Water Resources--Office of Water Quality
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The membrane filtration (MF) and most probable number
(MPN) methods are used for the presumptive identification,
confirmation, and enumeration of indicator bacteria. For general use,
the MF method is preferable to the MPN method. The MPN
method is preferred if toxic substances are present in the sample or
if, after filtration, a residue heavy enough to block the
micropores of the membrane filter is visible. The MPN method is
described in Standard Methods for the Examination of Water and
Wastewater, 18th edition (American Public Health Association and
others, 1992, p. 9-45 to 9-53) and in Britton and Greeson (1989).
Procedures for analyzing water samples by use of MF methods are
IDENTIFICATION AND ENUMERATION METHODS
Indicator bacteria for presumptive identification and
enumeration are cultured on selective media after filtration of several
different sample volumes onto gridded membrane filters.
Detailed confirmation, identification, and enumeration of these
bacteria require additional culturing and biochemical testing, the
details of which are beyond the scope of this manual. However,
additional confirmation procedures are needed under certain
circumstances, such as use of the data in support of environmental
regulation and enforcement.
The fecal indicator bacteria are operationally defined by
the method employed for identification and enumeration, as follows:
||The total coliform bacteria are defined as the organisms
that produce red colonies with a golden-green metallic
sheen within 24 ± 2 hours when incubated at 35.0 ± 0.5°C on
||The fecal coliform bacteria are defined as the organisms
that produce blue colonies in whole or part within 24 ± 2
hours when incubated at 44.5 ± 0.2°C on m-FC medium.|
||E. coli are defined as the organisms that produce yellow
or yellow-brown colonies that remain so when placed on a
filter pad saturated with urea substrate broth for 15
minutes after rescusitation at 35.0 ± 0.5°C for 2 hours and
incubation for 22 to 24 hours at 44.5 ± 0.2°C on m-TEC medium.|
||E. coli are defined as the organisms that produce a blue
fluorescent margin around a darker colony center within 4
hours when incubated at 35 ± 0.5°C on NA-MUG medium after
primary culturing as total coliform bacteria on m-Endo medium.|
||The fecal streptococci are defined as the organisms that
produce red or pink colonies within 48 ± 2 hours when
incubated at 35.0 ± 0.5°C on KF medium.|
||Enterococci are defined as the organisms that produce
pink to red colonies with a black or reddish-brown precipitate
after primary culture for 48 to 50 hours at 41.0 ± 0.5°C on
m-E medium followed by incubation for 20 minutes at 41.0°C
on EIA medium.|
MF analysis requires the use of several types of media and
reagents, the types being dependent on the indicator. The
necessary media and reagents include sterile buffered water, agar-
or broth-based selective and differential growth media, and
media and reagents for additional biochemical identification.
PREPARATION OF MEDIA AND REAGENTS
Sterile buffered water (buffer) is used to dilute samples and
to rinse the membrane-filtration apparatus and utensils.
Purchase sterile buffered water from the Quality of Water Service
Unit (QWSU). It is provided in 250-mL bottles and in 99-mL
dilution bottles. There are two types: phosphate buffer to be used for
total and fecal coliform, and fecal streptococci tests; and
saline buffer to be used for E. coli and enterococci tests. Buffer
exceeding the expiration date should not be used. When sterile
buffered water is not obtained from the QWSU, it can be
prepared ahead of time and sterilized by autoclaving. Preparation
instructions for sterile buffered water are described in Britton
and Greeson (1989, p. 18) and Standard Methods for the Examination
of Water and Wastewater (American Public Health Association
and others, 1992, p. 9-17).
Culture media for enumeration of fecal indicator
bacteria should be purchased in kits from the
QWSU. The QWSU provides instructions for media preparation with each kit. Otherwise, dehydrated media can be purchased from scientific
suppliers. Guidelines for storage of media and reagents are as follows:
||Store media kit (supplied by QWSU) and dehydrated,
commercially prepared media in a desiccator. Store other
reagents in a dust-free laboratory cabinet (not in a field vehicle).|
||Label all media with the date received, date opened,
and preparer's initials. Discard media and reagents with an
expired shelf life.|
||Refrigerate reagents when necessary. Use buffered
dilution water immediately after opening; discard any remainder.
Storing an opened bottle is not recommended.|
||Mark all plates to identify the media type, the
preparation date, and the preparer.|
||Store prepared petri dishes upside down in a plastic bag
before use and refrigerate.|
The preparation of selective and differential culture media
for indicator bacteria is an important part of analysis. Adhering
to proper preparation, storage, and holding-time requirements
will help ensure the quality of the analysis. Instructions for the
preparation of 100 mL of primary culture media for five MF tests
and additional confirmation media or broth for three MF
confirmation tests are described in section 7.1.5, entitled
"Instructions for Media Preparation."
PREPARATION, HOLDING TIMES, AND SPECIFICATIONS FOR CULTURE MEDIA
Quality control. Supplies of dehydrated media purchased
from the QWSU or through catalogs have been quality-control
tested. Media prepared fresh by the analyst must also be
quality-control tested. If sterile buffered water is prepared in the laboratory,
quality-control procedures must be used to ensure it will provide
a suitable medium for transfer of bacteria from samples to
filters. Sterile buffered water should be tested for sterility by use
of blanks of 100 mL, processed along with each set of
samples. Quality-control procedures applicable to microbiological
testing can be found in the 18th edition of "Standard Methods for
the Examination of Water and Wastewater" (American Public
Health Association and others, 1992, p. 9-7 to 9-13).
After collecting the sample and selecting the appropriate
sample volumes, label the petri dishes with the station number (or
other identifiers), the volume of sample filtered, date, and time.
Select those sample volumes that are anticipated to yield one or
two plates in the ideal colony count range. General information
on the concentrations of fecal indicator bacteria in surface
water and contaminated surface water is given in table 7.1-1.
MEMBRANE FILTRATION PROCEDURE
A suitable work area inside the field vehicle and out of
direct sunlight and wind is best.
||Before and after processing the samples, clean
countertops in field vehicles with an antibacterial cleaning solution;
for example, a 7-percent phenolic solution, 50 to 70 percent
isopropyl or ethyl alcohol; 5 percent bleach; or a 7-percent
ammonia solution. |
||Preheat incubators for at least 2 hours before beginning
analysis, according to specifications for each test (table
7.1-5). Portable heater-block incubators must not be left on in
closed, unventilated vehicles when the outside temperature is
less than 15°C or greater than 37°C.|
Technical Note: Review past analyses for the
site to help determine the number of sample volumes to be filtered. Where
past analyses of samples from a site have shown a small variation in the
number of fecal indicator bacteria, the filtration of as few as three or
four different sample volumes may suffice. However, where past analyses
have shown the variation to be large or where the variation is not known,
the filtration of five or more different
sample volumes is recommended.
The steps required for membrane filtration are depicted in
figure 7.1-2 (pages 28 and
29) and listed below. Quality-control
samples must be collected as part of the filtration procedure (see
Technical Note, step 16).
Steps to follow when filtering samples and making
colony counts are listed below (and summarized in fig.
Quality control. In addition to blanks, collect and
analyze samples in duplicate at a minimum frequency of 5 percent (1
in every 20 samples). Periodically purchase and analyze a pure
culture containing Escherichia coli or Enterococcus
faecalis (formerly Streptococcus faecalis to ensure that the test procedure is
- Select sample volumes (table 7.1-6) to result in at least
one filter having colonies in the ideal counting range. The ideal
range and number of sample volumes to filter depend on the test
and the expected bacterial concentrations. Record on the petri
dish and on the record sheet the site name, date, time of
sample collection, and sample volume. Record the time of sample
processing on the record sheet. Also label equipment and
procedure blanks and other quality-control samples.
- Assemble filtration equipment by inserting the base of the
filter-holder assembly into a flask. Vacuum is supplied by use of
a hand-held pump, vacuum, or battery-operated peristaltic
pump. If flame sterilization was used, rinse the inside of the
filtration apparatus with sterile buffered water to remove any residue
- Sterilize stainless steel forceps by immersing tips in a small
bottle or flask containing 70 or 90 percent ethanol; then pass
forceps through the open flame of an alcohol burner. Allow alcohol
to burn out and allow the forceps to cool for several seconds
to prevent heat damage to the membrane filter. Resterilize
forceps before each use. Return cooled forceps to alcohol container
between transfers. Do not set forceps on the
- Remove the sterilized funnel from the filtration apparatus.
Always hold the funnel in one hand while placing or removing
the membrane filter. (Placing the funnel on anything but the
filtration apparatus might result in contamination of the
- Using sterile forceps, place a sterile, gridded
membrane filter (47-mm diameter) on top of the
filter base, grid-side up. Be sure to use the correct
pore-size membrane filter for the test procedure (table 7.1-7).
- Carefully replace and secure the filter funnel on filter base. Avoid
tearing or creasing the membrane filter.
Rinse funnel with 100 mL of sterile buffered water before
filtering sample volumes to obtain a filtration assembly
equipment blank (filter blank).
Filter sample in order of smallest to largest sample volume.
- If the sample volume is less than 1.0 mL, prepare dilutions
with sterile buffered water in a 99-mL dilution bottle and
transfer appropriate volume of dilution to the membrane filter (fig.
7.1-3 and table 7.1-8).
- When preparing dilutions, use a sterile pipet to measure
each sample volume.
- After each sample-volume transfer, close and shake the
dilution bottle vigorously at least 25 times.
- Filter diluted samples within 20 minutes after
preparation. Keep dilution bottles out of sunlight and do not transfer
dilute sample volumes with pipets used to transfer
- Shake the sample vigorously at least 25 times before each
sample volume is withdrawn in order to break up particles and
ensure an even distribution of indicator bacteria in the sample
container. Proceeding from smallest to largest sample volume,
deliver the sample volume to the membrane filter by use of
a pipettor or pipet bulb with a valve for volume control.
- Allow the pipet to drain, and touch the tip to the inside
of the funnel to remove remaining sample. Pipets of the TD
(to deliver) type will have a small amount of liquid left in the
tip after dispensing the liquid.
- If the volume of sample to be filtered is 10 mL or
more--transfer the sample with a sterile pipet or graduated
cylinder directly into the funnel.
- If the volume of sample to be filtered is between 1.0
and 10.0 mL--pour about 20 mL of sterile buffered water into
the funnel before pipetting the sample to facilitate distribution
of bacteria on the membrane filter. Refer to table 7.1-6 for
appropriate sample volumes for each test.
- Apply vacuum with a hand, peristaltic, or vacuum pump.
To avoid damage to bacteria, do not exceed a pressure of about
5 lb/in2 (25 cm of mercury).
- Rinse inside of funnel twice with 20 to 30 mL of sterile
buffered water while applying vacuum. If a graduated cylinder was
used, rinse the cylinder with sterile buffered water and deliver
rinse water to the filtration apparatus.
- Remove the funnel and hold it in one hand. Do not set the
funnel on the countertop. Remove the membrane filter with
sterile forceps. Release the vacuum. Releasing the vacuum after
removing the filter prevents backflow of sample water onto the
filter. Unnecessarily wet filters promote confluent growth of
colonies and poor results. Replace funnel on filter base.
- Open petri dish and place membrane filter grid side up on
medium by use of a rolling action, starting at one edge. Avoid
trapping air bubbles under the membrane filter. If air is trapped,
use sterile forceps to remove the membrane filter and roll it
onto the medium again. Do not expose prepared plates to
- Close petri dish by pressing top firmly onto bottom. Invert
the petri dish. To avoid growth of interfering microorganisms,
incubate within 20 minutes.
- Continue to filter the other sample volumes in order, from
smallest to largest volume. Record on the field forms the
volumes filtered and time of processing.
- After filtrations are complete, place a sterile,
gridded-membrane filter on the funnel base and rinse the funnel with 100 mL
of sterile buffered water to obtain a procedure blank.
- After the sample volumes and blanks have been filtered,
place the inverted petri dishes in a preheated aluminum
heater-block or water-bath incubator. Incubate at the prescribed times
and temperatures (table 7.1-5). Wash, then flame sterilize or
autoclave filtration apparatus. Wash countertop between
each sample and wash hands with bacteriocidal soap.
- After incubation, remove the petri dishes from the
incubator. Count and record on the field forms, for each sample
volume filtered, the number of typical colonies (table 7.1-9).
Recount until results agree within 5 percent. Recounting is done by
turning the plate 90 degrees to obtain a slightly different
angle. Count by use of a preset plan (a side-to-side pattern along
grid lines is suggested) (fig. 7.1-4). Make the counts with the
aid of 5 to 15 magnifications and a fluorescent illuminator
placed as directly above the filter as possible.
- For total coliform colonies, enhance sheen production by
removing filters from media and placing them on
absorbent pads to dry for at least 1 minute before counting.
- If the optional NA-MUG test is done for
E. coli, transfer the total coliform filter onto NA-MUG plates and incubate for
4 hours at 35°C. Afterward, count under a long-wave
ultraviolet light in a completely darkened room (U.S.
Environmental Protection Agency, 1991b).
- For E. coli and enterococci, additional biochemical tests
are required by use of confirmation media. For E.
coli, transfer the filter to a filter pad saturated with urea-phenol reagent;
count only yellow colonies after 15 to 20 minutes at room
temperature (U.S. Environmental Protection Agency, 1985).
- For enterococci, transfer the filter to EIA media after
incubation for 20 minutes at 41°C; count colonies from the
underside of the plate placed over a fluorescent illuminator.
- Check quality-control blanks for colony growth, and report
results on the field forms.
- The presence of colonies on blanks indicates that results
of the bacterial analyses bracketed by positive blanks are
suspect and should not be reported.
- It is not valid to subtract colony counts on blanks from
results calculated for samples.
TECHNICAL NOTE: It is necessary to collect
equipment, filter, and procedure blanks. The equipment and
filter blanks measure the effectiveness of sterilization. One
or more colonies on this type of blank indicates
inadequate sterilization of either the equipment or the buffered
water. The procedure blank measures the effectiveness of
the analyst's rinsing technique. One or more colonies on
the procedure blank indicates either inadequate rinsing or
contamination of equipment or buffered water during
- Calculate the number of colonies per 100 mL of sample as
shown in section 7.1.4, "Calculation and Reporting of Fecal
- Put all plates to be discarded in an autoclavable bag. Freeze
or chill the plates to be discarded until they can be autoclaved
in the laboratory. Autoclave all cultures at 121°C for a minimum
of 30 minutes before discarding.
Section 7.1 Contents
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Last Modified: 1Feb99 imc