-
tively not greater
than 0.1 mg/L
None detected
< 500/mL
Ratio 0.8-3.0
Frequency
monthly
annually
mortthly
monthly
annually
QC
QC
QC
Prepare stock buffer solution or peptone water
according to Standard Methods, 16th Edition, p. 855
Stock buffer autoclaved or filter sterilized, labeled,
dated, and free of turbidity
10% peptone stock solution autoclaved or filter
sterilized, labeled, dated, and free of turbidity
Prepare dilution/rinse water by adding 1.25 mL volume of
stock buffer solution and 5 mL volume of MgCI2 stock solution
per liter of reagent water
Prepare Q.1% peptone water by adding 10 mL of 10%
stock solution per liter of reagent water
pH of stock phosphate buffer solution is 7.2 ± 0.2
pH of peptone water is 6.8 ± 0.2
Check dilution/rinse water for sterility
55
Change 1
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4.5 Glassware Washing
Use distilled or deionized water for final rinse •
QC Perform inhibitory residue test on clean glassware
4.6 Analytical Media
4.6.1 General J
Commercially available dehydrated or prepared media used
Dehydrated media stored in cool, dry location
Caked or discolored media discarded
Dehydrated media dated when received and when
initially opened
Dehydrated or commercially prepared media that have passed
manufacturer's expiration date are discarded
Open dehydrated media discarded after 6 months (12 months
if stored in desiccator).
QC Media preparation records include:
(a) Date of preparation
(b) Type of medium
(c) Lot number
(d) Sterilization time and temperature
(e) Rnal pH
(f) Technician's initials
QC For liquid media prepared commercially, the following are :
recorded:
(a) Date received
(b) Type of medium
(c) Lot number
(d) pH verification
QC Each commercial lot of medium and each batch of laboratory-
prepared medium is checked before use with positive and
negative controls, and results recorded
Prepared plates are refrigerated in sealed plastic bags or
containers
4.6.2 Membrane Filter Media (for total coliforms)
m-Endo broth or agar, fihal pH:.7.2± 0.1 or m-Endo LES agar,
final pH: 7.2 ± 0.2
Change 1 56
-------
Medium dissolved using:
(a) Boiling water bath
(b) Hot plate with stir bar, constantly attended
Media prepared in sterile flasks
Ethanol not denatured
MF broth refrigerated no longer than 96 hours
MF agar plates refrigerated no longer than 2 weeks
Ampouled m-Endo refrigerated in accordance with
manufacturer's expiration date
Uninoculated media discarded if growth or surface sheen
observed
4.6.3 Multiple Tube Fermentation (MTF or MPN) Media (for
total coliforms)
Lauryl tryptose (lauryl sulfate) or lactose broth used for
presumptive phase
Concentration adjusted so that medium is single strength
after sample addition
Autoclaved at 121 °C for 12-15 min. Final pH: 6.8 ± 0.2
Inverted vials in sterilized medium are one-third to
one-half covered by medium and free of gas bubbles
Brilliant green lactose bile broth used for confirmed phase
Autoclaved at 121 °C for 12-15 min. Final pH 7.2 ± 0.2
Sterilized media stored at 1 ° to 5°C in the dark
Refrigerated media in tubes/containers with loose-fitting
closures used within one week
If stored, broth media are refrigerated in screw cap tubes/
containers, and used within 3 months
Stored broth media is discarded if evaporation exceeds 10%
of original volume
Refrigerated sterile media are incubated overnight at 35 °C before
use, and tubes/containers showing growth or bubbles are discarded
m-Endo LES agar used for the completed phase
If stored, medium is refrigerated. Refrigerated medium used
within two weeks, and discarded if growth is observed. Protect
medium from light
Change 1
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4.6.4 Presence-Absence (P-A) Coliform Test Medium
Medium is autoclaved for 12 minutes at 121 °C, with space allowed
between bottles. Final pH: 6.8 ± 0.2
Space allowed between bottles
Stored medium is refrigerated in culture bottles in dark,
used within 3 months, and discarded if evaporation exceeds 10%
of original volume
4.6.5 EC Medium (for fecal conforms)
Autoclaved for 12-15 minutes at 121 °C. Final pH: 6.9 ± 0.2 ,'
Inverted tubes following sterilization are one-half to one-third
covered by the medium and free of air bubbles
If stored, sterile medium is refrigerated in tightly closed screw
cap tubes and used within 3 months
Stored sterile media incubated overnight at 35°C before use,
and tubes with growth and/or bubbles discarded
4.6.6 MMO-MUG Test Medium (for total coliforms)
Commercial preparation used
Medium protected from light
Medium is not autoclaved
4.6.7 EC Medium + MUG (for £ coli)
QC Each lot of commercially-prepared medium, or batch of
laboratory-prepared medium, is checked with culture controls,
and results recorded
Tubes and autoclaved medium observed for fluorescence before
use with 366-nm ultraviolet light. If weak fluorescence is observed,
either another lot of medium is used, or MUG-positive and
MUG-negative controls are used with analysis
Inverted vial in test tube is not used
Final MUG concentration: 50 pg/ml
RnalpH: 6.9 ± 0.2
If sterile medium is stored, it is refrigerated in tightly closed
screw cap tubes and used within 3 months
Stored media is incubated overnight at 44,5 °C before use, and
tubes with growth discarded
4.6.8 Nutrient Agar Medium + MUG (for £ coli)
QC Quality of medium lot/batch evaluated by spot-inoculating
control bacteria
Change 1 58.1
-------
Medium sterilized in 100-ml volumes at 121 °C for 15 minutes
Final pH 6.8 ± 0.2
Final MUG concentration: 100^g/ml
If media are stored in petri dishes, they are refrigerated in
plastic bag or tightly closed container, and used within 2 weeks
Refrigerated medium incubated overnight at 35 °C before use
and plates with growth discarded
4.6.9 Heterotrophic Plate Count (HPC) Medium
Autoclaved at 121 °C for 15 minutes. Final pH: Plate Count
Agar 7.0 ± 0.2; TGE Agar 6.8- 7.0; R2A Agar 7.2
Melted agar used within 3 hours, and agar tempered (44-46 °C)
before pouring
Sterile agar medium melted not more than once
If media are stored in petri dishes, they are refrigerated
in plastic bag or tightly closed container, and used within 2 weeks
(one week for R2A medium)
Refrigerated medium incubated overnight at 35°C before use
and plates with growth discarded
4.6.10 A-1 Medium (for fecal coliforms)
Medium is sterilized at 121 °C for 10 minutes. Final pH: 6.9 ± 0.1
Inverted tubes are one-third to one-half covered by the medium
and free of air bubbles
Sterilized medium is stored in dark at room temperature and
used within one week
4.6.11 Fecal Coliform Membrane Filter (M-FC) Broth/Agar
Sterilized by bringing to boiling point; not autoclaved. Final pH 7.4
If medium is stored, it is refrigerated and used within 96 hours
(if broth) or two weeks (if agar)
Refrigerated medium is incubated overnight at 44.5 °C before
use, and plates with growth discarded
5. Analytical Methodology
5.1 General
Only analytical methodology specified in the National Primary
Drinking Water Regulations (see Appendix H) is used
Laboratory is to be certified for at least one total coliform method
plus one fecal coliform or £ coli method
Laboratory is to be certified for HPC (Pour Plate Method), if it
conducts method for compliance purposes
58-2 Changel
-------
Absorbent pad, if used, is saturated with liquid medium
Water sample shaken vigorously before analyzing
QC Coliform test conducted monthly on known coliform-posjtive
and fecal- or £. co//-positive sample
5.2 Membrane Filter Technique (for total coliforms in drinking water)
Sample volume analyzed is 100 mL ± 2.5 mL
QC Sterility check conducted at beginning and end of each
filtration series. If control indicates contamination, all data
rejected and another sample obtained
Inoculated medium incubated at 35° ± 0.5°C for 22-24 hours
All samples with confluent or TNTC growth invalidated, unless
total coliform-positive, and new sample obtained
AH sheen colonies verified (up to at least 10 colonies)
Total coliform colonies tested for either fecal coliforms or
E co//, using approved medium and transfer technique
5.3 Multiple Tube Fermentation Technique (for total coliforms in
drinking water)
Concentration of inoculated medium is correct
Sample volume analyzed is 100 mL ±2.5 mL
Inoculated medium incubated at 35° ± 0.5°C for 24 ± 2 hours
If no gas is detected, incubate for another 24 hr
AH turbid gas-negative cultures are invalidated, and another
sample obtained
Cultures from gas-positive tubes incubated in BGLB .broth
QC Completed test performed on at least 10% of all coliform
positive samples/quarter. If no positive samples were observed,
then one obtained and method conducted through the
completed test
5.4 Presence-Absence (P-A) Coliform Test
Sample volume analyzed is 100 mL± 2.5 mL
Inoculated medium incubated at 35° ± 0.5°C and observed for
yellow color after 24 and (if necessary) 48 hours
Yellow cultures confirmed in BGLB broth
AH non-yellow turbid cultures are invalidated, and another
sample obtained
5.5 Fecal Coliform Test
(For distribution system samples) Positive culture from total
coliform medium is transferred to EC Medium, using an approved
Change 1 58.3
-------
transfer technique
(For source water samples) Positive culture from total coliform
medium is transferred to EC medium, using approved transfer
technique, or A-1 Medium is directly inoculated with a water sample.
Three sample volumes used, 5 or 10 tubes/sample volume
Water level of water bath is above upper level of medium in
culture tubes
EC Medium incubated at 44.5° + 0.2°C for 24 ± 2 hours.
A-1 Medium incubated at 35° ± 0.5°C for 3 hours, then at
44.5° ± 0.2°Cfor21 ±2 hours
Any gas detected in inverted vial of tube that has turbid growth
is considered fecal coliform-positive
5.6 MMO-MUG Test (for total coliforms in source water or drinking water)
If bulk medium is employed, a sterility control is used with each
analysis
(For source water) 5 or 10 tubes for each sample volume.
Dilution water, if used, is dechlorinated tap water, deionized
water or distilled water
(For drinking water) 10 tubes used, each containing 10-mL
water sample, or a single vessel containing 100-mL water sample
Inoculated medium incubated at 35° ± 0.5°C for 24 hours
If color indeterminate after 24 hours, medium incubated an
additional 4 hours. If color still questionable after 28 hours,
reference comparator used
If sample color remains indeterminate, sample declared invalid
and another sample requested from same site
Inoculated test not exposed to prolonged direct sunlight
MMO-MUG test is not used to verify/confirm coliforms on
membrane filters or in broth cultures (e.g., lauryl tryptose
broth)
Parallel testing performed for several months or over several
seasons between the MMO-MUG test and another EPA-approved
procedure (optional)
5.7 EC Medium + MUG Test (for £ coli)
Positive culture from total coliform presumptive medium is
transferred to EC Medium, using an approved transfer technique
Water level of water bath is maintained above upper level of
medium in culture tubes
Inoculated medium incubated at 44.5° ± 0.2°C for 24 ± 2 hours
Fluorescence examined with ultraviolet lamp (366-nm). MUG-
positive and MUG-negative controls used when needed
58.4
Change 1
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QC At least 5% of both MUG-positive results and turbid MUG-
negative results are verified for E. coli
5.8 Nutrient Agar + MUG Test (for £ coli)
Total coliform-positive membrane filters transferred to
Nutrient Agar + MUG
Each total coliform-positive colony marked before incubation
on Nutrient Agar + MUG
Inoculated medium incubated at 35° ± 0.5°C for 4 hours
Fluorescence examined with ultraviolet lamp (366-nm). Any
amount of fluorescence in a halo around a sheen colony is
considered E. co//-positive
QC At least 5% of both MUG-positive results and MUG-negative,
total coliform-positive results are verified for E. coli
QC Non-sheen colonies that fluoresce are verified for E. coli
5.9 Pour Plate Method (for heterotrophic bacteria)
Appropriate volume of sample added to plate
Agar tempered to 44-46°C before adding to plate
Sample and melted agar mixed carefully
At least two replicate plates prepared for each sample
Plates incubated in inverted position at 35° ± 0.5°C for
48 ± 3 hours (except for R2A Medium; see Standard Methods)
Plates stacked no more than four high
Sterile agar medium remelted only once
Colonies counted manually using a counting aid such as a
Quebec colony counter
Counts reported for plates having 30 - 300 colonies. (If 1.0 ml
of undiluted sample results in fewer than 30 colonies, that
count is acceptable)
Sterility check performed by pouring a final agar control plate.
Data rejected if control is contaminated
5.10 Spread Plate Method (for heterotrophic bacteria)
R2A agar medium used
Plates with solidified medium dried before use
Medium inoculated in accordance with Standard Methods
At least two replicate plates used for each sample
Plates incubated in inverted position at 28 °C for 7 days
Change 1 58.5
-------
Plates stacked no more than four high
Colonies counted manually using a counting aid such as a Quebec
colony counter
Counts reported for plates having 30 - 300 colonies. (If 1.0 mL
of undiluted sample results in fewer than 30 colonies, that
count is acceptable)
Sterility check performed on an uninoculated control plate.
Data rejected if control is contaminated
5.11 Membrane Filter Method (for heterotrophic bacteria)
Sample volume filtered yields filters with 20-200 colonies
Filter transferred to R2A agar medium
Plates incubated at 35 °C or lower for 5-7 days in a close fitting
box containing moistened paper towels
At least two replicate plates prepared for each sample
Stereoscopic microscope used to count colonies
Sterility check performed on a filter in a control plate.
Data rejected if control is contaminated
5.12 MF Procedure (for total coliforms in source water)
Sample volume filtered yields 80 or fewer colonies/membrane •
QC Sterility check conducted at beginning and end of each
filtration series. If control indicates contamination, all data
rejected and another sample obtained
Inoculated medium incubated at 35° ± 0.5°C for 22-24 hours
All samples with confluent or TNTC growth invalidated, and new
sample obtained
All sheen colonies verified (up to at least 10 colonies)
Initial counts adjusted, based upon verification data
QC If two or more analysts are available, each counts the total
coliform colonies on same membrane at least monthly.
Colony counts agree within 10%
5.13 Multiple Tube Fermentation Technique (for total coliforms in
source water)
Three sample volumes of source water (10 ml, 1 ml, 0.1 ml) used
Five or ten tubes/sample volume used
Inoculated medium incubated at 35° ± 0.5 °C for 24 ± 2 hours
Any sample which produces a turbid culture with no gas is
invalidated, and another sample collected or requested.
Alternatively, if confirmed test is conducted on each turbid,
58.6 Change 1
-------
gas-negative tube, and result is total coliform-positive for each
tube, then Most Probable Number is reported. If any turbid,
gas-negative tube is coliform-negative, sample is invalidated,
and another sample requested
QC Completed test is performed quarterly on coliform-positive
tube(s)/bottle
5.14 Fecal Coliform Membrane Filter Procedure (for fecal conforms in
source water)
Sample volumes used which will yield 60 or fewer fecal
coliform colonies/membrane (and preferably at least 20)
Inoculated medium incubated at 44.5° ± 0.2°C for 24 ± 2 hrs
QC Sterility check conducted at beginning and end of each filtration
series. If control indicates contamination, data rejected and
another sample obtained
QC If two or more analysts are available, each counts the total
coliform colonies on same membrane at least monthly. Colony
counts agree within 10%
6. Sample Collection, Handling, and Preservation ._
6.1 Follow sample procedures described in Standard Methods for the
Examination of Water and Wastewater or Microbiological
Methods for Monitoring the Environment,^.^. EPA-600/8-78-017
6.2 Sample collectors receive training
6.3 Samples representative of distribution system
6.4 Water taps free of any attachments and mixing type faucets
6.5 Water run to waste for at least two minutes
6.6 Sample volume is at least 100 ml_ with sufficient space for
mixing sample
6.7 Sample report form completed by collector
6.8 Samples iced when carrying samples directly to laboratory
6.9 Record date and time of sample arrival at laboratory and
date and time analysis begins
6.10 Transit time does not exceed 30 hours
If laboratory is required by State regulation to examine
samples after 30 hours and up to 48 hours, data are
indicated as possibly invalid
All samples arriving in laboratory after 48 hours are not
analyzed for compliance use
6.11 Compliance with State chain-of-custody regulations, if required
Change 1 58.7
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7. Quality Assurance Program
7.1 Written QA Plan implemented and available for review
7.2 Quality control records maintained for five years
QC 7.3 PE sample is satisfactorily analyzed annually (if available)
8. Data Reporting
8.1 Data entered on the sample report form is checked and initialed
8.2 Sample report forms are retained by laboratory or State program
for five years
Report forms include identification of sample, date and
time of sample receipt and analysis, laboratory and
person (s) responsible for performing analyses,
analytical method used and results of analysis
8.3 Results of analyses
Total coliform MPN data based on confirmed or completed
test (for broth media) and verified counts (for MF media)
9. Action Response by Laboratory
All total coliform-positive cultures tested for the presence
of either fecal coliforms or £ coli (for Total Coliform Rule only)
Proper authority notified of a positive total coliform, fecal
coliform, or £ coli result (for Total Coliform Rule)
Proper authority notified when results indicate that high levels of
noncoliforms may have interfered with the total coliform analysis
(for Total Coliform Rule)
58.8 Change 1
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Appendix E
Required Analytical Capability for Principal State Laboratory Systems^
(As of October 1, 1991)
Volatile Organic
141.24)
Benzene
Carbon tetrachloride
1,2-Dichloroethane
1,1 -Dichloroethylene
p-Dichlorobenzene
1,1,1 -Trichloroethane
Vinyl chloride
Trichloroethylene
trans-1,2-Dichloro-
ethylene
Ethylbenzene
Styrene
Chemicals (40 CFR
Toluene
Total Trihalomethanes
Chloroform
Bromodichloromethane
Dibromochloromethane
Bromoform
o-Dichlorobenzene
cis-1,2-Dichloroethylene
1,2-Dichloropropane
Monochlorobenzene
Tetrachloroethylene
Xylenes (total)
Organics other than VOCs (40 CFR
141.24)
Alachlor
Aldicarb
Aldicarb sulfone
Aldicarb sulfoxide
Atrazine
Carbofuran
Chlordane
Dibromochloro-
propane (DBCP)
Endrin
Ethylene dibromide
(EDB)
Heptachlor
Heptachlor epoxide
Lindane
Methoxychlor
PCBs
Pentachlorophenol
Toxaphene
2,4-D
2,4,5-TP (Silvex)
Inorganics (40 CFR 141.23, 141.89)
Asbestos Lead
Arsenic Mercury
Barium Nitrate-N
Cadmium Nitrite-N
Chromium Selenium
Copper Silver (until July 1992)
Fluoride
Radionuclides
Gross alpha
Gross beta
Radium 226
Radium 228
Tritium
Strontium 89
Strontium 90
(40 CFR 141.25)
Gamma radiation
Cesium 134
Cesium 137
Chromium 51
Cobalt 60
Iodine 131
Ruthenium 106
Zinc 65
Microorganisms (40 CFR 141.21)
Total coliforms
Escherichia do// or fecal conforms
Heterotrophic bacteria
1lf principal State laboratories or other laboratories analyze
compliance samples for sodium or §1445 chemicals, they must be
certified for these contaminants.
87
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Appendix F
Additional Contaminants Scheduled for Rules in 1992-1993
Volatile Organic Chemicals Inorganics
1,2,4-Trichlorobenzene Antimony
1,1,2-Trichloroethane Beryllium
Methylene chloride (Dichloromethane) Cyanide
Nickel
Thallium
Sulfate
Organics (other than VOCs)
Adipate, di(2-diethylhexyl)
Daiapon Radionuclides
Djnoseb Radon
E>ICLUaL „ Uranium
Endothall
Glyphosate
Hexachlorobenzene
Hexachlorocyclopentadiene
Oxamyl (vydate)
PAHs (benzo(a)pyrene)
Phthalates (2-diethylhexyl)
Picloram
Simazine
2,3,7,8-TCDD (dioxin)
89 Change 1
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Appendix G
§1445 Unregulated Chemicals to be Monitored
40 CFR 141.40 (final rule July 8, 1987)
Bromobenzene
Bromodichloromethane
Bromoform
Bromomethane
Chlorodibromomethane
Chloroethane
Chloroform
Chloromethane
o-Chlorotoluene
p-Chlorotoluene
Dibromomethane
m-Dichlorobenzene
1,1-Dichloroethane
Dichloromethane
1,3-Dichloropropane
2,2-Dichloropropane
1,1 -Dichloropropene
1,3-Dichloropropene
1,1,2,2-Tetrachloroethane
1,1,1,2-Tetrachloroethane
1,1,2-Trichloroethane
1,2,3-Trichloropropane
40 CFR 141.40 (final rule
January 30, 1991)
Monitoring is required for the following contaminants.
If the State determines the system is not vulnerable
to contamination, the system can receive a waiver.
Synthetic Organics
Aldrin
Hexachlorobenzene*
Dalapon*
Dinoseb*
Picloram*
Oxamyl (vydate)*
Simazine*
Glyphosate*
Hexachlorocyclopentadiene*
Benzo (a) pyrene*
Metolachlor
di(2-ethylhexyl) Phthalate*
2,3,7,8-TCDD (Dioxin)*
Inorganics
Antimony*
Beryllium*
Cyanide*
Nickel*
Sulfate*
Thallium*
Diquat*
Dieldrin
Dicamba
Carbaryl
3-Hydroxycarbofuran
Methomyl
Butachlor
Endothall*
Metribuzin
Propachlor
di(2-ethylhexyl)
Adipate*
* Being considered for regulation in 1992.
91
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Appendix H
Analytical Methods for Microbiology
1. Total Coliform Rule (40 CFR 141.21(f)>
(1) The standard sample volume required for total
coliform analysis, regardless of analytical method
used, is 100 ml.
(2) Public water systems need only determine the
presence or absence of total coliforms; a
• determination of total coliform density is not
required.
(3) Public water systems must conduct total coliform
analyses in accordance with one of the following
analytical methods:
(i) Multiple-Tube Fermentation (MTF) Technique,
as set forth in Standard Methods for the
Examination of Water and Wastewater, 1985,
American Public Health Association et al.,
16th edition, Method 908, 908A, and 908B-
pp. 870-878, except that 10 fermentation
tubes must be used; or Microbiological
Methods for Monitoring the Environment,
Water and Wastes, U.S. EPA, Environmental
Monitoring and Support Laboratory,
Cincinnati, Ohio 45268 (EPA-600/8-78-017,
December 1978, available from ORD
Publications, CERI, U.S. EPA, Cincinnati,
Ohio 45268), Part III, Section B.4.1-4.6.4, pp.
114-118 (Most Probable Number Method),
except that 10 fermentation tubes must be
used; or
(ii) Membrane Filter (MF) Technique, as set forth
in Standard Methods for the Examination of
Water and Wastewater, 1985, American Pub-
lic Health Association, et al., 16th edition,
Method 909, 909A and 909B-pp. 886-896; or
Microbiological Methods for Monitoring the
Environment, Water and Wastes, U.S. EPA,
Environmental Monitoring and Support
Laboratory, Cincinnati, Ohio 45268 (EPA-
600/8-78-017, December 1978, available from
ORD Publications, CERI, U.S. EPA,
Cincinnati, Ohio 45268), Part III, Section
B.2.1-2.6, pp. 108-112; or
(iii) Presence-Absence (P-A) Coliform Test, as
set forth in Standard Methods for the
Examination of Water and Wastewater, 1985,
American Public Health Association et al.,
16th edition, Method 908E-pp. 882-886; or
(iv) Minimal Medium ONPG-MUG (MMO-MUG)
Test as set forth in the article "National Field
Evaluation of a Defined Substrate Method for
the Simultaneous Detection of Total Coliforms
and Escherichia coli from Drinking Water:
Comparison with Presence-Absence
Techniques" (Edberg et al.), Applied and
Environmental Microbiology, Volume 55, pp.
1003-1008, April 1989. (Note: The MMO-
MUG Test is sometimes referred to as the
Autoanalysis Colilert System.)
(4) In lieu of the 10-tube MTF Technique specified in
paragraph (f)(3)(i) of this section, a public water
system may use the MTF Technique using either
five tubes (20-ml sample portions) or a single
culture bottle containing the culture medium for
the MTF Technique, i.e., lauryl tryptose broth
(formulated as described in Standard Methods for
the Examination of Water and Wastewater, 1985,
American Public Health Association et al., 16th
edition, Method 908A-pp. 872), as long as a 100-
ml water sample is used in the analysis.
(5) Public water systems must conduct fecal coliform
analysis in accordance with the following
procedure. When the MTF Technique or
Presence-Absence (P-A) Coliform Test is used to
test for total coliforms, shake the lactose-positive
presumptive tube or P-A vigorously and transfer
the growth with a sterile 3-mm loop or sterile
applicator stick into brilliant green lactose bile
broth and EC medium to determine the presence
of total and fecal coliforms, respectively. For EPA-
approved analytical methods which use a
membrane filter, transfer the total coliform-
positive culture by one of the following methods:
remove the membrane containing the total
coliform colonies from the substrate with sterile
forceps and carefully curl and insert the
membrane into a tube of EC medium (the
laboratory may first remove a small portion of
93
Change 1
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selected colonies for verification), swab the entire
membrane filter surface with a sterile cotton swab
and transfer the inoculum to EC medium (do not
leave the cotton swab in the EC medium), or
inoculate individual total coliform-positive colonies
into EC medium. Gently shake the inoculated
tubes of EC medium to insure adequate mixing
and incubate in a water bath at 44.5 ± 0. 2°C for
24 ± 2 hours. Gas production of any amount in
the inner fermentation tube of the EC medium
indicates a positive fecal coliform test. The
preparation of EC medium is described in
Standard Methods for the Examination of Water
and Wastewater, 1985, American Public Health
Association, 16th edition, Method 908C--p. 879,
paragraph 1a. Public water systems need only
determine the presence or absence of fecal
coliforms; a determination of fecal coliform
density is not required.
(6) Public water systems must conduct analysis of
Escherichia coli in accordance with one of the
following analytical methods:
(i) EC medium supplemented with 50 pg/ml of 4-
methylumbelliferyl-beta-D-glucuronide (MUG)
(final concentration). EC medium is described
in Standard Methods for the Examination of
Water and Wastewater, 1985, American Pub-
lic Health Association et al., 16th edition, p.
879. MUG may be added to EC medium
before autoclaving. EC medium supple-
mented with 50 ng/ml °f MUG is
commercially available. At least 10 ml of EC
medium supplemented with MUG must be
used. The inner inverted fermentation tube
may be omitted. The procedure for
transferring a total coliform-positive culture to
EC medium supplemented with MUG shall be
as specified in paragraph (f)(5) of this section
for transferring a total coliform-positive culture
to EC medium. Observe fluorescence with an
ultraviolet light (366 nm) in the dark after
incubating tube at 44.5 ± 0.2°C for 24 ± 2
hours; or
(ii) Nutrient agar supplemented with 100 ug/ml 4-
methylumbelliferyl-beta-D-glucuronide (MUG)
(final concentration). Nutrient Agar is
described in Standard Methods for the
Examination of Water and Wastewater, 1985,
American Public Health Association et al.,
16th edition, p. 874. This test is used to
determine if a total coliform-positive sample,
as determined by the Membrane Filter
Technique or any other method in which a
membrane filter is used, contains E coli.
Transfer the membrane filter containing a
total coliform colony(ies) to nutrient agar
supplemented with 100 ug/ml (final
concentration) of MUG. After incubating the
agar plate at 35°C for 4 hours, observe the
colony(ies) under ultraviolet light (366 nm) in
the dark for fluorescence. If fluorescence is
visible, E. coli are present.
2. Surface Water Treatment
Requirements (40 CFR 141.74(a))
Only the analytical method(s) specified in this
paragraph, or otherwise approved by EPA, may be
used to demonstrate compliance with the require-
ments of §§141.71, 141.72, and 1141.73. Measure-
ments for pH, temperature, turbidity, and residual
disinfectant concentrations must be conducted by a
party approved by the State. Measurements for total
coliforms, fecal coliforms, and HPC must be
conducted by a laboratory certified by the State or
EPA to do such analysis. Until laboratory certification
criteria are developed for the analysis of HPC and
fecal coliforms, any laboratory certified for total
coliform analysis by EPA is deemed certified for HPC
and fecal coliform analysis. The following procedures
shall be performed in accordance with the
publications listed in the following section. This
incorporation by reference was approved by the
Director of the Federal Register in accordance with 5
U.S.C. 552(a) and 1 CFR Part 51. Copies of the
methods published in Standard Methods for the
Examination of Water and Wastewater may be
obtained from the American Public Health Association
et al., 1015 Fifteenth Street, NW, Washington, DC
20005; copies of the Minimal Medium ONPG-MUG
Method as set forth in the article "National Field
Evaluation of a Defined Substrate Method for the
Simultaneous Enumeration of Total Coliforms and
Escherichia coli from Drinking Water: Comparison
with the Standard Multiple Tube Fermentation
Method" (Edberg et al.), Applied and Environmental
Microbiology, Volume 54, pp. 1595-1601, June 1988
(as amended under Erratum, Applied and
Environmental Microbiology, Volume 54, p. 3197,
December, 1988), may be obtained from the
American Water Works Association Research
Foundation, 6666 West Quincy Avenue, Denver,
Colorado 80235; and copies of the Indigo Method as
set forth in the article "Determination of Ozone in
Water by the Indigo Method" (Bader and Hoigne),
may be obtained from Ozone Science & Engineering,
Pergamon Press Ltd., Fairview Park, Elmsford, New
York 10523. Copies may be inspected at the U.S.
Environmental Protection Agency, Room EB15, 401
M Street, SW., Washington, DC 20460 or at the
Office of the Federal Register, 1100 L Street NW,
Room 8401, Washington, DC.
(1) Fecal coliform concentration-Method 908C (Fecal
Coliform MPN Procedures), pp. 878-880, Method
908D (Estimation of Bacterial Density), pp. 880-
882, or Method 909C (Fecal Coliform Membrane
Filter Procedure), pp. 896-898, as set forth in
Standard Methods for the Examination of Water
Change 1
94
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and Wastewater, 1985, American Public Health
Association et al., 16th edition.
(2) Total coliform concentration-Method 908A
(Standard Total Coliform Multiple-Tube
(MPN) Tests), pp. 872-876, Method 908B
(Application of Tests to Routine Examina-
tions), pp. 876-878, Method 908D (Estimation
of Bacterial Density), pp. 880-882, Method
909A (Standard Total Coliform Membrane
Filter Procedure), pp. 887-894, or Method
909B (Delayed-lncubation Total Coliform
Procedure), pp. 894-896, as set forth in
Standard Methods for the Examination of
Water and Wastewater, 1985, American
Public Health Association et al., 16th edition;
Minimal Medium ONPG-MUG Test, as set
forth in the article "National Field Evaluation
of a Defined Substrate Method for the
Simultaneous Enumeration of Total Coliforms
and Escherichia coli from Drinking Water:
Comparison with the Standard Multiple Tube
Fermentation Method" (Edberg et al.), Applied
and Environmental Microbiology, Volume 54,
pp. 1595-1601, June 1988 (as amended
under Erratum, Volume 54, p. 3197,
December, 1988).
(Note: The Minimal Medium ONPG-MUG Test is
sometimes referred to as the Autoanalysis Colilert
System.) Systems may use a five-tube test or a ten-
tube test.
(3) Heterotrophic Plate Count-Method 907A
(Pour Plate Method), pp. 864-866, as set
forth in Standard Methods for the Examination
of Water and Wastewater, 1985, American
Public Health Association et al, 16th edition.
95
Change 1
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REPORT DOCUMENTATION PAGE
I
Form Approved
OMB No. 0704-0)88
Puoiic reporting Burden for this collection of information is estimated to average 1 hour per response, including the time for revewing instructions searchinq existma data sources
gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this
collection of information including suggestions for reducing this burden, to Washmgton Headquarters Services. Directorate for information Operations and Reports. 1215 Jefferson
Davis Highway. Suite 1204. Arlington. VA 22202-4302. and to the Office of Management and Budget. Paperwork Reduction Project (0704-0188) Washington DC 20503 *"
1. AGENCY USE ONLY (Leave blank)
2. REPORT DATE
October 1991
I 3. REPORT TYPE AND DATES COVERED
4. TITLE AND SUBTITLE
Manual for the Certification of
Laboratories Analyzing Drinking Water: Criteria
and Procedures; Quality Assurance - Change 1
6. AUTHUR(S)
5. FUNDING NUMBERS
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)
8. PERFORMING ORGANIZATION
REPORT NUMBER
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES)
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
401 M St., SW
Washington, DC 20/f60
10. SPONSORING/MONITORING
AGENCY REPORT NUMBER
570/9-90-008A
11. SUPPLEMENTARY NOTES
12a. DISTRIBUTION/AVAILABILITY STATEMENT
12b. DISTRIBUTION CODE
13. ABSTRACT (Maximum 200 words)
Since 1978, the U.S. Environmental Protection Agency (EPA) has had
a program for certifying regional laboratories, principal state
laboratories in primacy states, and local laboratories in non-
primacy states performing drinking water analyses required by
regulations issued pursuant to the Safe Drinking Water Act. The
manual describing the program's implementation procedures and
technical criteria, THE MANUAL FOR THE CERTIFICATION OF
LABORATORIES ANALYZING DRINKING MATER, is now in its third edition
(April 1990, EPA 570/9-90-008).
This document is change 1 to the 1990 MANUAL FOR THE CERTIFICATION
OP LABORATORIES ANALYZING DRINKING WATER. Change 1 revises Chapter
V. This revision includes the certification criteria for the
revised Total Coliform Rule and Surface Water Treatment
Requirements. The updated- Chapter V is to be inserted into the
1990 manual and the previous edition of Chapter V completely
removed, along with the other pages indicated on page ii of this
document.
U. SUBJECT TERMS
Drinking Water, Laboratory Certification,
Quality- Control
15. NUMBER OF PAGES
52
16. PRICE CODE
17. SECURITY CLASSIFICATION
OF REPORT
unclassified
18. SECURITY CLASSIFICATION
OF THIS PAGE
unclassified
19. SECURITY CLASSIFICATION
OF ABSTRACT
unclassified
20. LIMITATION OF ABSTRACT
NSN 7510-01-280-5500
Standard Form 298 (Rev 2-89)
Prescribed by ANSI Std 239-18 —
-------
TECHNICAL RFPORT DATA .
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA 570/9-90/008
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Manual for the Certification of Laboratories
Analyzing Drinking Water
5. REPORT DATE
April 1990
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFOR
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Office of Drinking Water (WH-550D)
US EPA
401 M Street, SW
Washington, DC 20460
10. PROGR
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Same as #9
13. TYPE OF REPORT AND PERIOD CO'
14. SPONSORING AGENCY'CODE
15. SUPPLEMENTARY NOTES
This manual describes the operational and technical criteria and
zocedures EPA will use to evaluate a laboratory for its ability to
properly analyze a regulated microbiological, chemical, or
radiochemical drinking water contaminant. The certification program
described in this manual extends to the EPA Regional laboratories,
principal State laboratories in States which have primary enforcement
responsibility (primacy), and to all laboratories that perform analyses
under the SDWA in the few States without primacy. The vast majority
of primacy States have thir own laboratory certification programs.
Although many of them use the EPA's program as presented in this manual,
individual State programs should be contacted to insure equivalency
with State requirements.
This document is the third edition of the manual, and supersedes
EPA 570/9-82-002, of the same title, which was issued in 1982,,
7.
KEY WORDS AND DOCUMENT ANALYSIS
a.
DESCRIPTORS
Laboratory
Certification ,
Safe Drinking Water''Act (SDWA)
18. DISTRIBUTION STATEMENT
Release to Public
b-IDENTlFIERS/OPEN ENDED TERMS
Water Supply
Drinking Water
Quality Assurance
Sanitary Microbiolo^
Chemistry
Radiochemistry
19. SECURITY CLASS (ThisReport)
Unclassified
20. SECURITY CLASS (Tills page)
Unclassified
c. COSATI Field/Group
22. PRICE
EPA Form 2220-1 (Rov. 4-77) PREVIOUS EDITION is OBSOLETE
-U.S. GOVERNMENT PRINTING OFFIC&1990-717-003/28006
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