9010 Introduction*
The following sections describe procedures for examining a
water sample’s microbiological content. These methods were
developed primarily to permit rapid examination of water
samples, so analysts often assume they only apply to routine
examinations. However, they are suitable for both compliance
monitoring and research studies of ambient, drinking, waste, and
marine waters. The methods are used by many laboratories and
are the best techniques currently available, but to be effective,
their limitations must be thoroughly understood and appropriate
quality control practices must be followed. Likewise, all tech-
niques should be investigated to establish their specificity, im-
prove their procedural details, and expand their uses in analyzing
various types of water.
Keep in mind that routine bacteriological analyses do not
provide a complete water quality picture. Always consider bac-
teriological results in light of information available about sani-
tary conditions in and around the sample source. Water quality
evaluations based on test results of one sample from a given
source are inadequate. When possible, base water quality eval-
uations on the results of a series of samples collected and
analyzed over a known, protracted period.
Also, experience has shown that when un-iced samples are
mailed, noticeable changes may occur in the types or numbers of
bacteria (even if shipping time is brief). So, samples should be
refrigerated during transportation—particularly when ambient
air temperature exceeds 13°C—and minimal time should elapse
between collection and analysis.
One of the principal indicators of a water’s suitability for
domestic, industrial, or other uses is the coliform group of
bacteria, as herein defined. Experience has established the sig-
nificance of coliform density—in particular, that of Escherichia
coli or thermotolerant coliforms (previously called fecal coli-
forms) as a water quality criterion, and the group’s cultural
reactions and characteristics have been studied extensively.
Tests for detecting and enumerating indicator organisms and
pathogens are presented, including two for coliforms: the mem-
brane filter technique and the multiple-tube fermentation test.
The membrane filter technique involves direct plating to detect
and estimate coliform densities. Procedural modifications—
particularly the culture medium—have made its results compa-
rable to those of the multiple-tube fermentation procedure.
Although the membrane filter technique has limited applications,
it is equivalent when used appropriately. Either procedure can be
used to evaluate water quality or treatment process effectiveness.
Coliform density can be reported as a most probable number
(MPN) index or membrane filter count per 100 mL. It is cus-
tomary to report results of the multiple-tube fermentation pro-
cedure as MPN, which is not an actual enumeration but rather an
index of the number of coliform bacteria most likely to give the
results produced during testing. In contrast, the membrane filter
procedure and other direct plating methods enable analysts to
actually count coliform colonies.
Not all sample types require a quantitative assessment of coliform
bacteria, so qualitative, presence–absence tests are included.
* Approved by Standard Methods Committee, 2008.
Joint Task Group: 22nd Edition—Margo E. Hunt.
https://doi.org/10.2105/SMWW.2882.179
Pathogen contamination also can be indicated by fecal strep-
tococci and enterococci. Methods for their detection and enu-
meration include multiple-tube dilution and membrane filter
procedures.
Methods for differentiating coliforms are included. Generally,
differentiation is of limited value in assessing drinking water
quality because the mere presence of coliform bacteria—partic-
ularly thermotolerant coliforms or E. coli—renders the water
potentially unsatisfactory and unsafe. However, identification
may confirm the validity of coliform results or show how a
distribution system is colonized. Differentiation also yields valu-
able information about the possible source of water pollution—
especially its remoteness—because nonfecal coliforms may be
expected to survive longer than thermotolerant coliforms in
water (an unfavorable environment). Procedures for thermotol-
erant coliforms and E. coli include a 24-h, multiple-tube test
using A-1 medium; a rapid (7-h) method; and chromogenic sub-
strate coliform tests. Genetic testing also is becoming available.
Unlike other coliforms, those found in the guts and feces of
warm-blooded animals generally include organisms capable of
producing gas and/or acid from lactose in a suitable culture
medium at 44.5 ⫾ 0.2°C. Both the multiple-tube dilution tech-
nique and the membrane filter procedure have been modified to
incorporate incubation in confirmatory tests at 44.5°C so ana-
lysts can estimate the density of thermotolerant organisms.
Heterotrophic plate counts may be done via pour plate, spread
plate, membrane filter, or multiwell-enzyme substrate methods.
The methods provide an approximation of viable bacteria that
may yield useful information about water quality and indicate
the significance of coliform test results. They may be used to
judge the efficiency of various treatment processes, application
as an in-plant control test, and to check water quality in the
laboratory’s reagent-grade water system or in distribution sys-
tems (by indicating microbial colonization and sediment buildup
in slow-flow sections and dead ends).
Procedures for isolating certain pathogenic bacteria and pro-
tozoa are presented. These procedures are tedious, complicated,
and followed by laboratory personnel with related expertise.
Likewise, tentative procedures for enteric viruses are included,
but their routine use is not advocated. However, these methods
are useful in investigating waterborne diseases, characterizing
watersheds, or determining groundwater quality.
Pollution in tidal estuaries and other bodies of saline water has
raised concerns about whether existing bacteriological tech-
niques need to be modified to analyze such waters effectively.
However, the methods used to analyze fresh waters generally are
also satisfactory when testing saline waters.
Methods for examining waters in swimming pools and other
bathing places are included. The standard plate count procedures
for thermotolerant coliforms and streptococci are identical to
those used for other waters. Procedures for Staphylococcus and
Pseudomonas aeruginosa— organisms commonly associated
with the skin or upper respiratory tract—also are included, as are
procedures for aquatic fungi and actinomycetes.
Sections on rapid coliform tests and on recovering stressed
organisms are included. The quality control section has been
expanded because of increased interest in this topic.
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