BACTERIOLOGICAL ANALYSIS OF WATER
Spread Plate Method
The PNSDW 2017 or Philippine National
Standards for Drinking Water requires the
combination of physicochemical,
microbiological, and radiological tests to
determine the potability of water.
For microbiological:
Heterotrophic Plate Count
Total Coliform Count
Fecal Coliform Count
E. coli / E. coli Coun
HETEROTROPHIC PLATE COUNT
The heterotrophic plate count (HPC),
formerly known as the
standard plate count, is a procedure for
estimating the number of
live, culturable heterotrophic bacteria
in water and for measuring
changes in swimming pools or during
water treatment and
distribution.
Colonies may arise from pairs, chains,
clusters, or single cells—all
of which are included in the term
colony forming units (CFU). The
final count also depends on interaction
among developing
colonies.
Methods:
Pour Plate Method
Membrane Filtration Method
CULTURE MEDIA AND REAGENTS
Plate Count Agar - This medium can
be used to examine heterotrophic
bacteria in a wide variety of matrices. It
can be used for both pour- and spread-
plate methods. This high-nutrient agar,
widely used in the past, may give lower
counts than R2A or NWRI agar.
M-HPC Agar - This can be used to
count heterotrophic bacteria in
waters with low levels of heterotrophs,
such as treated potable
water. This high-nutrient medium is
used only with the membranefilter
method
R2A Agar - Use for pour-plate, spread-
plate, and membrane-filter
methods. This low-nutrient agar was
developed for use with potable
treated water to monitor trends in
potable water production; this
agar and a longer incubation period can
improve the recovery of
stressed and chlorine-tolerant bacteria.
This medium may yield
higher counts than high-nutrient
formulations.
NWRI Agar - is an alternate low-
nutrient medium to R2A agar for
detection of HPC. This agar typically is
not available in dehydrated
form; it must be prepared from
individual ingredients, making its
use less desirable.
Dilution blanks:
99 mL sterile Butterfield’s
Phosphate Buffer
GLASSWARE AND
OTHER MATERIALS
Petri dish (Pyrex glass or disposable
plastic)
Dilution bottles
 Micropipette (1 and 0.1 mL)
Micropipette 10 mL
Sterile pipette tips
Biosafety Cabinet
Incubator
Colony counter
SAMPLE
PREPARATION
Thoroughly mix all samples or
dilutions by shaking vigorously
for 7 s by hand (approximately
25 times with a 1-ft movement)
or via a mechanical shaker for
15 s at low speed. Analytical
results depend on adequate
sample mixing; if the sample is
not adequately shaken before
aliquots are removed, the
actual bacterial density could
be underestimated.
COLONY COUNTING
LABORATORY ANALYSIS
Label or Mark each plate with sample
number, dilution, date, and any other
necessary information before
examination.
After homogenizing the water sample
by hand shaking, aseptically pipette
duplicate 1 mL and 0.1 mL portions of
undiluted water samples in two sterile
petri dish.
Prepare serial dilution of water
sample by transferring 1 mL of
undiluted sample to 99 mL of
Butterfield’s Phosphate Buffer.
Homogenize by vigorous hand shaking
for 7 seconds.
Aseptically pipette duplicate 1 mL and
0.1 mL portions of diluted water
samples in two sterile petri dish.
Pour 20 mL of sterile liquefied
tempered agar and incubate at
recommended incubation requirement.
Incubation - The media described
should be incubated as follows:
Plate count agar (PCA): Incubate at
35°C for 48 ± 3 h.
m-HPC agar: Incubate at 35°C for 48 ±
3 h.
R2A agar: Incubate at 20–28°C for 5 to
7 d.
NWRI agar: Incubate at 20°C for 7 d.
Count all colonies on selected plates
promptly after incubation. If counting
must be delayed temporarily,
store plates refrigerated for <24 h, but
avoid this as routine practice. Record
results of sterility controls
on the report for each lot of samples.
Count colonies manually using a dark-
field colony counter, such as a Quebec
colony counter or equivalent. If such
equipment is unavailable, then
other counters can be used on non-
compliance samples so long as they
provide equivalent magnification.
When preparing plates, pipet sample
volumes that will yield between 30 and
300 colonies/plate. The aim is to have
at least one dilution yield colony counts
within these limits, except as provided
below.
Otherwise, use only plates containing
30 to 300 colonies when determining
plate count.
Compute bacterial count per milliliter as follows: When bacterial counts on crowded plates are
100 colonies/cm2, report results as 6500
divided by the smallest sample volume plated
for glass plates or 5700 divided by the
smallest sample volume plated for plastic
If no plate contains 30 to 300
plates. Report as estimated CFU/mL.
colonies, and one or more plates have >
65 𝑋 100
300 colonies, use the plate(s)
1 𝑋 0.1
whose count is nearest 300 colonies.
= > 65 000 𝑒𝑠𝑡. 𝐶𝐹𝑈/𝑚�
Compute the
TOTAL COLIFORM
count as above and report as estimated
AND FECAL
CFU/mL.
COLIFORM COUNT
If plates from all dilutions of a
sample have no colonies, report
Total coliform bacteria are commonly
the count as < 1 divided by the
found in the environment (e.g., soil or
corresponding largest sample
vegetation) and are generally
volume used. For example, if no
harmless. If only total coliform bacteria
colonies develop from the 0.01
are detected in drinking water, the
mL sample volume, report the
source is probably environmental.
count as < 100 CFU/mL
Traditionally called fecal coliforms,
thermotolerant coliforms (those that
ferment lactose to produce gas at
44.5°C) have been documented in
organically rich waters or tropical
climates in the absence of recent fecal
contamination. So, when looking for
evidence of fecal contamination,
testing for E. coli—a more specific
indicator—is recommended.
PRESUMPTIVE Durham tube indicate a positive tube for
ANALYSIS coliform.
Method: Multiple Tube Count the number of coliform positive tubes
Fermentation Technique and use the
Culture medium: Lauryl Tryptose 5 tube MPN table to get the equivalent MPN
Broth value.
Glassware:
Screw-capped test tube
maximum capacity of 30 – 40
mL
Durham tubes
Volume of sample on each test
tube: 20 mL
Incubation: 35°C for 48 hours

STANDARD COLIFORM FERMENTATION

TECHNIQUE
Brilliant Green Bile Broth 2% is one of the
most widely
used medium for the detection of coliform
bacteria in
water, wastewater, foods, and milk and dairy
products.
This medium is formulated as per APHA for the
presumptive identification and confirmation of
coliform
bacteria This medium is also recommended by THERMOTOLERANT (FECAL) COLIFORM TEST
the ISO The thermotolerant coliform test using EC
Committee for enumeration of coliforms by medium is applicable to investigations of
most probable drinking
number technique water, stream pollution, unfiltered raw water
Peptone serves as a source of essential sources, wastewater treatment systems,
nutrients. bathing
Lactose is the fermentable carbohydrate. Bile waters, seawaters, and general water-quality
inhibits monitoring.
gram-positive bacteria whereas the gram- Tryptone provides nitrogenous and
negative carbonaceous
bacteria are inhibited by brilliant green. compounds, long chain amino acids and other
Production of gas essential growth nutrients. Lactose is the
from lactose fermentation is detected by fermentable sugar. Bile salts mixture inhibit
incorporating grampositive bacteria especially bacilli and fecal
inverted Durham's tube. Streptococci. Phosphates control the pH during
Transfer loopful of sample of positive tubes fermentation of lactose. Gas production in a
from fermentation tube within 24 hour or less is a
presumptive test. presumptive evidence of the presence of
Incubation: 35°C for 48 hours coliform
Formation of effervescence or gas/bubble bacteria. This medium can be used at 37°C for
inside the the
detection of coliform organisms or at 44.5°C for
the
isolation of Escherichia coli from water and
shellfish)
or 45.5°C for foods.
Transfer loopful of sample of positive tubes
from
presumptive test.
Incubation: 44.5 ± 0.2°C for 24 hours
Formation of effervescence or gas/bubble
inside the
Durham tube indicate a positive tube for fecal
coliform.
Count the number of fecal coliform positive
tubes
and use the 5 tube MPN table to determine the
equivalent MPN value.