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Standard Methods for the Examination of Water and Wastewater
Part 9000 MICROBIOLOGICAL EXAMINATION
9010 INTRODUCTION*#(1)
The following sections describe procedures for making microbiological examinations of
water samples to determine sanitary quality. The methods are intended to indicate the degree of
contamination with wastes. They are the best techniques currently available; however, their
limitations must be understood thoroughly.
Tests for detection and enumeration of indicator organisms, rather than of pathogens, are
used. The coliform group of bacteria, as herein defined, is the principal indicator of suitability of
a water for domestic, industrial, or other uses. The cultural reactions and characteristics of this
group of bacteria have been studied extensively.
Experience has established the significance of coliform group density as a criterion of the
degree of pollution and thus of sanitary quality. The significance of the tests and the
interpretation of results are well authenticated and have been used as a basis for standards of
bacteriological quality of water supplies.
The membrane filter technique, which involves a direct plating for detection and estimation
of coliform densities, is as effective as the multiple-tube fermentation test for detecting bacteria
of the coliform group. Modification of procedural details, particularly of the culture medium, has
made the results comparable with those given by the multiple-tube fermentation procedure.
Although there are limitations in the application of the membrane filter technique, it is equivalent
when used with strict adherence to these limitations and to the specified technical details. Thus,
two standard methods are presented for the detection and enumeration of bacteria of the coliform
group.
It is customary to report results of the coliform test by the multiple-tube fermentation
procedure as a Most Probable Number (MPN) index. This is an index of the number of coliform
bacteria that, more probably than any other number, would give the results shown by the
laboratory examination; it is not an actual enumeration. By contrast, direct plating methods such
as the membrane filter procedure permit a direct count of coliform colonies. In both procedures
coliform density is reported conventionally as the MPN or membrane filter count per 100 mL.
Use of either procedure permits appraising the sanitary quality of water and the effectiveness of
treatment processes. Because it is not necessary to provide a quantitative assessment of coliform
bacteria for all samples, a qualitative, presence-absence test is included.
Fecal streptococci and enterococci also are indicators of fecal pollution and methods for their
detection and enumeration are given. A multiple-tube dilution and a membrane filter procedure
are included.
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Standard Methods for the Examination of Water and Wastewater
Methods for the differentiation of the coliform group are included. Such differentiation
generally is considered of limited value in assessing drinking water quality because the presence
of any coliform bacteria renders the water potentially unsatisfactory and unsafe. Speciation may
provide information on colonization of a distribution system and further confirm the validity of
coliform results.
Coliform group bacteria present in the gut and feces of warm-blooded animals generally
include organisms capable of producing gas from lactose in a suitable culture medium at 44.5 ±
0.2°C. Inasmuch as coliform organisms from other sources often cannot produce gas under these
conditions, this criterion is used to define the fecal component of the coliform group. Both the
multiple-tube dilution technique and the membrane filter procedure have been modified to
incorporate incubation in confirmatory tests at 44.5°C to provide estimates of the density of fecal
organisms, as defined. Procedures for fecal coliforms and Escherichia coli include a 24-h
multiple-tube test using A-1 medium, a 7-h rapid method, and chromogenic substrate coliform
tests. This differentiation yields valuable information concerning the possible source of pollution
in water, and especially its remoteness, because the nonfecal members of the coliform group may
be expected to survive longer than the fecal members in the unfavorable environment provided
by the water.
The heterotrophic plate count may be determined by pour plate, spread plate, or membrane
filter method. It provides an approximate enumeration of total numbers of viable bacteria that
may yield useful information about water quality and may provide supporting data on the
significance of coliform test results. The heterotrophic plate count is useful in judging the
efficiency of various treatment processes and may have significant application as an in-plant
control test. It also is valuable for checking quality of finished water in a distribution system as
an indicator of microbial regrowth and sediment buildup in slow-flow sections and dead ends.
Experience in the shipment of un-iced samples by mail indicates that noticeable changes may
occur in type or numbers of bacteria during such shipment for even limited periods of time.
Therefore, refrigeration during transportation is recommended to minimize changes, particularly
when ambient air temperature exceeds 13°C.
Procedures for the isolation of certain pathogenic bacteria and protozoa are presented. These
procedures are tedious and complicated and are not recommended for routine use. Likewise,
tentative procedures for enteric viruses are included but their routine use is not advocated.
Examination of routine bacteriological samples cannot be regarded as providing complete
information concerning water quality. Always consider bacteriological results in the light of
information available concerning the sanitary conditions surrounding the sample source. For a
water supply, precise evaluation of quality can be made only when the results of laboratory
examinations are interpreted in the light of sanitary survey data. Consider inadequate the results
of the examination of a single sample from a given source. When possible, base evaluation of
water quality on the examination of a series of samples collected over a known and protracted
period of time.
Pollution problems of tidal estuaries and other bodies of saline water have focused attention
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Standard Methods for the Examination of Water and Wastewater
on necessary modification of existing bacteriological techniques so that they may be used
effectively. In the following sections, applications of specific techniques to saline water are not
discussed because the methods used for fresh waters generally can be used satisfactorily with
saline waters.
Methods for examination of the waters of swimming pools and other bathing places are
included. The standard procedures for the plate count, fecal coliforms, and fecal streptococci are
identical with those used for other waters. Procedures for Staphylococcus and Pseudomonas
aeruginosa, organisms commonly associated with the upper respiratory tract or the skin, are
included.
Procedures for aquatic fungi and actinomycetes are included.
Sections on rapid methods for coliform testing and on the recovery of stressed organisms are
included. Because of increased interest and concern with analytical quality control, this section
continues to be expanded.
The bacteriological methods in Part 9000, developed primarily to permit prompt and rapid
examination of water samples, have been considered frequently to apply only to routine
examinations. However, these same methods are basic to, and equally valuable in, research
investigations in sanitary bacteriology and water treatment. Similarly, all techniques should be
the subject of investigations to establish their specificity, improve their procedural details, and
expand their application to the measurement of the sanitary quality of water supplies or polluted
waters.
9020 QUALITY ASSURANCEQUALITY CONTROL*#(2)
9020 A. Introduction
1. General Considerations
The growing emphasis on microorganisms in water quality standards and enforcement
activities and their continuing role in research, process control, and compliance monitoring
require the establishment and effective operation of a quality assurance (QA) program to
substantiate the validity of analytical data.
A laboratory quality assurance program is the integration of intralaboratory and
interlaboratory quality control (QC), standardization, and management practices into a formal,
documented program with clearly defined responsibilities and duties to ensure that the data are of
the type, quality, and quantity required.
The program must be practical and require only a reasonable amount of time or it will be
bypassed. Generally, about 15% of overall laboratory time should be spent on different aspects of
a quality assurance program. However, more time may be needed for more important analytical
data, e.g., data for enforcement actions. When properly administered, a balanced, conscientiously
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Standard Methods for the Examination of Water and Wastewater
applied QA program will optimize data quality without adversely affecting laboratory
productivity.
Because microbiological analyses measure constantly changing living organisms, they are
inherently variable. Some quality control tools used by chemists, such as reference standards,
instrument calibration, and quality control charts, may not be available to the microbiologist.
Because QA programs vary among laboratories as a result of differences in organizational
mission, responsibilities, and objectives; laboratory size, capabilities, and facilities; and staff
skills and training, this provides only general guidance. Each laboratory should determine the
appropriate QA level for its purpose.
2. Guidelines for a Quality Assurance Program
Develop a QA program to meet the laboratory’s specific needs and the planned use of the
data. Emphasis on the use of data is particularly important where significant and costly decisions
depend on analytical results. An effective QA program will confirm the quality of results and
increase confidence in the data.
a. Management responsibilities: Management must recognize the need for quality assurance,
commit monetary and personnel resources, assume a leadership role, and involve staff in
development and operation of the QA program. Management should meet with the laboratory
supervisor and staff to develop and maintain a comprehensive program and establish specific
responsibility for management, supervisors, and analysts.
b. Quality assurance officer: In large laboratories, a QA officer has the authority and
responsibility for application of the QA program. Ideally, this person should have a staff position
reporting directly to upper management, not a line position. The QA officer should have a
technical education, be acquainted with all aspects of laboratory work, and be familiar with
statistical techniques for data evaluation. The QA officer is responsible for initiating the program,
convincing staff of its value, and providing necessary information and training to the staff. Once
the QA program is functioning, the coordinator conducts frequent (weekly to monthly) reviews
with the laboratory supervisor and staff to determine the current status and accomplishments of
the program and to identify and resolve problems. The QA officer also reports periodically to
management to secure backing in actions necessary to correct problems that threaten data quality.
c. Staff: Laboratory and field staffs participate with management in planning the QA
program, preparing standard operating procedures, and most importantly, implementing the QC
program in their daily tasks of collecting samples, conducting analyses, performing quality
control checks, and calculating and reporting results. Because the staffs are the first to see
potential problems, they should identify them and work with the supervisor to correct and avoid
them. It is critical to the success of the QA program that staff understand and actively support it.
3. Quality Assurance Program Objectives
The objectives of a QA program include providing data of known quality, ensuring a high
quality of laboratory performance, maintaining continuing assessment of laboratory operations,
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Standard Methods for the Examination of Water and Wastewater
identifying weaknesses in laboratory operations, detecting training needs, and improving
documentation and recordkeeping.
4. Elements of a Quality Assurance Program
Each laboratory should develop and implement a written QA plan describing the QA
program and QC activities of the laboratory. The plan should address the following basic
common aspects:
a. Statement of objectives, describing the specific goals of the laboratory.
b. Sampling procedures, including selection of representative sites and specified holding
time and temperature conditions. If data may be subjected to litigation, use chain-of- custody
procedures.
c. Personnel policies, describing specific qualification and training requirements for
supervisors and analysts.
d. Equipment and instrument requirements, providing calibration procedures and frequency
and maintenance requirements.
e. Specifications for supplies, to ensure that reagents and supplies are of high quality and are
tested for acceptability.
f. Analytical methods, i.e., standardized methods established by a standards-setting
organization and validated. Ideally, these laboratory methods have documented precision, bias,
sensitivity, selectivity, and specificity.
g. Analytical quality control measures, including such analytical checks as duplicate
analyses, positive and negative controls, sterility checks, and verification tests.
h. Standard operating procedures (SOPs), i.e., written statement and documentation of all
routine laboratory operations.
i. Documentation requirements, concerning data acquisition, recordkeeping, traceability, and
accountability.
j. Assessment requirements:
1) Internal audits of the laboratory operations, performed by the QA officer and supervisor.
2) On-site evaluations by outside experts to ensure that the laboratory and its personnel are
following an acceptable QA program.
3) Performance evaluation studies, in which the QA officer works with the supervisor to
incorporate unknown challenge samples into routine analytical runs and laboratories are
encouraged to participate in state and national proficiency testing and accreditation programs.
The collaborative studies confirm the abilities of a laboratory to generate acceptable data
comparable to those of other laboratories and identify potential problems.
k. Corrective actions: When problems are identified by the staff, supervisor, andor QA
coordinator, use standard stepwise procedures to determine the causes and correct them.
Nonconformances identified by external laboratory evaluation are corrected, recorded, and signed
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Standard Methods for the Examination of Water and Wastewater
off by the laboratory manager and QA officer.
Detailed descriptions of quality assurance programs are available.
1-4

The QC guidelines discussed in Section 9020B and Section 9020C are recommended as
useful source material, but all elements need to be addressed in developing a QA program.
5. References
1.
GASKIN, J.E.
1992. Quality Assurance in Water Quality Monitoring. Inland Water
Directorate, Conservation & Protection, Ottawa, Ont., Canada.
2.
RATLIFF, T.A., JR.
1990. The Laboratory Quality Assurance System. A Manual of
Quality Procedures with Related Forms. Van Nostrand Reinhold, New York, N.Y.
3.
GARFIELD, F.M.
1984. Quality Assurance Principles of Analytical Laboratories. Assoc.
Official Analytical Chemists, Arlington, Va.
4.
DUX, J.P.
1983. Quality assurance in the analytical laboratory. Amer. Lab. 26:54.
9020 B. Intralaboratory Quality Control Guidelines

All laboratories have some intralaboratory QC practices that have evolved from common
sense and the principles of controlled experimentation. A QC program applies practices
necessary to minimize systematic and random errors resulting from personnel, instrumentation,
equipment, reagents, supplies, sampling and analytical methods, data handling, and data
reporting. It is especially important that laboratories performing only a limited amount of
microbiological testing exercise strict QC. A listing of key QC practices is given in Table 9020:I.
Other sources of QC practices are available.
1-3
These practices and guidelines will assist
laboratories in establishing and improving QC programs. Laboratories should address all of the
QC guidelines discussed herein, but the depth and details may differ for each laboratory.
1. Personnel
Microbiological testing should be performed by a professional microbiologist or technician
trained in environmental microbiology whenever possible. If not, a professional microbiologist
should be available for guidance. Train and evaluate the analyst in basic laboratory procedures.
The supervisor periodically should review procedures of sample collecting and handling, media
and glassware preparation, sterilization, routine analytical testing, counting, data handling, and
QC techniques to identify and eliminate problems. Management should assist laboratory
personnel in obtaining additional training and course work to advance their skills and career.
2. Facilities
a. Ventilation: Plan well- ventilated laboratories that can be maintained free of dust, drafts,
and extreme temperature changes. Whenever possible, laboratories should have air conditioning
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Standard Methods for the Examination of Water and Wastewater
to reduce contamination, permit more stable operation of incubators, and decrease moisture
problems with media and instrumentation.
b. Space utilization: Design and operate the laboratory to minimize through traffic and
visitors, with a separate area for preparing and sterilizing media, glassware, and equipment. Use a
vented laminar- flow hood for dispensing and preparing sterile media, transferring microbial
cultures, or working with pathogenic materials. In smaller laboratories it may be necessary,
although undesirable, to carry out these activities in the same room.
c. Laboratory bench areas: Provide at least 2 m of linear bench space per analyst and
additional areas for preparation and support activities. For stand-up work, typical bench
dimensions are 90 to 97 cm high and 70 to 76 cm deep. For sit-down activities such as
microscopy and plate counting, benches are 75 to 80 cm high. Specify bench tops of stainless
steel, epoxy plastic, or other smooth, impervious surface that is inert and corrosion-resistant, has
a minimum number of seams, and has adequate sealing of any crevices. Install even, glare-free
lighting with about 1000 lux (100 ft-candles) intensity at the working surface.
d. Walls and floors: Assure that walls are covered with a smooth finish that is easily cleaned
and disinfected. Specify floors of smooth concrete, vinyl, asphalt tile, or other impervious, sealed
washable surfaces.
e. Work-area monitoring: Maintain high standards of cleanliness in work areas. Monitor air,
at least monthly, with air density plates. The number of colonies on the air density plate test
should not exceed 160m
2
15 min exposure (15 coloniesplate15 min).
Plate or the swab method
1
can be used weekly or more frequently to monitor bench surface
contamination. Although uniform limits for bacterial density have not been set, each laboratory
can use these tests to establish a base line and take action on a significant increase.
f. Laboratory cleanliness: Regularly clean laboratory rooms and wash benches, shelves,
floors, and windows. Wet-mop floors and treat with a disinfectant solution; do not sweep or
dry-mop. Wipe bench tops and treat with a disinfectant before and after use. Do not permit
laboratory to become cluttered.
3. Laboratory Equipment and Instrumentation
Verify that each item of equipment meets the user’s needs for precision and minimization of
bias. Perform equipment maintenance on a regular basis as recommended by the manufacturer or
obtain preventive maintenance contracts on autoclave, balances, microscopes, and other
equipment. Directly record all quality control checks in a permanent log book.
Use the following quality control procedures:
a. Thermometertemperature-recording instruments: Check accuracy of thermometers or
temperature- recording instruments semiannually against a certified National Institute of
Standards and Technology (NIST) thermometer or one traceable to NIST and conforming to
NIST specifications. For general purposes use thermometers graduated in increments of 0.5°C or
less. Maintain in water or glycerol for air incubators and refrigerators and glycerol for freezers
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Standard Methods for the Examination of Water and Wastewater
and seal in a flask. For a 44.5°C water bath, use a submersible thermometer graduated to 0.2°C
or less. Record temperature check data in a quality control log. Mark the necessary NIST
calibration corrections on each thermometer and incubator, refrigerator, or freezer. When
possible, equip incubators and water baths with temperature-recording instruments that provide a
continuous record of operating temperature.
b. Balances: Follow manufacturer’s instructions in operation and routine maintenance of
analytical and top- loading balances. Balances should be serviced and recalibrated by a
manufacturer technician annually or more often as conditions change or problems occur. In
weighing 2 g or less, use an analytical balance with a sensitivity less than 1 mg at a 10-g load.
For larger quantities use a pan balance with sensitivity of 0.1 g at a 150-g load.
Wipe balance before use with a soft brush. Clean balance pans after use and wipe spills up
immediately with a laboratory tissue. Inspect weights with each use and replace if corroded. Use
only a plastic-tip forceps to handle weights. Check balance and working weights monthly against
a set of reference weights (ANSIASTM Class 1 or NIST Class S) for accuracy, precision, and
linearity.
4
Record results.
c. pH meter: Use a meter graduated in 0.1 pH units or less, that includes temperature
compensation. Preferably use digital meters and commercial buffer solutions. With each use,
standardize meter with two buffers that bracket the pH of interest and record. Date buffer
solutions when opened and check monthly against another pH meter. Discard solution after each
use and replace buffer supply before expiration date. For full details of pH meter use and
maintenance, see Section 4500-H
+
.
d. Water purification system: Commercial systems are available that include some
combination of prefiltration, activated carbon, mixed-bed resins, and reverse-osmosis with final
filtration to produce a reagent-grade water. The life of such systems can be extended greatly if
the source water is pretreated by distillation or by reverse osmosis to remove dissolved solids.
Such systems tend to produce the same quality water until resins or activated carbon are near
exhaustion and quality abruptly becomes unacceptable. Some deionization components are
available now that automatically regenerate the ion exchange resins. Do not store reagent water
unless a commercial UV irradiation device is installed and is confirmed to maintain sterility.
Monitor reagent water continuously or daily with a calibrated conductivity meter and analyze
at least annually for trace metals. Replace cartridges at intervals recommended by the
manufacturer based on the estimated usage and source water quality. Do not wait for column
failure. If bacteria-free water is desired, include aseptic final filtration with a 0.22-µm-pore
membrane filter and collect in a sterile container. Monitor treated water for contamination and
replace the filter as necessary.
e. Water still: Stills produce water of a good grade that characteristically deteriorates slowly
over time as corrosion, leaching, and fouling occur. These conditions can be controlled with
proper maintenance and cleaning. Stills efficiently remove dissolved substances but not dissolved
gases or volatile organic chemicals. Freshly distilled water may contain chlorine and ammonia
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Standard Methods for the Examination of Water and Wastewater
(NH
3
). On storage, additional NH
3
and CO
2
are absorbed from the air. Use softened water as the
source water to reduce frequency of cleaning the still. Drain and clean still and reservoir
according to manufacturer’s instructions and usage.
f. Media dispensing apparatus: Check accuracy of volumes dispensed with a graduated
cylinder at start of each volume change and periodically throughout extended runs. If the unit is
used more than once per day, pump a large volume of hot reagent water through the unit to rinse
between runs. Correct leaks, loose connections, or malfunctions immediately. At the end of the
work day, break apparatus down into parts, wash, rinse with reagent water, and dry. Lubricate
parts according to manufacturer’s instructions or at least once per month.
g. Hot-air oven: Test performance monthly with commercially available Bacillus subtilis
spore strips or spore suspensions. Monitor temperature with a thermometer accurate in the 160 to
180°C range and record results. Use heat-indicating tape to identify supplies and materials that
have been exposed to sterilization temperatures.
h. Autoclave: Record items sterilized, temperature, pressure, and time for each run.
Optimally use a recording thermometer. Check and record operating temperature weekly with a
minimummaximum thermometer. Test performance with Bacillus stearothermophilus spore
strips, suspensions, or capsules monthly. Use heat- indicating tape to identify supplies and
materials that have been sterilized.
i. Refrigerator: Maintain temperature at 1 to 4°C. Check and record temperature daily and
clean monthly. Identify and date materials stored. Defrost as required and discard outdated
materials quarterly.
j. Freezer: Maintain temperature at −20°C to −30°C. Check and record temperature daily. A
recording thermometer and alarm system are highly desirable. Identify and date materials stored.
Defrost and clean semiannually; discard outdated materials.
k. Membrane filtration equipment: Before use, assemble filtration units and check for leaks.
Discard units if inside surfaces are scratched. Wash and rinse filtration assemblies thoroughly
after use, wrap in nontoxic paper or foil, and sterilize.
l. Ultraviolet lamps: Disconnect lamps monthly and clean bulbs with a soft cloth moistened
with ethanol. Test lamps quarterly with an appropriate (short- or long- wave) UV light meter*#(3)
and replace bulbs if output is less than 70% of the original. For short-wave lamps used in
disinfecting work areas, expose plate count agar spread plates containing 200 to 300 organisms
of interest, for 2 min. Incubate plates at 35°C for 48 h and count colonies. Replace bulb if count
is not reduced 99%.

CAUTION:
Although short- wave (254-nm) UV light is known to be more dangerous than
long-wave UV (365-nm), both types of UV light can damage eyes and skin and potentially are
carcinogenic.
5
Protect eyes and skin from exposure to UV light. (See Section 1090B .)
m. Biohazard hood: Once per month expose plate count agar plates to air flow for 1 h.
Incubate plates at 35°C for 48 h and examine for contamination. A properly operating biohazard
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Standard Methods for the Examination of Water and Wastewater
hood should produce no growth on the plates. Disconnect UV lamps and clean monthly by
wiping with a soft cloth moistened with ethanol. Check lamps’ efficiency as specified above.
Inspect cabinet for leaks and rate of air flow quarterly. Use a pressure monitoring device to
measure efficiency of hood performance. Have laminar-flow safety cabinets containing HEPA
filters serviced by the manufacturer. Maintain hoods as directed by the manufacturer.
n. Water bath incubator: Verify that incubators maintain test temperature, such as 35 ±
0.5°C or 44.5 ± 0.2°C. Keep an appropriate thermometer (¶ 3a, above) immersed in the water
bath; monitor and record temperature twice daily (morning and afternoon). For optimum
operation, equip water bath with a gable cover. Use only stainless steel, plastic-coated, or other
corrosion-proof racks. Clean bath as needed.
o. Incubator (air, water jacketed, or aluminum block): Verify that incubators maintain
appropriate test temperatures. Also, verify that cold samples are incubated at the test temperature
for the required time. Check and record temperature twice daily (morning and afternoon) on the
shelves in use. If a glass thermometer is used, submerge bulb and stem in water or glycerine to
the stem mark. For best results use a recording thermometer and alarm system. Place incubator in
an area where room temperature is maintained between 16 and 27°C (60 to 80°F).
p. Microscopes: Use lens paper to clean optics and stage after each use. Cover microscope
when not in use.
Permit only trained technicians to use fluorescence microscope and light source. Monitor
fluorescence lamp with a light meter and replace when a significant loss in fluorescence is
observed. Log lamp operation time, efficiency, and alignment. Periodically check lamp
alignment, particularly when the bulb has been changed; realign if necessary. Use known positive
4 + fluorescence slides as controls.
4. Laboratory Supplies
a. Glassware: Before each use, examine glassware and discard items with chipped edges or
etched inner surfaces. Particularly examine screw-capped dilution bottles and flasks for chipped
edges that could leak and contaminate the analyst and the area. Inspect glassware after washing
for excessive water beading and rewash if necessary. Make the following tests for clean
glassware as necessary:
1) pH check—Because some cleaning solutions are difficult to remove completely, spot
check batches of clean glassware for pH reaction, especially if soaked in alkali or acid. To test
clean glassware for an alkaline or acid residue add a few drops of 0.04% bromthymol blue (BTB)
or other pH indicator and observe the color reaction. BTB should be blue-green (in the neutral
range).
To prepare 0.04% bromthymol blue indicator solution, add 16 mL 0.01N NaOH to 0.1 g BTB
and dilute to 250 mL with reagent water.
2) Test for inhibitory residues on glassware and plasticware—Certain wetting agents or
detergents used in washing glassware may contain bacteriostatic or inhibiting substances that
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Standard Methods for the Examination of Water and Wastewater
require 6 to 12 rinsings to remove all traces and insure freedom from residual bacteriostatic
action. Perform this test annually and before using a new supply of detergent. If prewashed,
presterilized plasticware is used, test it for inhibitory residues. Although the following procedure
describes testing of petri dishes for inhibitory residue, it is applicable to other glass or
plasticware.
a) Procedure—Wash and rinse six petri dishes according to usual laboratory practice and
designate as Group A.
Wash six petri dishes as above, rinse 12 times with successive portions of reagent water, and
designate as Group B.
Rinse six petri dishes with detergent wash water (in use concentration), and air-dry without
further rinsing, and designate as Group C.
Sterilize dishes in Groups A, B, and C by the usual procedure.
For presterilized plasticware, set up six plastic petri dishes and designate them as Group D.
Prepare and sterilize 200 mL plate count agar and hold in a 44 to 46°C water bath.
Prepare a culture of E. aerogenes known to contain 50 to 150 colony-forming unitsmL.
Preliminary testing may be necessary to achieve this count range. Inoculate three dishes from
each test group with 0.1 mL and the other three dishes from each group with 1 mL culture.
Analyze the four sets of six plates each, following heterotrophic plate count method (Section
9215B), and incubate at 35°C for 48 h. Count plates with 30 to 300 colonies and record results as
CFU mL.
b) Interpretation of results—Difference in averaged counts on plates in Groups A through D
should be less than 15% if there are no toxic or inhibitory effects.
Differences in averaged counts of less than 15% between Groups A and B and greater than
15% between Groups A and C indicate that the cleaning detergent has inhibitory properties that
are eliminated during routine washing. Differences between B and D greater than 15% indicate
an inhibitory residue.
b. Utensils and containers for media preparation: Use utensils and containers of borosilicate
glass, stainless steel, aluminum, or other corrosion-resistant material (see Section 9030). Do not
use copper utensils.
c. Dilution water bottles: Use scribed bottles made of nonreactive borosilicate glass or
plastic with screwcaps containing inert liners. Clean before use. Disposable plastic bottles
prefilled with dilution water are available commercially and are acceptable. Before use of each
lot, check pH and volume and examine sterile bottles of dilution water for a precipitate; discard if
present. Reclean bottles with acid if necessary, and remake the dilution water. If precipitate
repeats, procure a different source of bottles.
d. Reagent- grade water quality: The quality of water obtainable from a water purification
system differs with the system used and its maintenance. See ¶ 3d and ¶ 3e above.
Recommended limits for reagent water quality are given in Table 9020:II. If these limits are not
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Standard Methods for the Examination of Water and Wastewater
met, investigate and correct or change water source. Although pH measurement of reagent water
is characterized by drift, extreme readings are indicative of chemical contamination.
e. Use test for evaluation of reagent water, media, and membranes: When a new lot of
culture medium, membrane filters, or a new source of reagent-grade water is to be used make
comparison tests, at least quarterly, of the current lot in use (reference lot) against the new lot
(test lot).
1) Procedure—Use a single batch of control water (redistilled or distilled water polished by
deionization), glassware, membrane filters, or other needed materials to control all variables
except the one factor under study. Make parallel pour or spread plate or membrane filter plate
tests on reference lot and test lot, according to procedures in Section 9215 and Section 9222. As
a minimum, make single analyses on five different water samples positive for the target
organism. Replicate analyses and additional samples can be tested to increase the sensitivity of
detecting differences between reference and test lots.
When conducting the use test on reagent water, perform the quantitative bacterial tests in
parallel using a known high-quality water as a control water. Prepare dilutionrinse water and
media with new source of reagent and control water. Test water for all uses (dilution, rinse,
media preparation, etc.).
2) Counting and calculations—After incubation, compare bacterial colonies from the two
lots for size and appearance. If colonies on the test lot plates are atypical or noticeably smaller
than colonies on the reference lot plates, record the evidence of inhibition or other problem,
regardless of count differences. Count plates and calculate the individual count per 1 mL or per
100 mL. Transform the count to logarithms and enter the log-transformed results for the two lots
in parallel columns. Calculate the difference, d, between the two transformed results for each
sample, including the + or − sign, the mean, and the standard deviation s
d
of these differences
(see Section 1010B).
Calculate Student’s t statistic, using the number of samples as n:
These calculations may be made with various statistical software packages available for
personal computers.
3) Interpretation—Use the critical t value, from a Student’s t table for comparison against the
calculated value. At the 0.05 significance level this value is 2.78 for five samples (four degrees of
freedom). If the calculated t value does not exceed 2.78, the lots do not produce significantly
different results and the test lot is acceptable. If the calculated t value exceeds 2.78, the lots
produce significantly different results and the test lot is unacceptable.
If the colonies are atypical or noticeably smaller on the test lot or the Student’s t exceeds
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Standard Methods for the Examination of Water and Wastewater
2.78, review test conditions, repeat the test, andor reject the test lot and obtain another one.
f. Reagents: Because reagents are an integral part of microbiological analyses, their quality
must be assured. Use only chemicals of ACS or equivalent grade because impurities can inhibit
bacterial growth, provide nutrients, or fail to produce the desired reaction. Date chemicals and
reagents when received and when first opened for use. Make reagents to volume in volumetric
flasks and transfer for storage to good-quality inert plastic or borosilicate glass bottles with
borosilicate, polyethylene, or other plastic stoppers or caps. Label prepared reagents with name
and concentration, date prepared, and initials of preparer. Include positive and negative control
cultures with each series of cultural or biochemical tests.
g. Dyes and stains: In microbiological analyses, organic chemicals are used as selective
agents (e.g., brilliant green), as indicators (e.g., phenol red), and as microbiological stains (e.g.,
Gram stain). Dyes from commercial suppliers vary from lot to lot in percent dye, dye complex,
insolubles, and inert materials. Because dyes for microbiology must be of proper strength and
stability to produce correct reactions, use only dyes certified by the Biological Stain Commission.
Check bacteriological stains before use with at least one positive and one negative control culture
and record results.
h. Membrane filters and pads: The quality and performance of membrane filters vary with
the manufacturer, type, brand, and lot. These variations result from differences in manufacturing
methods, materials, quality control, storage conditions, and application.
1) Membrane filters and pads for water analyses should meet the following specifications:
a) Filter diam 47 mm, mean pore diam 0.45 µm. Alternate filter and pore sizes may be used
if the manufacturer provides data verifying performance equal to or better than that of
47-mm-diam, 0.45-µm-pore size filter. At least 70% of filter area must be pores.
b) When filters are floated on reagent water, the water diffuses uniformly through the filters
in 15 s with no dry spots on the filters.
c) Flow rates are at least 55 mLmincm
2
at 25°C and a differential pressure of 93 kPa.
d) Filters are nontoxic, free of bacterial-growth- inhibiting or stimulating substances, and free
of materials that directly or indirectly interfere with bacterial indicator systems in the medium;
ink grid is nontoxic. The arithmetic mean of five counts on filters must be at least 90% of the
arithmetic mean of the counts on five agar spread plates using the same sample volumes and agar
media.
e) Filters retain the organisms from a 100-mL suspension of Serratia marcescens containing
1 × 10
3
cells.
f) Water-extractables in filter do not exceed 2.5% after the membrane is boiled in 100 mL
reagent water for 20 min, dried, cooled, and brought to constant weight.
g) Absorbent pad has diam 47 mm, thickness 0.8 mm, and is capable of absorbing 2.0 ± 0.2
mL Endo broth.
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Standard Methods for the Examination of Water and Wastewater
h) Pads release less than 1 mg total acidity calculated as CaCO
3
when titrated to the
phenolphthalein end point with 0.02N NaOH.
i) If filter and absorbent pad are not sterile, they should not be degraded by sterilization at
121°C for 10 min. Confirm sterility by absence of growth when a membrane filter is placed on a
pad saturated with tryptone glucose extract broth or tryptone glucose extract agar and incubated
at 35 ±0.5°C for 24 h.
j) Some lots of membrane filters yield low recoveries, poor differentiation, or malformation
of colonies due to toxicity, chemical composition, or structural defects.
6
Perform the use test (¶
4e) on new lots of filters.
2) Standardized tests:
Standardized tests are available for evaluating retention, recovery, extractables, and flow rate
characteristics of membrane filters.
7

Some manufacturers provide information beyond that required by specifications and certify
that their membranes are satisfactory for water analysis. They report retention, pore size, flow
rate, sterility, pH, percent recovery, and limits for specific inorganic and organic chemical
extractables. Although the standard membrane filter evaluation tests were developed for the
manufacturers, a laboratory can conduct its own tests.
To maintain quality control inspect each lot of membranes before use and during testing to
insure they are round and pliable, with undistorted gridlines after autoclaving. After incubation,
colonies should be well-developed with well-defined color and shape as defined by the test
procedure. The gridline ink should not channel growth along the ink line nor restrict colony
development. Colonies should be distributed evenly across the membrane surface.
i. Culture media: Because cultural methods depend on properly prepared media, use the best
available materials and techniques in media preparation, storage, and application. For control of
quality, use commercially prepared media whenever available but note that such media may vary
in quality among manufacturers and even from lot to lot from the same manufacturer.
Order media in quantities to last no longer than 1 year. Use media on a first- in, first-out
basis. When practical, order media in quarter pound (114 g) multiples rather than one pound (454
g) bottles, to keep the supply sealed as long as possible. Record kind, amount, and appearance of
media received, lot number, expiration date, and dates received and opened. Check inventory
quarterly for reordering.
Store dehydrated media at an even temperature in a cool dry place, away from direct sunlight.
Discard media that cake, discolor, or show other signs of deterioration. If expiration date is given
by manufacturer, discard unused media after that date. A conservative time limit for unopened
bottles is 2 years at room temperature. Compare recovery of newly purchased lots of media
against proven lots, using recent pure-culture isolates and natural samples.
Use opened bottles of media within 6 months. Dehydrated media are hygroscopic. Protect
opened bottles from moisture. Close bottles as tightly as possible, immediately after use. If
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Standard Methods for the Examination of Water and Wastewater
caking or discoloration of media occurs, discard media. Store opened bottles in a dessicator.
1) Preparation of media—Prepare media in containers that are at least twice the volume of
the medium being prepared. Stir media, particularly agars, while heating. Avoid scorching or
boil- over by using a boiling water bath for small batches of media and by continually attending to
larger volumes heated on a hot plate or gas burner. Preferably use hot plate-magnetic stirrer
combinations. Label and date prepared media. Prepare media in reagent water. Measure water
volumes and media with graduates or pipets conforming to NIST and APHA standards,
respectively. Do not use blow-out pipets. After preparation and storage, remelt agar media in
boiling water or flowing steam.
Check and record pH of a portion of each medium after sterilization and cooling. Check pH
of solid medium with a surface probe. Record results. Make minor adjustments in pH (<0.5 pH
units) with 1N NaOH or HCl solution to the pH specified in formulation. If the pH difference is
larger than 0.5 units, discard the batch and check preparation instructions and pH of reagent
water to resolve the problem. Incorrect pH values may be due to reagent water quality, medium
deterioration, or improper preparation. Review instructions for preparation and check water pH.
If water pH is unsatisfactory, prepare a new batch of medium using water from a new source (see
Section 9020B.3d and e). If water is satisfactory, remake medium and check; if pH is again
incorrect, prepare medium from another bottle.
Record pH problems in the media record book and inform the manufacturer if the medium is
indicated as the source of error. Examine prepared media for unusual color, darkening, or
precipitation and record observations. Consider variations of sterilization time and temperature as
possible causes for problems. If any of the above occur, discard the medium.
2) Sterilization—Sterilize media at 121 to 124°C for the minimum time specified. A
double-walled autoclave permits maintenance of full pressure and temperature in the jacket
between loads and reduces chance for heat damage. Follow manufacturer’s directions for
sterilization of specific media. The required exposure time varies with form and type of material,
type of medium, presence of carbohydrates, and volume. Table 9020:III gives guidelines for
typical items. Do not expose media containing carbohydrates to the elevated temperatures for
more than 45 min. Exposure time is defined as the period from initial exposure to removal from
the autoclave.
Some currently available autoclave models are automatic and include features such as vertical
sliding, self-sealing and opening doors, programmable sterilization cycles, and continuous
multipoint monitoring of chamber temperature and pressure. These units also may incorporate
solution cooling and vapor removal features. When sterilizer design includes heat exchangers and
solution cooling features as part of a factory-programmed liquid cycle, strict adherence to the
45-min total elapsed time in the autoclave is not necessary provided that printout records verify
normal cycle operation and chamber cooling during exhaust and vapor removal.
Remove sterilized media from autoclave as soon as chamber pressure reaches zero, or, if a
fully automatic model is used, as soon as the door opens. Do not reautoclave media.
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Standard Methods for the Examination of Water and Wastewater
Check effectiveness of sterilization weekly by placing Bacillus stearothermophilus spore
suspensions or strips (commercially available) inside glassware. Sterilize at 121°C for 15 min.
Place in trypticase soy broth tubes and incubate at 55°C for 48 h. If growth of the autoclaved
spores occurs after incubation at 55°C, sterilization was inadequate. A small, relatively
inexpensive 55°C incubator is available commercially.†#(4)
Sterilize heat-sensitive solutions or media by filtration through a 0.22-µm-pore-diam filter in
a sterile filtration and receiving apparatus. Filter and dispense medium in a safety cabinet or
biohazard hood if available. Sterilize glassware (pipets, petri dishes, sample bottles) in an
autoclave or an oven at 170°C for 2 h. Sterilize equipment, supplies, and other solid or dry
materials that are heat-sensitive, by exposing to ethylene oxide in a gas sterilizer. Use
commercially available spore strips or suspensions to check dry heat and ethylene oxide
sterilization.
3) Use of agars and broths—Temper melted agars in a water bath at 44 to 46°C until used
but do not hold longer than 3 h. To monitor agar temperature, expose a bottle of water or medium
to the same heating and cooling conditions as the agar. Insert a thermometer in the monitoring
bottle to determine when the temperature is 45 to 46°C and suitable for use in pour plates. If
possible, prepare media on the day of use. After pouring agar plates for streaking, dry agar
surfaces by keeping dish slightly open for at least 15 min in a bacteriological hood to avoid
contamination. Discard unused liquid agar; do not let harden or remelt for later use.
Handle tubes of sterile fermentation media carefully to avoid entrapping air in inner tubes,
thereby producing false positive reactions. Examine freshly prepared tubes to determine that gas
bubbles are absent.
4) Storage of media—Prepare media in amounts that will be used within holding time limits
given in Table 9020:IV. Protect media containing dyes from light; if color changes occur, discard
the media. Refrigerate poured agar plates not used on the day of preparation. Seal agar plates
with loose-fitting lids in plastic bags if held more than 2 d. Prepare broth media that will be
stored for more than 2 weeks in screw-cap tubes, other tightly sealed tubes, or in loose-capped
tubes placed in a sealed plastic bag or other tightly sealed container to prevent evaporation.
Mark liquid level in several tubes and monitor for loss of liquid. If loss is 10% or more,
discard the batch. If media are refrigerated, incubate overnight at test temperature before use and
reject the batch if false positive responses occur. Prepared sterile broths and agars available from
commercial sources may offer advantages when analyses are done intermittently, when staff is
not available for preparation work, or when cost can be balanced against other factors of
laboratory operation. Check performance of these media as described in ¶ 5 below.
5) Quality control of prepared media—Maintain in a bound book a complete record of each
prepared batch of medium with name of preparer and date, name and lot number of medium,
amount of medium weighed, volume of medium prepared, sterilization time and temperature, pH
measurements and adjustments, and preparations of labile components. Compare quantitative
recoveries of new lots with previously acceptable ones. Include sterility and positive and negative
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Standard Methods for the Examination of Water and Wastewater
control culture checks on all media as described below.
5. Standard Operating Procedures (SOPs)
SOPs are the operational backbone of an analytical laboratory. SOPs describe in detail all
laboratory operations such as preparation of reagents, reagent water, standards, culture media,
proper use of balances, sterilization practices, and dishwashing procedures, as well as methods of
sampling, analysis, and quality control. The SOPs are unique to the laboratory. They describe the
tasks as performed on a day- to-day basis, tailored to the laboratory’s own equipment,
instrumentation, and sample types. The SOPs guide routine operations by each analyst, help to
assure uniform operations, and provide a solid training tool.
6. Sampling
a. Planning: Microbiologists should participate in the planning of monitoring programs that
will include microbial analyses. They can provide valuable expertise on the selection of sampling
sites, number of samples and analyses needed, workload, and equipment and supply needs. For
natural waters, knowledge of the probable microbial densities, and the impact of season, weather,
tide and wind patterns, known sources of pollution, and other variables, are needed to formulate
the most effective sampling plan.
b. Methods: Sampling plans must be specific for each sampling site. Prior sampling guidance
can be only general in nature, addressing the factors that must be considered for each site.
Sampling SOPs describe sampling equipment, techniques, frequency, holding times and
conditions, safety rules, etc., that will be used under different conditions for different sites. From
the information in these SOPs sampling plans will be drawn up.
7. Analytical Methods
a. Method selection: Because minor variations in technique can cause significant changes in
results, microbiological methods must be standardized so that uniform data result from multiple
laboratories. Select analytical methods appropriate for the sample type from Standard Methods or
other source of standardized methods and ensure that methods have been validated in a
multi-laboratory study with the sample types of interest.
b. Data objectives: Review available methods and determine which produce data to meet the
program’s needs for precision, bias, specificity, selectivity, and detection limit. Ensure that the
methods have been demonstrated to perform within the above specifications for the samples of
interest.
c. Internal QC: The written analytical methods should contain required QC checks of
positive and negative control cultures, sterile blank, replicate analyses (precision), and a known
quantitative culture, if available.
d. Method SOPs: As part of the series of SOPs, provide each analyst with a copy of the
analytical methods written in step-wise fashion exactly as they are to be performed and specific
to the sample type, equipment, and instrumentation used in the laboratory.
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Standard Methods for the Examination of Water and Wastewater
8. Analytical Quality Control Procedures
a. General quality control procedures:
1) New methods—Conduct parallel tests with the standard procedure and a new method to
determine applicability and comparability. Perform at least 100 parallel tests across seasons of
the year before replacement with the new method for routine use.
2) Comparison of plate counts—For routine performance evaluation, repeat counts on one or
more positive samples at least monthly and compare the counts with those of other analysts
testing the same samples. Replicate counts for the same analyst should agree within 5% and
those between analysts should agree within 10%. See Section 9020B.10b for a statistical
calculation of data precision.
3) Control cultures—For each lot of medium check analytical procedures by testing with
known positive and negative control cultures for the organism(s) under test. See Table 9020:V
for examples of test cultures.
4) Duplicate analyses—Perform duplicate analyses on 10% of samples and on at least one
sample per test run. A test run is defined as an uninterrupted series of analyses. If the laboratory
conducts less than 10 testsweek, make duplicate analyses on at least one sample each week.
5) Sterility checks—For membrane filter tests, check sterility of media, membrane filters,
buffered dilution and rinse water, pipets, flasks and dishes, and equipment as a minimum at the
end of each series of samples, using sterile reagent water as the sample. If contaminated, check
for the source. For multiple-tube and presence-absence procedures, check sterility of media,
dilution water, and glassware. To test sterility of media, incubate a representative portion of each
batch at an appropriate temperature for 24 to 48 h and observe for growth. Check each batch of
buffered dilution water for sterility by adding 20 mL water to 100 mL of a nonselective broth.
Alternatively, aseptically pass 100 mL or more dilution water through a membrane filter and
place filter on growth medium suitable for heterotrophic bacteria. Incubate at 35 ± 0.5°C for 24 h
and observe for growth. If any contamination is indicated, determine the cause and reject
analytical data from samples tested with these materials. Request immediate resampling and
reanalyze.
b. Precision of quantitative methods: Calculate precision of duplicate analyses for each
different type of sample examined, for example, drinking water, ambient water, wastewater, etc.,
according to the following procedure:
1) Perform duplicate analyses on first 15 positive samples of each type, with each set of
duplicates analyzed by a single analyst. If there is more than one analyst, include all analysts
regularly running the tests, with each analyst performing approximately an equal number of tests.
Record duplicate analyses as D
1
and D
2
.
2) Calculate the logarithm of each result. If either of a set of duplicate results is <1, add 1 to
both values before calculating the logarithms.
3) Calculate the range (R) for each pair of transformed duplicates as the mean (î ) of these
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Standard Methods for the Examination of Water and Wastewater
ranges.
See sample calculation in Table 9020:VI.
4) Thereafter, analyze 10% of routine samples in duplicate. Transform the duplicates as in ¶
2) and calculate their range. If the range is greater than 3.27 R, there is greater than 99%
probability that the laboratory variability is excessive. Determine if increased imprecision is
acceptable; if not, discard all analytical results since the last precision check (see Table
9020:VII). Identify and resolve the analytical problem before making further analyses.
5) Update the criterion used in ¶ 4) by periodically repeating the procedures of ¶s 1) through
3) using the most recent sets of 15 duplicate results.
9. Verification
For the most part, the confirmationverification procedures for drinking water differ from
those for other waters because of specific regulatory requirements.
a. Multiple-tube fermentation (MTF) methods:
1) Total coliform procedure (Section 9221B)
a) Drinking water—Carry samples through confirmed phase only. Verification is not
required. For QC purposes, if normally there are no positive results, analyze at least one positive
source water quarterly to confirm that the media produce appropriate responses. For samples with
a history of heavy growth without gas in presumptive-phase tubes, carry the tubes through the
confirmed phase to check for false negative responses for coliform bacteria. Verify any positives
for fecal coliforms or E. coli.
b) Other water types—Verify by performing the completed MTF Test on 10% of samples
positive through the confirmed phase.
2) Enzyme substrate coliform test (total coliformE. coli) (Section 9223B)
a) Drinking water—Verify at least 5% of total coliform positive results from enzyme
substrate coliform tests by inoculating growth from a known positive sample and testing for
lactose fermentation or for β-
D
-galactopyranosidase by the o-nitrophen yl-β-
D
-galactopyranoside
(ONPG) test and indophenol by the cytochrome oxidase (CO) test. See Section 9225D for these
tests. Coliforms are ONPG-positive and cytochrome- oxidase-negative. Verify E. coli using the
EC MUG test (see Section 9221F).
b) Other water types—Verify at least 10% of total coliform positive samples as in ¶ 2a
above.
3) Fecal streptococci procedure—Verify as in 9230C.5. Growth of catalase-negative,
gram-positive cocci on bile esculin agar at 35°C and in brain- heart infusion broth at 45°C verifies
the organisms as fecal streptococci. Growth at 45°C and in 6.5% NaCl broth indicates the
streptococci are members of the enterococcus group.
4) Include known positive and negative pure cultures as a QC check.
b. Membrane filter methods:
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Standard Methods for the Examination of Water and Wastewater
1) Total coliform procedures
a) Drinking water—Pick all, up to 5 typical and 5 atypical (nonsheen) colonies from positive
samples on M-Endo medium and verify as in Section 9222B.5 f. Also verify any positives for
fecal coliforms or E. coli. If there are no positive samples, test at least one known positive source
water quarterly.
b) Other water types—Verify positives monthly by picking at least 10 sheen colonies from a
positive water sample as in Section 9222B.5 f. Adjust counts based on percent verification.
c) To determine false negatives, pick representative atypical colonies of different
morphological types and verify as in Section 9222B.5 f.
2) Fecal coliform procedure
a) Verify positives monthly by picking at least 10 blue colonies from one positive sample.
Verify in lauryl tryptose broth and EC broth as in Section 9221B.3 and Section 9221E. Adjust
counts based on percent verification.
b) To determine false negatives, pick representative atypical colonies of different
morphological types and verify as in Section 9221B.3 and Section 9221E.
3) Escherichia coli procedure
a) Drinking water—Verify at least 5% of MUG-positive and MUG- negative results. Pick
from well-isolated sheen colonies that fluoresce on nutrient agar with MUG (NA MUG), taking
care not to pick up medium, which can cause a false positive response. Also verify nonsheen
colonies that fluoresce. Verify by performing the citrate test and the indole test as described in
Section 9225D, but incubate indole test at 44.5°C. E. coli are indole-positive and yield no growth
on citrate.
b) Other water types—Verify one positive sample monthly as in ¶ a) above. Adjust counts
based on percentage of verification.
4) Fecal streptococci procedure—Pick to verify monthly at least 10 isolated esculin-positive
red colonies from m-Enterococcus agar to brain heart infusion (BHI) media. Verify as described
in Section 9230C. Adjust counts based on percentage of verification.
5) Enterococci procedures—Pick to verify monthly at least 10 well-isolated pink to red
colonies with black or reddish-brown precipitate from EIA agar. Transfer to BHI media as
described in Section 9230C. Adjust counts based on percentage of verification.
6) Include known positive and negative pure cultures as a quality control check.
9. Documentation and Recordkeeping
a. QA plan: The QA program documents management’s commitment to a QA policy and
sets forth the requirements needed to support program objectives. The program describes overall
policies, organization, objectives, and functional responsibilities for achieving the quality goals.
In addition, the program should develop a project plan that specifies the QC requirements for
each project. The plan specifies the QC activities required to achieve the data representativeness,
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
completeness, comparability, and compatibility. Also, the QA plan should include a program
implementation plan that ensures maximum coordination and integration of QC activities within
the overall program (sampling, analyses, and data handling).
b. Sampling records: A written SOP for sample handling records sample collection, transfer,
storage, analyses, and disposal. The record is most easily kept on a series of printed forms that
prompt the user to provide all the necessary information. It is especially critical that this record
be exact and complete if there is any chance that litigation may occur. Such record systems are
called chain of custody. Because laboratories do not always know whether analytical results will
be used in future litigation, some maintain chain-of-custody on all samples. Details on chain of
custody are available in Section 1060B and elsewhere.
1
c. Recordkeeping: An acceptable recordkeeping system provides needed information on
sample collection and preservation, analytical methods, raw data, calculations through reported
results, and a record of persons responsible for sampling and analyses. Choose a format agreeable
to both the laboratory and the customer (the data user). Ensure that all data sheets are signed and
dated by the analyst and the supervisor. The preferable record form is a bound and
page- numbered notebook, with entries in ink and a single line drawn through any change with the
correction entered next to it.
Keep records of microbiological analyses for at least 5 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 sample collector; identification of sample;
date of receipt of sample and analysis; person(s) responsible for performing analysis; analytical
method used; the raw data and the calculated results of analysis. Verify that each result was
entered correctly from the bench sheet and initialed by the analyst. If an information storage and
retrieval system is used, double check data on the printouts.
10. Data Handling
a. Distribution of bacterial populations: In most chemical analyses the distribution of
analytical results follows the Gaussian curve, which has symmetrical distribution of values about
the mean (see Section 1010B). Microbial distributions are not necessarily symmetrical. Bacterial
counts often are characterized as having a skewed distribution because of many low values and a
few high ones. These characteristics lead to an arithmetic mean that is considerably larger than
the median. The frequency curve of this distribution has a long right tail, such as that shown in
Figure 9020:1, and is said to display positive skewness.
Application of the most rigorous statistical techniques requires the assumption of
symmetrical distributions such as the normal curve. Therefore it usually is necessary to convert
skewed data so that a symmetrical distribution resembling the normal distribution results. An
approximately normal distribution can be obtained from positively skewed data by converting
numbers to their logarithms, as shown in Table 9020:VIII. Comparison of the frequency tables
for the original data (Table 9020:IX) and their logarithms (Table 9020:X) shows that the
logarithms approximate a symmetrical distribution.
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
b. Central tendency measures of skewed distribution: If the logarithms of numbers from a
positively skewed distribution are approximately normally distributed, the original data have a
log- normal distribution. The best estimate of central tendency of log-normal data is the geometric
mean, defined as:

and
that is, the geometric mean is equal to the antilog of the arithmetic mean of the logarithms. For
example, the following means calculated from the data in Table 9020:VIII are drastically
different.
geometric mean
and arithmetic mean
Therefore, although regulations or tradition may require or cause microbiological data to be
reported as the arithmetic mean or median, the preferred statistic for summarizing
microbiological monitoring data is the geometric mean. An exception may be in the evaluation of
data for risk assessment. The arithmetic mean may be a better measure for this purpose because it
may generate a higher central tendency value and possibly provide a greater safety factor.
8

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Standard Methods for the Examination of Water and Wastewater
c. ‘‘Less than’’ (<) values: There has always been uncertainty as to the proper way to
include ‘‘less than’’ values in calculation and evaluation of microbiological data because such
values cannot be treated statistically without modification. Proposed modifications involve
changing such numbers to zero, choosing values halfway between zero and the ‘‘less than’’
value, or assigning the ‘‘less than’’ value itself, i.e., changing <1 values to 1,
1

2
, or 0.
There are valid reasons for not including < values, whether modified or not. If the database is
fairly large with just a few < values, the influence of these uncertain values will be minimal and
of no benefit. If the database is small or has a relatively large number of < values, inclusion of
modified < values would exert an undue influence on the final results and could result in an
artificial negative or positive bias. Including < values is particularly inappropriate if the < values
are <100, <1000, or higher because the unknown true values could be anywhere from 0 to 99, 0
to 999, etc. When < values are first noted, adjust or expand test volumes. The only exception to
this caution would be regulatory testing with defined compliance limits, such as the <1100 mL
values reported for drinking water systems where the 100-mL volume is required.
11. References
1.
BORDNER, R.H., J.A. WINTER & P.V. SCARPINO,
eds. 1978. Microbiological Methods for
Monitoring the Environment, Water and Wastes. EPA-6008-78-017, Environmental
Monitoring & Support Lab., U.S. Environmental Protection Agency, Cincinnati, Ohio.
2.
AMERICAN SOCIETY FOR TESTING AND MATERIALS.
1995. Standard guide for good
laboratory practices in laboratories engaged in sampling and analysis of water.
D-3856-95, Annual Book of ASTM Standards, Vol. 11.01, American Soc. Testing &
Materials, Philadelphia, Pa.
3.
AMERICAN SOCIETY FOR TESTING AND MATERIALS.
1996. Standard practice for writing
quality control specifications for standard test methods for water analysis. D-5847-96,
Annual Book of ASTM Standards, Vol. 11.01, American Soc. Testing & Materials,
West Conshohocken, Pa.
4.
AMERICAN SOCIETY FOR TESTING AND MATERIALS.
1993. Annual Book of ASTM
Standards, Vol. 14.02, General Methods and Instrumentation. E-319-86 (reapproved
1993), Standard Practice for Evaluation of Single-Pan Mechanical Balances, and
E-898-88 (reapproved 1993), Standard Method of Testing Top-Loading,
Direct-Reading Laboratory Scales and Balances. American Soc. Testing & Materials,
Philadelphia, Pa.
5.
SCHMITZ, S., C. GARBE, B. TEBBE & C. ORFANOS.
1994. Long wave ultraviolet radiation
(UVA) and skin cancer. Hautarzt 45:517.
6.
BRENNER, K. & C.C. RANKIN.
1990. New screening test to determine the acceptability of
0.45 µm membrane filters for analysis of water. Appl. Environ. Bacteriol. 56:54.
7.
AMERICAN SOCIETY FOR TESTING AND MATERIALS.
1977. Annual Book of ASTM
Standards. Part 31, Water. American Soc. Testing & Materials, Philadelphia, Pa.
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
8.
HAAS, C.N.
1996. How to average microbial densities to characterize risk. Water Res.
30:1036.
9020 C. Interlaboratory Quality Control
1. Background
Interlaboratory QC programs are a means of establishing an agreed-upon, common
performance criteria system that will assure an acceptable level of data quality and comparability
among laboratories with similar interests andor needs.
These systems may be volunteer, such as that for the cities in the Ohio River Valley Water
Sanitation Commission (ORSANCO), or regulatory, such as the Federal Drinking Water
Laboratory Certification Program (see below). Often, the term ‘‘accreditation’’ is used
interchangeably with certification. Usually, interlaboratory quality control programs have three
elements: uniform criteria for laboratory operations, external review of the program, and
external proficiency testing.
2. Uniform Criteria
Interlaboratory quality control programs begin as a volunteer or mandatory means of
establishing uniform laboratory standards for a specific purpose. The participants may be from
one organization or a group of organizations having common interests or falling under common
regulations. Often one group or person may agree to draft the criteria. If under regulation, the
regulating authority may set the criteria for compliance-monitoring analyses.
Uniform sampling and analytical methods and quality control criteria for personnel, facilities,
equipment, instrumentation, supplies, and data handling and reporting are proposed, discussed,
reviewed, modified if necessary, and approved by the group for common use. Criteria identified
as necessary for acceptable data quality should be mandatory. A formal document is prepared and
provided to all participants.
The QAQC responsibilities of management, supervisors, and technical staff are described in
9020A. In large laboratories, a QA officer is assigned as a staff position but may be the
supervisor or other senior person in smaller laboratories.
After incorporation into laboratory operations and confirmation that the QA program has
been adapted and is in routine use, the laboratory supervisor and the QA officer conduct an
internal program review of all operations and records for acceptability, to identify possible
problems and assist in their resolution. If this is done properly, there should be little concern that
subsequent external reviews will find major problems.
3. External Program Review
Once a laboratory has a QA program in place, management informs the organization and a
qualified external QA person or team arranges an on-site visit to evaluate the QA program for
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Standard Methods for the Examination of Water and Wastewater
acceptability and to work with the laboratory to solve any problems. An acceptable rating
confirms that the laboratory’s QA program is operating properly and that the laboratory has the
capability of generating valid defensible data. Such on-site evaluations are repeated and may be
announced or unannounced.
4. External Proficiency Testing
Whenever practical, the external organization conducts formal performance evaluation
studies among all participant laboratories. Challenge samples are prepared and sent as unknowns
on a set schedule for analyses and reporting of results. The reported data are coded for
confidentiality and evaluated according to an agreed-upon scheme. The results are summarized
for all laboratories and individual laboratory reports are sent to participants. Results of such
studies indicate the quality of routine analyses of each laboratory as compared to group
performance. Also, results of the group as a whole characterize the performance that can be
expected for the analytical methods tested.
5. Example Program
In the Federal Drinking Water Laboratory Certification Program, public water supply
laboratories must be certified according to minimal criteria and procedures and quality assurance
described in the EPA manual on certification:
1
criteria are established for laboratory operations
and methodology; on- site inspections are required by the certifying state agency or its surrogate
to verify minimal standards; annually, laboratories are required to perform acceptably on
unknown samples in formal studies, as samples are available; the responsible authority follows
up on problems identified in the on-site inspection or performance evaluation and requires
corrections within a set period of time. Individual state programs may exceed the federal criteria.
On-site inspections of laboratories in the present certification program show that primary
causes for discrepancies in drinking water laboratories have been inadequate equipment,
improperly prepared media, incorrect analytical procedures, and insufficiently trained personnel.
6. References
1.
U.S. ENVIRONMENTAL PROTECTION AGENCY.
1997. Manual for the Certification of
Laboratories Analyzing Drinking Water, 4th ed. EPA-814B-92-002, U.S.
Environmental Protection Agency, Cincinnati, Ohio.
9030 LABORATORY APPARATUS*#(5)
9030 A. Introduction
This section contains specifications for microbiological laboratory equipment. For testing
and maintenance procedures related to quality control, see Section 9020.
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Standard Methods for the Examination of Water and Wastewater
9030 B. Equipment Specifications
1. Incubators
Incubators must maintain a uniform and constant temperature at all times in all areas, that is,
they must not vary more than ±0.5°C in the areas used. Obtain such accuracy by using a
water-jacketed or anhydric-type incubator with thermostatically controlled low- temperature
electric heating units properly insulated and located in or adjacent to the walls or floor of the
chamber and preferably equipped with mechanical means of circulating air.
Incubators equipped with high-temperature heating units are unsatisfactory, because such
sources of heat, when improperly placed, frequently cause localized overheating and excessive
drying of media, with consequent inhibition of bacterial growth. Incubators so heated may be
operated satisfactorily by replacing high-temperature units with suitable wiring arranged to
operate at a lower temperature and by installing mechanical air- circulation devices. It is
desirable, where ordinary room temperatures vary excessively, to keep laboratory incubators in
special rooms maintained at a few degrees below the recommended incubator temperature.
Alternatively, use special incubating rooms well insulated and equipped with properly
distributed heating units, forced air circulation, and air exchange ports, provided that they
conform to desired temperature limits. When such rooms are used, record the daily temperature
range in areas where plates or tubes are incubated. Provide incubators with open metal wire or
perforated sheet shelves so spaced as to assure temperature uniformity throughout the chamber.
Leave a 2.5-cm space between walls and stacks of dishes or baskets of tubes.
Maintain an accurate thermometer, traceable to the National Institute of Standards and
Technology (NIST), with the bulb immersed in liquid (glycerine, water, or mineral oil) on each
shelf in use within the incubator and record daily temperature readings (preferably morning and
afternoon). It is desirable, in addition, to maintain a maximum and minimum registering
thermometer within the incubator on the middle shelf to record the gross temperature range over
a 24-h period. At intervals, determine temperature variations within the incubator when filled to
maximum capacity. Install a recording thermometer whenever possible, to maintain a continuous
and permanent record of temperature.
Ordinarily, a water bath with a gabled cover to reduce water and heat loss, or a solid heat sink
incubator, is required to maintain a temperature of 44.5 ± 0.2°C. If satisfactory temperature
control is not achieved, provide water recirculation. Keep water depth in the incubator sufficient
to immerse tubes to upper level of media.
2. Hot-Air Sterilizing Ovens
Use hot-air sterilizing ovens of sufficient size to prevent internal crowding; constructed to
give uniform and adequate sterilizing temperatures of 170 ± 10°C; and equipped with suitable
thermometers. Optionally use a temperature-recording instrument.
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Standard Methods for the Examination of Water and Wastewater
3. Autoclaves
Use autoclaves of sufficient size to prevent internal crowding; constructed to provide
uniform temperatures within the chambers (up to and including the sterilizing temperature of
121°C); equipped with an accurate thermometer the bulb of which is located properly on the
exhaust line so as to register minimum temperature within the sterilizing chambers
(temperature-recording instrument is optional); equipped with pressure gauge and properly
adjusted safety valves connected directly with saturated-steam supply lines equipped with
appropriate filters to remove particulates and oil droplets or directly to a suitable special steam
generator (do not use steam from a boiler treated with amines for corrosion control); and capable
of reaching the desired temperature within 30 min. Confirm, by chemical or toxicity tests, that
the steam supply has not been treated with amines or other corrosion-control chemicals that will
impart toxicity.
Use of a vertical autoclave or pressure cooker is not recommended because of difficulty in
adjusting and maintaining sterilization temperature and the potential hazard. If a pressure cooker
is used in emergency or special circumstances, equip it with an efficient pressure gauge and a
thermometer the bulb of which is 2.5 cm above the water level.
4. Gas Sterilizers
Use a sterilizer equipped with automatic controls capable of carrying out a complete
sterilization cycle. As a sterilizing gas use ethylene oxide (C
AUTION
: Ethylene oxide is
toxic—avoid inhalation, ingestion, and contact with the skin. Also, ethylene oxide forms an
explosive mixture with air at 3-80% proportion.) diluted to 10 to 12% with an inert gas. Provide
an automatic control cycle to evacuate sterilizing chamber to at least 0.06 kPa, to hold the
vacuum for 30 min, to adjust humidity and temperature, to charge with the ethylene oxide
mixture to a pressure dependent on mixture used, to hold such pressure for at least 4 h, to vent
gas, to evacuate to 0.06 kPa, and finally, to bring to atmospheric pressure with sterile air. The
humidity, temperature, pressure, and time of sterilizing cycle depend on the gas mixture used.
Store overnight sample bottles with loosened caps that were sterilized by gas, to allow last
traces of gas mixture to dissipate. Incubate overnight media sterilized by gas, to insure
dissipation of gas.
In general, mixtures of ethylene oxide with chlorinated hydrocarbons such as freon are
harmful to plastics, although at temperatures below 55°C, gas pressure not over 35 kPa, and time
of sterilization less than 6 h, the effect is minimal. If carbon dioxide is used as a diluent of
ethylene oxide, increase exposure time and pressure, depending on temperature and humidity that
can be used.
Determine proper cycle and gas mixture for objects to be sterilized and confirm by sterility
tests.
5. Optical Counting Equipment
a. Pour and spread plates: Use Quebec-type colony counter, dark-field model preferred, or
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Standard Methods for the Examination of Water and Wastewater
one providing equivalent magnification (1.5 diameters) and satisfactory visibility.
b. Membrane filters: Use a binocular microscope with magnification of 10 to 15×. Provide
daylight fluorescent light source at angle of 60 to 80° above the colonies; use low-angle lighting
for nonpigmented colonies.
c. Mechanical tally.
6. pH Equipment
Use electrometric pH meters, accurate to at least 0.1 pH units, for determining pH values of
media.
7. Balances
Use balances providing a sensitivity of at least 0.1 g at a load of 150 g, with appropriate
weights. Use an analytical balance having a sensitivity of 1 mg under a load of 10 g for weighing
small quantities (less than 2 g) of materials. Single-pan rapid-weigh balances are most
convenient.
8. Media Preparation Utensils
Use borosilicate glass or other suitable noncorrosive equipment such as stainless steel. Use
glassware that is clean and free of residues, dried agar, or other foreign materials that may
contaminate media.
9. Pipets and Graduated Cylinders
Use pipets of any convenient size, provided that they deliver the required volume accurately
and quickly. The error of calibration for a given manufacturer’s lot must not exceed 2.5%. Use
pipets having graduations distinctly marked and with unbroken tips. Bacteriological transfer
pipets or pipets conforming to APHA standards may be used. Do not pipet by mouth; use a pipet
aid.
Use graduated cylinders meeting ASTM Standards (D-86 and D-216) and with accuracy
limits established by NIST where appropriate.
10. Pipet Containers
Use boxes of aluminum or stainless steel, end measurement 5 to 7.5 cm, cylindrical or
rectangular, and length about 40 cm. When these are not available, paper wrappings for
individual pipets may be substituted. To avoid excessive charring during sterilization, use
best-quality sulfate pulp (kraft) paper. Do not use copper or copper alloy cans or boxes as pipet
containers.
11. Refrigerator
Use a refrigerator maintaining a temperature of 1 to 4.4°C to store samples, media, reagents,
etc. Do not store volatile solvents, food, or beverages in a refrigerator with media. Frost- free
refrigerators may cause excessive media dehydration on storage longer than 1 week.
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Standard Methods for the Examination of Water and Wastewater
12. Temperature-Monitoring Devices
Use glass or metal thermometers graduated to 0.5°C to monitor most incubators and
refrigerators. Use thermometers graduated to 0.1°C for incubators operated above 40°C. Use
continuous recording devices that are equally sensitive. Verify accuracy by comparison with a
NIST-certified thermometer, or equivalent.
13. Dilution Bottles or Tubes
Use bottles or tubes of resistant glass, preferably borosilicate glass, closed with glass
stoppers or screw caps equipped with liners that do not produce toxic or bacteriostatic
compounds on sterilization. Do not use cotton plugs as closures. Mark graduation levels indelibly
on side of dilution bottle or tube. Plastic bottles of nontoxic material and acceptable size may be
substituted for glass provided that they can be sterilized properly.
14. Petri Dishes
For the plate count, use glass or plastic petri dishes about 100 × 15 mm. Use dishes the
bottoms of which are free from bubbles and scratches and flat so that the medium will be of
uniform thickness throughout the plate. For the membrane filter technique use loose-lid glass or
plastic dishes, 60 × 15 mm, or tight-lid dishes, 50 × 12 mm. Sterilize petri dishes and store in
metal cans (aluminum or stainless steel, but not copper), or wrap in paper—preferably
best-quality sulfate pulp (kraft)—before sterilizing. Presterilized petri dishes are commercially
available.
15. Membrane Filtration Equipment
Use filter funnel and membrane holder made of seamless stainless steel, glass, or
autoclavable plastic that does not leak and is not subject to corrosion. Field laboratory kits are
acceptable but standard laboratory filtration equipment and procedures are required.
16. Fermentation Tubes and Vials
Use fermentation tubes of any type, if their design permits conforming to medium and
volume requirements for concentration of nutritive ingredients as described subsequently. Where
tubes are used for a test of gas production, enclose a shell vial, inverted. Use tube and vial of
such size that the vial will be filled completely with medium, at least partly submerged in the
tube, and large enough to make gas bubbles easily visible.
17. Inoculating Equipment
Use wire loops made of 22- or 24-gauge nickel alloy*#(6) or platinum-iridium for flame
sterilization. Use loops at least 3 mm in diameter. Sterilize by dry heat or steam. Single- service
hardwood or plastic applicators also may be used. Make these 0.2 to 0.3 cm in diameter and at
least 2.5 cm longer than the fermentation tube; sterilize by dry heat and store in glass or other
nontoxic containers.
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Standard Methods for the Examination of Water and Wastewater
18. Sample Bottles
For bacteriological samples, use sterilizable bottles of glass or plastic of any suitable size
and shape. Use bottles capable of holding a sufficient volume of sample for all required tests and
an adequate air space, permitting proper washing, and maintaining samples uncontaminated until
examinations are completed. Ground-glass- stoppered bottles, preferably wide-mouthed and of
resistant glass, are recommended. Plastic bottles of suitable size, wide-mouthed, and made of
nontoxic materials such as polypropylene that can be sterilized repeatedly are satisfactory as
sample containers. Presterilized plastic bags, with or without dechlorinating agent, are available
commercially and may be used. Plastic containers eliminate the possibility of breakage during
shipment and reduce shipping weight.
Metal or plastic screw-cap closures with liners may be used on sample bottles provided that
no toxic compounds are produced on sterilization.
Before sterilization, cover tops and necks of sample bottles having glass closures with
aluminium foil or heavy kraft paper.
19. Bibliography

COLLINS, W.D. & H.B. RIFFENBURG.
1923. Contamination of water samples with material
dissolved from glass containers. Ind. Eng. Chem. 15:48.

CLARK, W.M.
1928. The Determination of Hydrogen Ion Concentration, 3rd ed. Williams &
Wilkins, Baltimore, Md.

ARCHAMBAULT, J., J. CUROT & M.H. MCCRADY.
1937. The need of uniformity of conditions for
counting plates (with suggestions for a standard colony counter). Amer. J. Pub. Health
27:809.

BARKWORTH, H. & J.O. IRWIN.
1941. The effect of the shape of the container and size of gas tube
in the presumptive coliform test. J. Hyg. 41:180.

RICHARDS, O.W. & P.C. HEIJN.
1945. An improved dark-field Quebec colony counter. J. Milk
Technol. 8:253.

COHEN, B.
1957. The measurement of pH, titratable acidity, and oxidation-reduction potentials.
In Manual of Microbiological Methods. Society of American Bacteriologists. McGraw-Hill
Book Co., New York, N.Y.

MORTON, H.E.
1957. Stainless-steel closures for replacement of cotton plugs in culture tubes.
Science. 126:1248.

MCGUIRE, O.E.
1964. Wood applicators for the confirmatory test in the bacteriological analysis of
water. Pub. Health Rep. 79:812.

BORDNER, R.H., J.A. WINTER & P.V. SCARPINO,
eds. 1978. Microbiological Methods for
Monitoring the Environment, Water and Wastes. EPA-6008-78-017, Environmental
Monitoring & Support Lab., U.S. Environmental Protection Agency, Cincinnati, Ohio.

AMERICAN PUBLIC HEALTH ASSOCIATION.
1993. Standard Methods for the Examination of
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Standard Methods for the Examination of Water and Wastewater
Dairy Products, 16th ed. American Public Health Assoc., Washington, D.C.
9040 WASHING AND STERILIZATION*#(7)
Cleanse all glassware thoroughly with a suitable detergent and hot water, rinse with hot
water to remove all traces of residual washing compound, and finally rinse with laboratory-pure
water. If mechanical glassware washers are used, equip them with influent plumbing of stainless
steel or other nontoxic material. Do not use copper piping to distribute water. Use stainless steel
or other nontoxic material for the rinse water system.
Sterilize glassware, except when in metal containers, for not less than 60 min at a
temperature of 170°C, unless it is known from recording thermometers that oven temperatures
are uniform, under which exceptional condition use 160°C. Heat glassware in metal containers to
170°C for not less than 2 h.
Sterilize sample bottles not made of plastic as above or in an autoclave at 121°C for 15 min.
For plastic bottles loosen caps before autoclaving to prevent distortion.
9050 PREPARATION OF CULTURE MEDIA*#(8)
9050 A. General Procedures
1. Storage of Culture Media
Store dehydrated media (powders) in tightly closed bottles in the dark at less than 30°C in an
atmosphere of low humidity. Do not use them if they discolor or become caked and lose the
character of a free-flowing powder. Purchase dehydrated media in small quantities that will be
used within 6 months after opening. Additionally, use stocks of dehydrated media containing
selective agents such as sodium azide, bile salts or derivatives, antibiotics, sulfur-containing
amino acids, etc., of relatively current lot number (within a year of purchase) so as to maintain
optimum selectivity. See also Section 9020.
Prepare culture media in batches that will be used in less than 1 week. However, if the media
are contained in screw-capped tubes they may be stored for up to 3 months. See Table 9020:IV
for specific details. Store media out of direct sun and avoid contamination and excessive
evaporation.
Liquid media in fermentation tubes, if stored at refrigeration or even moderately low
temperatures, may dissolve sufficient air to produce, upon incubation at 35°C, a bubble of air in
the tube. Incubate fermentation tubes that have been stored at a low temperature overnight before
use and discard tubes containing air.
Fermentation tubes may be stored at approximately 25°C; but because evaporation may
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Standard Methods for the Examination of Water and Wastewater
proceed rapidly under these conditions—resulting in marked changes in concentration of the
ingredients—do not store at this temperature for more than 1 week. Discard tubes with an
evaporation loss exceeding 1 mL.
2. Adjustment of Reaction
State reaction of culture media in terms of hydrogen ion concentration, expressed as pH.
The decrease in pH during sterilization will vary slightly with the individual sterilizer in use,
and the initial reaction required to obtain the correct final reaction will have to be determined.
The decrease in pH usually will be 0.1 to 0.2 but occasionally may be as great as 0.3 in
double-strength media. When buffering salts such as phosphates are present in the media, the
decrease in pH value will be negligible.
Make tests to control adjustment to required pH with a pH meter. Measure pH of prepared
medium as directed in Section 4500-H
+
. Titrate a known volume of medium with a solution of
NaOH to the desired pH. Calculate amount of NaOH solution that must be added to the bulk
medium to reach this reaction. After adding and mixing thoroughly, check reaction and adjust if
necessary. The required final pH is given in the directions for preparing each medium. If a
specific pH is not prescribed, adjustment is unnecessary.
The pH of reconstituted dehydrated media seldom will require adjustment if made according
to directions. Such factors as errors in weighing dehydrated medium or overheating reconstituted
medium may produce an unacceptable final pH. Measure pH, especially of rehydrated selective
media, regularly to insure quality control and media specifications.
3. Sterilization
After rehydrating a medium, dispense promptly to culture vessels and sterilize within 2 h. Do
not store nonsterile media.
Sterilize all media, except sugar broths or broths with other specifications, in an autoclave at
121°C for 15 min after the temperature has reached 121°C. When the pressure reaches zero,
remove medium from autoclave and cool quickly to avoid decomposition of sugars by prolonged
exposure to heat. To permit uniform heating and rapid cooling, pack materials loosely and in
small containers. Sterilize sugar broths at 121°C for 12 to 15 min. The maximum elapsed time
for exposure of sugar broths to any heat (from time of closing loaded autoclave to unloading) is
45 min. Preferably use a double- walled autoclave to permit preheating before loading to reduce
total needed heating time to within the 45-min limit. Presterilized media may be available
commercially.
4. Bibliography

BUNKER, G.C. & H. SCHUBER.
1922. The reaction of culture media. J. Amer. Water Works Assoc.
9:63.

RICHARDSON, G.H
., ed. 1985. Standard Methods for the Examination of Dairy Products, 15th ed.
American Public Health Assoc., Washington, D.C.
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater

BALOWS, A., W.J. HAUSLER, JR., K.L. HERRMANN, H.D. ISENBERG & H.J. SHADOMY,
eds. 1991.
Manual of Clinical Microbiology, 5th ed. American Soc. Microbiology, Washington, D.C.
9050 B. Water
1. Specifications
To prepare culture media and reagents, use only distilled or demineralized reagent-grade
water that has been tested and found free from traces of dissolved metals and bactericidal or
inhibitory compounds. Toxicity in distilled water may be derived from fluoridated water high in
silica. Other sources of toxicity are silver, lead, and various unidentified organic complexes.
Where condensate return is used as feed for a still, toxic amines or other boiler compounds may
be present in distilled water. Residual chlorine or chloramines also may be found in distilled
water prepared from chlorinated water supplies. If chlorine compounds are found in distilled
water, neutralize them by adding an equivalent amount of sodium thiosulfate or sodium sulfite.
Distilled water also should be free of contaminating nutrients. Such contamination may be
derived from flashover of organics during distillation, continued use of exhausted carbon filter
beds, deionizing columns in need of recharging, solder flux residues in new piping, dust and
chemical fumes, and storage of water in unclean bottles. Store distilled water out of direct
sunlight to prevent growth of algae and turn supplies over as rapidly as possible. Aged distilled
water may contain toxic volatile organic compounds absorbed from the atmosphere if stored for
prolonged periods in unsealed containers. Good housekeeping practices usually will eliminate
nutrient contamination.
See Section 9020.
2. Bibliography

STRAKA, R.P. & J.L. STOKES.
1957. Rapid destruction of bacteria in commonly used diluents and
its elimination. Appl. Microbiol. 5:21.

GELDREICH, E.E. & H.F. CLARK.
1965. Distilled water suitability for microbiological applications.
J. Milk Food Technol. 28:351.

MACLEOD, R.A., S.C. KUO & R. GELINAS.
1967. Metabolic injury to bacteria. II. Metabolic injury
induced by distilled water or Cu
++
in the plating diluent. J. Bacteriol. 93:961.
9050 C. Media Specifications
The need for uniformity dictates the use of dehydrated media. Never prepare media from
basic ingredients when suitable dehydrated media are available. Follow manufacturer’s directions
for rehydration and sterilization. Commercially prepared media in liquid form (sterile ampule or
other) also may be used if known to give equivalent results. See Section 9020 for quality- control
specifications.
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Standard Methods for the Examination of Water and Wastewater
The terms used for protein source in most media, for example, peptone, tryptone, tryptose,
were coined by the developers of the media and may reflect commercial products rather than
clearly defined entities. It is not intended to preclude the use of alternative materials provided
that they produce equivalent results.
N
OTE
—The term ‘‘percent solution’’ as used in these directions is to be understood to mean
‘‘grams of solute per 100 mL solution.’’
1. Dilution Water
a. Buffered water: To prepare stock phosphate buffer solution, dissolve 34.0 g potassium
dihydrogen phosphate (KH
2
PO
4
), in 500 mL reagent-grade water, adjust to pH 7.2 ± 0.5 with 1N
sodium hydroxide (NaOH), and dilute to 1 L with reagent- grade water.
Add 1.25 mL stock phosphate buffer solution and 5.0 mL magnesium chloride solution (81.1
g MgCl
2
⋅6H
2
OL reagent-grade water) to 1 L reagent-grade water. Dispense in amounts that will
provide 99 ± 2.0 mL or 9 ± 0.2 mL after autoclaving for 15 min.
b. Peptone water: Prepare a 10% solution of peptone in distilled water. Dilute a measured
volume to provide a final 0.1% solution. Final pH should be 6.8.
Dispense in amounts to provide 99 ± 2.0 mL or 9 ± 0.2 mL after autoclaving for 15 min.
Do not suspend bacteria in any dilution water for more than 30 min at room temperature
because death or multiplication may occur.
2. Culture Media
Specifications for individual media are included in subsequent sections. Details are provided
where use of a medium first is described.
9060 SAMPLES*#(9)
9060 A. Collection
1. Containers
Collect samples for microbiological examination in nonreactive borosilicate glass or plastic
bottles that have been cleansed and rinsed carefully, given a final rinse with deionized or distilled
water, and sterilized as directed in Section 9030 and Section 9040. For some applications
samples may be collected in presterilized plastic bags.
2. Dechlorination
Add a reducing agent to containers intended for the collection of water having residual
chlorine or other halogen unless they contain broth for direct planting of sample. Sodium
thiosulfate (Na
2
S
2
O
3
) is a satisfactory dechlorinating agent that neutralizes any residual halogen
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Standard Methods for the Examination of Water and Wastewater
and prevents continuation of bactericidal action during sample transit. The examination then will
indicate more accurately the true microbial content of the water at the time of sampling.
For sampling chlorinated wastewater effluents add sufficient Na
2
S
2
O
3
to a clean sterile
sample bottle to give a concentration of 100 mgL in the sample. In a 120-mL bottle 0.1 mL of a
10% solution of Na
2
S
2
O
3
will neutralize a sample containing about 15 mgL residual chlorine.
For drinking water samples, the concentration of dechlorinating agent may be reduced: 0.1 mL of
a 3% solution of Na
2
S
2
O
3
in a 120-mL bottle will neutralize up to 5 mgL residual chlorine.
Cap bottle and sterilize by either dry or moist heat, as directed (Section 9040). Presterilized
plastic bags or bottles containing Na
2
S
2
O
3
are available commercially.
Collect water samples high in metals, including copper or zinc (>1.0 mgL), and wastewater
samples high in heavy metals in sample bottles containing a chelating agent that will reduce
metal toxicity. This is particularly significant when such samples are in transit for 4 h or more.
Use 372 mgL of the disodium salt of ethylenediaminetetraacetic acid (EDTA). Adjust EDTA
solution to pH 6.5 before use. Add EDTA separately to sample bottle before bottle sterilization
(0.3 mL 15% solution in a 120-mL bottle) or combine it with the Na
2
S
2
O
3
solution before
addition.
3. Sampling Procedures
When the sample is collected, leave ample air space in the bottle (at least 2.5 cm) to facilitate
mixing by shaking, before examination. Collect samples that are representative of the water
being tested, flush or disinfect sample ports, and use aseptic techniques to avoid sample
contamination.
Keep sampling bottle closed until it is to be filled. Remove stopper and cap as a unit; do not
contaminate inner surface of stopper or cap and neck of bottle. Fill container without rinsing,
replace stopper or cap immediately, and if used, secure hood around neck of bottle.
a. Potable water: If the water sample is to be taken from a distribution-system tap without
attachments, select a tap that is supplying water from a service pipe directly connected with the
main, and is not, for example, served from a cistern or storage tank. Open tap fully and let water
run to waste for 2 or 3 min, or for a time sufficient to permit clearing the service line. Reduce
water flow to permit filling bottle without splashing. If tap cleanliness is questionable, choose
another tap. If a questionable tap is required for special sampling purposes, disinfect the faucet
(inside and outside) by applying a solution of sodium hypochlorite (100 mg NaOClL) to faucet
before sampling; let water run for additional 2 to 3 min after treatment. Do not sample from
leaking taps that allow water to flow over the outside of the tap. In sampling from a mixing
faucet remove faucet attachments such as screen or splash guard, run hot water for 2 min, then
cold water for 2 to 3 min, and collect sample as indicated above.
If the sample is to be taken from a well fitted with a hand pump, pump water to waste for
about 5 to 10 min or until water temperature has stabilized before collecting sample. If an
outdoor sampling location must be used, avoid collecting samples from frost-proof hydrants. If
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Standard Methods for the Examination of Water and Wastewater
there is no pumping machinery, collect a sample directly from the well by means of a sterilized
bottle fitted with a weight at the base; take care to avoid contaminating samples by any surface
scum. Other sterile sampling devices, such as a trip bailer, also may be used.
In drinking water evaluation, collect samples of finished water from distribution sites selected
to assure systematic coverage during each month. Carefully choose distribution system sample
locations to include dead-end sections to demonstrate bacteriological quality throughout the
network and to ensure that localized contamination does not occur through cross-connections,
breaks in the distribution lines, or reduction in positive pressure. Sample locations may be public
sites (police and fire stations, government office buildings, schools, bus and train stations,
airports, community parks), commercial establishments (restaurants, gas stations, office
buildings, industrial plants), private residences (single residences, apartment buildings, and
townhouse complexes), and special sampling stations built into the distribution network.
Preferably avoid outdoor taps, fire hydrants, water treatment units, and backflow prevention
devices. Establish sampling program in consultation with state and local health authorities.
b. Raw water supply: In collecting samples directly from a river, stream, lake, reservoir,
spring, or shallow well, obtain samples representative of the water that is the source of supply to
consumers. It is undesirable to take samples too near the bank or too far from the point of
drawoff, or at a depth above or below the point of drawoff.
c. Surface waters: Stream studies may be short-term, high-intensity efforts. Select
bacteriological sampling locations to include a baseline location upstream from the study area,
industrial and municipal waste outfalls into the main stream study area, tributaries except those
with a flow less than 10% of the main stream, intake points for municipal or industrial water
facilities, downstream samples based on stream flow time, and downstream recreational areas.
Dispersion of wastewaters into the receiving stream may necessitate preliminary cross-section
studies to determine completeness of mixing. Where a tributary stream is involved, select the
sampling point near the confluence with the main stream. Samples may be collected from a boat
or from bridges near critical study points. Choose sampling frequency to be reflective of
changing stream or water body conditions. For example, to evaluate waste discharges, sample
every 4 to 6 h and advance the time over a 7- to 10-d period.
To monitor stream and lake water quality establish sampling locations at critical sites.
Sampling frequency may be seasonal for recreational waters, daily for water supply intakes,
hourly where waste treatment control is erratic and effluents are discharged into shellfish
harvesting areas, or even continuous.
d. Bathing beaches: Sampling locations for recreational areas should reflect water quality
within the entire recreational zone. Include sites from upstream peripheral areas and locations
adjacent to drains or natural contours that would discharge stormwater collections or septic
wastes. Collect samples in the swimming area from a uniform depth of approximately 1 m.
Consider sediment sampling of the water-beach (soil) interface because of exposure of young
children at the water’s edge.
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
To obtain baseline data on marine and estuarine bathing water quality include sampling at
low, high, and ebb tides.
Relate sampling frequency directly to the peak bathing period, which generally occurs in the
afternoon. Preferably, collect daily samples during the recognized bathing season; as a minimum
include Friday, Saturday, Sunday, and holidays. When limiting sampling to days of peak
recreational use, preferably collect a sample in the morning and the afternoon. Correlate
bacteriological data with turbidity levels or rainfall over the watershed to make rapid assessment
of water quality changes.
e. Sediments and biosolids: The bacteriology of bottom sediments is important in water
supply reservoirs, in lakes, rivers, and coastal waters used for recreational purposes, and in
shellfish-growing waters. Sediments may provide a stable index of the general quality of the
overlying water, particularly where there is great variability in its bacteriological quality.
Sampling frequency in reservoirs and lakes may be related more to seasonal changes in water
temperatures and stormwater runoff. Bottom sediment changes in river and estuarine waters may
be more erratic, being influenced by stormwater runoff, increased flow velocities, and sudden
changes in the quality of effluent discharges.
Microbiological examination of biosolids from water and wastewater treatment processes is
desirable to determine the impact of their disposal into receiving waters, ocean dumping, land
application, or burial in landfill operations.
Collect and handle biosolids with less than 7% total solids using the procedures discussed for
other water samples. Biosolids with more than 7% solids and exhibiting a ‘‘plastic’’ consistency
or ‘‘semisolid’’ state typical of thickened sludges require a finite shear stress to cause them to
flow. This resistance to flow results in heterogeneous distribution of biosolids in tanks and
lagoons. Use cross-section sampling of accumulated biosolids to determine distribution of
organisms within these impoundments. Establish a length-width grid across the top of the
impoundment, and sample at intercepts. A thief sampler that samples only the solids layer may
be useful. Alternatively use weighted bottle samplers that can be opened up at a desired depth to
collect samples at specific locations.
Processed biosolids having no free liquids are best sampled when they are being transferred.
Collect grab samples across the entire width of the conveyor and combine into a composite
sample. If solids are stored in piles, classification occurs. Exteriors of uncovered piles are subject
to various environmental stresses such as precipitation, wind, fugitive dusts, and fecal
contamination from scavengers. Consequently, surface samples may not reflect the
microbiological quality of the pile. Therefore, use cross-section sampling of these piles to
determine the degree of heterogeneity within the pile. Establish a length- width grid across the top
of the pile, and sample intercepts. Sample augers and corers may prove to be ineffective for
sampling piles of variable composition. In such cases use hand shovels to remove overburden.
f. Nonpotable samples (manual sampling): Take samples from a river, stream, lake, or
reservoir by holding the bottle near its base in the hand and plunging it, neck downward, below
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
the surface. Turn bottle until neck points slightly upward and mouth is directed toward the
current. If there is no current, as in the case of a reservoir, create a current artificially by pushing
bottle forward horizontally in a direction away from the hand. When sampling from a boat,
obtain samples from upstream side of boat. If it is not possible to collect samples from these
situations in this way, attach a weight to base of bottle and lower it into the water. In any case,
take care to avoid contact with bank or stream bed; otherwise, water fouling may occur.
g. Sampling apparatus: Special apparatus that permits mechanical removal of bottle stopper
below water surface is required to collect samples from depths of a lake or reservoir. Various
types of deep sampling devices are available. The most common is the ZoBell J-Z sampler,
1
which uses a sterile 350-mL bottle and a rubber stopper through which a piece of glass tubing has
been passed. This tubing is connected to another piece of glass tubing by a rubber connecting
hose. The unit is mounted on a metal frame containing a cable and a messenger. When the
messenger is released, it strikes the glass tubing at a point that has been slightly weakened by a
file mark. The glass tube is broken by the messenger and the tension set up by the rubber
connecting hose is released and the tubing swings to the side. Water is sucked into the bottle as a
consequence of the partial vacuum created by sealing the unit at time of autoclaving. Commercial
adaptations of this sampler and others are available.
Bottom sediment sampling also requires special apparatus. The sampler described by Van
Donsel and Geldreich
2
has been found effective for a variety of bottom materials for remote
(deep water) or hand (shallow water) sampling. Construct this sampler preferably of stainless
steel and fit with a sterile plastic bag. A nylon cord closes the bag after the sampler penetrates the
sediment. A slide bar keeps the bag closed during descent and is opened, thereby opening the
bag, during sediment sampling.
For sampling wastewaters or effluents the techniques described above generally are adequate;
in addition see Section 1060.
4. Size of Sample
The volume of sample should be sufficient to carry out all tests required, preferably not less
than 100 mL.
5. Identifying Data
Accompany samples by complete and accurate identifying and descriptive data. Do not
accept for examination inadequately identified samples.
6. References
1.
ZOBELL, C.E
. 1941. Apparatus for collecting water samples from different depths for
bacteriological analysis. J. Mar. Res. 4:173.
2.
VAN DONSEL, D.J. & E.E. GELDREICH
. 1971. Relationships of Salmonellae to fecal
coliforms in bottom sediments. Water Res. 5:1079.
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
7. Bibliography

PUBLIC HEALTH LABORATORY SERVICE WATER SUB-COMMITTEE.
1953. The effect of sodium
thiosulphate on the coliform and Bacterium coli counts of non-chlorinated water samples. J.
Hyg. 51:572.

SHIPE, E.L. & A. FIELDS
. 1956. Chelation as a method for maintaining the coliform index in water
samples. Pub. Health Rep. 71:974.

HOATHER, R.C
. 1961. The bacteriological examination of water. J. Inst. Water Eng. 61:426.

COLES, H.G
. 1964. Ethylenediamine tetra-acetic acid and sodium thiosulphate as protective
agents for coliform organisms in water samples stored for one day at atmospheric
temperature. Proc. Soc. Water Treat. Exam. 13:350.

DAHLING, D.R. & B.A. WRIGHT
. 1984. Processing and transport of environmental virus samples.
Appl. Environ. Microbiol. 47:1272.

U.S. ENVIRONMENTAL PROTECTION AGENCY.
1992. Environmental Regulations and Technology
Control of Pathogens and Vector Attraction in Sewage Sludge. EPA-625R-92-013.
Washington, D.C.
9060 B. Preservation and Storage
1. Holding Time and Temperature
a. General: Start microbiological analysis of water samples as soon as possible after
collection to avoid unpredictable changes in the microbial population. For most accurate results,
ice samples during transport to the laboratory, if they cannot be processed within 1 h after
collection. If the results may be used in legal action, employ special means (rapid transport,
express mail, courier service, etc.) to deliver the samples to the laboratory within the specified
time limits and maintain chain of custody. Follow the guidelines and requirements given below
for specific water types.
b. Drinking water for compliance purposes: Preferably hold samples at <10°C during transit
to the laboratory. Analyze samples on day of receipt whenever possible and refrigerate overnight
if arrival is too late for processing on same day. Do not exceed 30 h holding time from collection
to analysis for coliform bacteria. Do not exceed 8 h holding time for heterotrophic plate counts.
c. Nonpotable water for compliance purposes: Hold source water, stream pollution,
recreational water, and wastewater samples below 10°C during a maximum transport time of 6 h.
Refrigerate these samples upon receipt in the laboratory and process within 2 h. When transport
conditions necessitate delays in delivery of samples longer than 6 h, consider using either field
laboratory facilities located at the site of collection or delayed incubation procedures.
d. Other water types for noncompliance purposes: Hold samples below 10°C during
transport and until time of analysis. Do not exceed 24 h holding time.
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
2. Bibliography

CALDWELL, E.L. & L.W. PARR
. 1933. Present status of handling water samples—Comparison of
bacteriological analyses under varying temperatures and holding conditions, with special
reference to the direct method. Amer. J. Pub. Health 23:467.

COX, K.E. & F.B. CLAIBORNE
. 1949. Effect of age and storage temperature on bacteriological
water samples. J. Amer. Water Works Assoc. 41: 948.

PUBLIC HEALTH LABORATORY SERVICE WATER SUB- COMMITTEE
. 1952. The effect of storage
on the coliform and Bacterium coli counts of water samples. Overnight storage at room and
refrigerator temperatures. J. Hyg. 50:107.

PUBLIC HEALTH LABORATORY SERVICE WATER SUB-COMMITTEE
. 1953. The effect of storage
on the coliform and Bacterium coli counts of water samples. Storage for six hours at room
and refrigerator temperatures. J. Hyg. 51:559.

MCCARTHY, J.A
. 1957. Storage of water sample for bacteriological examinations. Amer. J. Pub.
Health 47:971.

LONSANE, B.K., N.M. PARHAD & N.U. RAO
. 1967. Effect of storage temperature and time on the
coliform in water samples. Water Res. 1: 309.

LUCKING, H.E
. 1967. Death rate of coliform bacteria in stored Montana water samples. J.
Environ. Health 29:576.

MCDANIELS, A.E. & R.H. BORDNER
. 1983. Effect of holding time and temperature on coliform
numbers in drinking water. J. Amer. Water Works Assoc. 75:458.

MCDANIELS, A.E
. et al. 1985. Holding effects on coliform enumeration in drinking water
samples. Appl. Environ. Microbiol. 50:755.
9213 RECREATIONAL WATERS*#(10)
9213 A. Introduction
1. Microbiological Indicators
Recreational waters include freshwater swimming pools, whirlpools, and naturally occurring
fresh and marine waters. Many local and state health departments require microbiological
monitoring of recreational waters. Historically, the most common microbiological tests to assess
sanitary quality have been heterotrophic counts and total and fecal coliform tests. Total coliform
tests and heterotrophic counts usually are performed on treated waters and fecal coliform tests
performed on untreated waters. Although detection of coliform bacteria in water indicates that it
may be unsafe to drink, other bacteria have been isolated from recreational waters that may
suggest health risks through body contact, ingestion, or inhalation. Other bacteria suggested as
indicators of recreational water quality include Pseudomonas aeruginosa, fecal streptococci,
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
enterococci, and staphylococci. Ideally, recreational water quality indicators are microorganisms
for which densities in the water can be related quantitatively to potential health hazards resulting
from recreational use, particularly where upper body orifices are exposed to water. The ideal
indicator is the one with the best correlation between density and the health hazards associated
with a given type of pollution. The most common potential sources of infectious agents in
recreational waters include untreated or poorly treated municipal and industrial effluents or
sludge, sanitary wastes from seaside residences, fecal wastes from pleasure craft, drainage from
sanitary landfills, stormwater runoff, and excretions from animals. In addition, the source of
infectious agents may be the aquatic environment itself. The potential health hazards from each
of these sources are not equal. Exposure to untreated or inadequately treated human fecal wastes
is considered the greatest health hazard. The presence of microbiological indicators in treated
swimming pools or whirlpools indicate possible insufficient water exchange, disinfection, and
maintenance. Bather density is a major factor in determining the probability of
swimmer-associated illnesses with swimming pools, particularly when there is insufficient
disinfection and water circulation. The bathers themselves may be the source of pollution by
shedding organisms associated with the mouth, nose, and skin.
2. Infectious Diseases from Water Exposure
In general, infections or disease associated with recreational water contact fall into two
categories. The first group is gastroenteritis resulting from unintentional ingestion of water
contaminated with fecal wastes. Enteric microorganisms that have been shown to cause
gastroenteritis from recreational water contact include Giardia, Cryptosporidium, Shigella,
Salmonella, E. coli 0157:H7, Hepatitis A, Coxsackie A and B, and Norwalk virus. Leptospirosis
is not an enteric infection but also is transmitted through contact with waters contaminated with
human or animal wastes. The second group or category of infections or disease is associated
mainly with microorganisms that are indigenous to the environment, which include the
following: Pseudomonas aeruginosa, Staphylococcus sp., Legionella sp., Naegleria fowleri,
Mycobacterium sp., and Vibrio sp. The illnesses or waterborne diseases caused by these
organisms include dermatitis or folliculitis, otitis externa, Pontiac fever, granulomas, primary
amebic meningoencephalitis (PAM), and conjunctivitis. Commonly occurring illnesses or
infections associated with recreational water contact are dermatitis caused by Pseudomonas
aeruginosa and otitis externa, ‘‘swimmer’s ear,’’ frequently caused by Pseudomonas aeruginosa
and Staphylococcus aureus.
3. Microbiological Monitoring Limitations
Routine examination for pathogenic microorganisms is not recommended except for
investigations of water-related illness and special studies; in such cases, focus microbiological
analyses on the known or suspected pathogen. Methods for several of these pathogens are given
in Section 9260, Detection of Pathogenic Bacteria, Section 9510, Detection of Enteric Viruses,
and Section 9711, Pathogenic Protozoa. Because some pathogenic organisms such as Giardia,
Cryptosporidium, Mycobacterium, and Naegleria are more resistant to changes in environmental
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
conditions than indicator bacteria, routine monitoring may not always reflect the risk of infection
from these organisms. Described below are recommended methods for microbial indicators of
recreational water quality. Consider the type(s) of water examined in selecting the
microbiological method(s) or indicator(s) to be used. No single procedure is adequate to isolate
all microorganisms from contaminated water. While bacterial indicators may not adequately
reflect risk of viral, fungal, or parasitic infection from recreational waters, available technology
limits monitoring for such organisms in routine laboratory operations.
4. Bibliography

CABELLI, V.J.
1977. Indicators of recreational water quality. In Bacterial IndicatorsHealth
Hazards Associated with Waters. STP 635, American Soc. Testing & Materials, Philadelphia,
Pa.

DUFOUR, A.P.
1986. Diseases caused by water contact. In Waterborne Diseases in the United
States. CRC Press Inc., Boca Raton, Fla.

MOE, C.L.
1996. Waterborne transmission of infectious agents. In Manual of Environmental
Microbiology. American Soc. Microbiology, ASM Press, Washington, D.C.
9213 D. Natural Bathing Beaches
1. General Discussion
a. Characteristics: A natural bathing beach is any area of a stream, lake, ocean,
impoundment, or hot spring that is used for recreation. A wide variety of pathogenic
microorganisms can be transmitted to humans through use of natural fresh and marine
recreational waters contaminated by wastewater.
1,2
These include enteric pathogens such as
Salmonella, Shigella, enteroviruses, protozoa, multicellular parasites, and ‘‘opportunists’’ such
as P. aeruginosa, Klebsiella sp., Vibrio sp., and Aeromonas hydrophila, which can multiply in
recreational waters with sufficient nutrients. Other organisms of concern are those associated
with the skin, mouth, or nose of bathers, such as Staphylococcus aureus and other organisms,
e.g., nontuberculous mycobacteria and leptospira, and Naegleria sp..
3-9
b. Monitoring requirements: Historically, fecal coliforms have been recommended as the
indicator of choice for evaluating the microbiological quality of recreational waters. Many states
have adopted use of this indicator in their water quality standards. Recent studies have
demonstrated that E. coli and enterococci showed a stronger correlation with
swimming-associated gastroenteritis than do fecal coliforms, and that both indicators were
equally acceptable for monitoring fresh-water quality. For marine water, enterococci showed the
strongest relationship of density to gastroenteritis. The recommended densities of these indicator
organisms were calculated to approximate the degree of protection previously accepted for fecal
coliforms. EPA- recommended water quality criteria are based on these findings.
10
While the
primary indicators of water quality are E. coli and enterococci, the enumeration of P. aeruginosa,
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
Aeromonas hydrophila, and Klebsiella sp. in recreational waters may be useful in cases of
discharge of pulp and paper wastes and effluents from textile finishing plants into receiving
waters.
2. Samples
a. Containers: Collect samples as directed in Section 9060A. The size of the container varies
with the number and variety of tests to be performed. Adding Na
2
S
2
O
3
to the bottle is
unnecessary.
b. Sampling procedure: Collect samples 0.3 m below the water surface in the areas of
greatest bather load. Take samples over the range of environmental and climatic conditions,
especially during times when maximal pollution can be expected, i.e., periods of tidal, current,
and wind influences, stormwater runoff, wastewater treatment bypasses. See Section 9213B.2b
for methods of sample collection and Section 9222 for suggested sample volumes.
c. Sample storage: See Section 9060B.
3. Tests for Escherichia coli
a. Media:
1) mTEC agar:*#(11)
Proteose peptone
Yeast extract
Lactose
Sodium chloride, NaCl
Dipotassium phosphate, K
2
HPO
4
Monopotassium phosphate KH
2
PO
4
Sodium lauryl sulfate
Sodium desoxycholate
Bromcresol purple
Bromphenol red
Agar
Reagent-grade water
5.0
3.0
10.0
7.5
3.3
0.2
0.1
0.08
0.08
15.0
1
g
g
g
g
g
g
g
g
g
g
L
1.0 g
Sterilize by autoclaving; pH should be 7.3 ± 0.2. Pour 4 to 5 mL liquefied agar into culture
dishes (50 × 10 mm). Store in refrigerator.
2) Urea substrate:* #(12)
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
Urea
Phenol red
Reagent-grade water
2.0g
10mg
100mL
Adjust pH to between 3 and 4. Store at 2 to 8°C. Use within 1 week.
b. Procedure: Filter sample through a membrane filter (see Section 9222), place membrane
on mTEC agar, incubate at 35 ± 0.5°C for 2 h to rejuvenate injured or stressed bacteria, and then
incubate at 44.5 ± 0.2°C for 22 h. Transfer filter to a filter pad saturated with urea substrate.
After 15 min, count yellow or yellow-brown colonies, using a fluorescent lamp and a magnifying
lens. E. coli produces yellow or yellow-brown colonies. Verify a portion of these differentiated
colonies with a commercial multi-test system [see Section 9222B.5 f2)b)].
4. Tests for Enterococci
Perform tests for enterococci by the multiple-tube technique (Section 9230B) or membrane
filter technique (Section 9230C).
5. Tests for Pseudomonas aeruginosa
Perform tests for P. aeruginosa as directed in Section 9213E and Section 9213F. Use the
multiple-tube test with samples but note that the procedures may not be applicable to marine
samples.
6. Tests for SalmonellaShigella
See Section 9260.
7. References
1.
CABELLI, V.J
. 1980. Health Effects Criteria for Marine Recreational Waters.
EPA-6001-80-031, U.S. Environmental Protection Agency, Research Triangle Park,
N.C.
2.
DUFOUR, A.P
. 1984. Health Effects Criteria for Fresh Recreational Waters.
EPA-6001-84-004, U.S. Environmental Protection Agency, Research Triangle Park,
N.C.
3.
KESWICK, B.H., C.P. GERBA & S.M. GOYAL
. 1981. Occurrence of enteroviruses in
community swimming pools. Amer. J. Pub. Health 71: 1026.
4.
DUTKA, B.J. & K.K. KWAN
. 1978. Health indicator bacteria in water surface microlayers.
Can. J. Microbiol. 24:187.
5.
CABELLI, V.J., H. KENNEDY & M.A. LEVIN
. 1976. Pseudomonas aeruginosa and fresh
recreational waters. J. Water Pollut. Control Fed. 48: 367.
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
6.
SHERRY, J.P., S.R. KUCHMA & B.J. DUTKA
. 1979. The occurrence of Candida albicans in
Lake Ontario bathing beaches. Can. J. Microbiol. 25:1036.
7.
STEVENS, A.R., R.L. TYNDALL, C.C. COUTANT & E. WILLAERT
. 1977. Isolation of the
etiological agent of primary amoebic meningoencephalitis from artificially heated
waters. Appl. Environ. Microbiol. 34:701.
8.
WELLINGS, F.M., P.T. AMUSO, S.L. CHANG & A.L. LEWIS
. 1977. Isolation and
identification of pathogenic Naegleria from Florida lakes. Appl. Environ. Microbiol.
34:661.
9.
N’DIAYE, A., P. GEORGES, A. N’GO & B. FESTY
. 1985. Soil amoebas as biological markers
to estimate the quality of swimming pool waters. Appl. Environ. Microbiol. 49:1072.
10.
U.S. ENVIRONMENTAL PROTECTION AGENCY.
1986. Ambient Water Quality Criteria for
Bacteria—1986. EPA-4405-84-002, U.S. Environmental Protection Agency,
Washington, D.C.
8. Bibliography

OLIVIERI, V.P., C.W. DRUSE & K. KAWATA
. 1977. Microorganisms in Urban Stormwater.
EPA-6002-77-087, U.S. Environmental Protection Agency, Cincinnati, Ohio.

RICE, E.W., T.C. COVERT, D.K. WILD, D. BERMAN, S.A. JOHNSON & C.H. JOHNSON.
1993.
Comparative resistance of Escherichia coli and Enterococci to chlorination. J. Environ.
Health. A28:89.
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

Standard Methods for the Examination of Water and Wastewater
Endnotes
1 (Popup - Footnote)
* APPROVED BY STANDARD METHODS COMMITTEE, 1993.
2 (Popup - Footnote)
* APPROVED BY STANDARD METHODS COMMITTEE, 1997.
3 (Popup - Footnote)
* Fisher Scientific, short wave meter (Cat. No. 11-924-54) and long wave meter (Cat. No.
11-984-53), Pittsburgh, PA 15219-4785, or equivalent.
4 (Popup - Footnote)
† 3M Health Care, St. Paul, MN 55144, or equivalent.
5 (Popup - Footnote)
* APPROVED BY STANDARD METHODS COMMITTEE, 1993.
6 (Popup - Footnote)
* Chromel, nichrome, or equivalent.
7 (Popup - Footnote)
* APPROVED BY STANDARD METHODS COMMITTEE, 1993.
8 (Popup - Footnote)
* APPROVED BY STANDARD METHODS COMMITTEE, 1993.
9 (Popup - Footnote)
* APPROVED BY STANDARD METHODS COMMITTEE, 1997.
10 (Popup - Footnote)
* APPROVED BY STANDARD METHODS COMMITTEE, 1997.
11 (Popup - Footnote)
* Difco or equivalent.
12 (Popup - Footnote)
* Difco or equivalent.
© Copyright 1999 by American Public Health Association, American Water Works Association, Water Environment Federation

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