MICROBIOLOGICAL ANALYSIS OF WATER

SCOPE
Estimation of microbial parameters of drinking water as per IS 10500

INSTRUMENT, MATERIALS AND GLASSWARE

a) Membrane Filtration assembly/ apparatus with Membrane Filters with pore size 0.45
µm.
b) Biosafety Cabinet Level II
c) Laminar Air Flow Chamber
d) Universal incubator
e) BOD incubator
f) Autoclave
g) Weighing balance
h) pH meter
i) Hot plate
j) Water bath
k) Hot air Oven
l) Colony Counter
m) Microscope
n) Vaccum pump
o) Petri plates
p) Inoculation Loops
q) Forceps
r) Pipettes (100-1000µl)
s) Durham tubes

AEROBIC MICROBIAL COUNT

Principle:
Plate count agar (PCA), a common growth medium, to evaluate "total" or "viable" bacterial
growth in water samples. The number of colonies grown on a PCA plate from a particular
dilution is used to determine the number of microorganisms per millilitre of sample. Using a
certain culture medium and a specific amount of the sample, poured plates are prepared. The
plates are incubated aerobically for 24 hours at 37°C.
Culture media:
 Plate count Agar (PCA)
Procedure:
• Disinfect the surface of the container holding the sample with 70% ethanol. Shake the
sample vigorously to obtain even distribution.
 • Use a sterile pipette to aseptically inoculate 1 ml of the water sample into two
separate sets of sterile petri plates. The sample number, date, and any other desired
information should be labelled on the petri plates.
• Pour 20 mL of the molten, sterilised PCA medium (cooled to 44–47°C) into each
plate. Be careful not to pour molten media right onto the inoculum. To ensure a
uniform dispersion of inoculum throughout the medium, gently swirl the media and
inoculum in both directions.
• Allow to cool and solidify. 
• Following complete solidification, invert the prepared plates and incubate for 24
hours at 37°C.
• Count all colonies, including pinpoint colonies, after the required incubation period. 
Calculation & Expression of results:
Count all colonies and determine the colony count per ml of sample used for analysis.
Count/ml = No of colonies
Volume of sample taken in ml
References:
 IS 5402:2012
 PITON, C., GRAPPIN, R. A model for statistical evaluation of precision parameters
of microbiological methods: Application to dry rehydratable film methods and IDG
reference methods for enumeration of total aerobic mesophilic flora and coliforms in
raw milk. J. AOAC, 74, 1991, pp. 92-103.
 SCOTTER, S., ALDRIDGE, M., BACK, J., WOOd, R. Validation of European
Community methods for microbiological and chemical analysis of raw and heat-
treated milk. J. Assoc. Publ. Analyst., 29, 1993, pp. 1-32.
 DAHMS, S., WEISS, H. Estimation of precision values for microbiological reference
methods: Standardized pour plate technique. Milchwiss., 53, 1988, pp. 555-559.

MULTIPLE TUBE FERMENTATION TECHNIQUE FOR COLIFORM BACTERIA

(MPN TEST)
Principle:
In the multiple-tube approach, test portions of a water sample are inoculated into a series of
tubes with a suitable selective broth culture medium (lactose-broth or MacConkey broth).
Each tube exhibiting gas generation is defined as "presumptive positive" after a specific
incubation period at a specific temperature since the gas suggests the potential presence of
coliforms. A follow-up confirmation test is crucial since other species may also create gas.
For the confirmatory test, sample taken from the positive tubes is inoculated into a more
selective culture medium (bright green bile broth). The tubes are once again examined for the
development of gas after the incubation period. The most probable number (MPN) of bacteria
present can then be estimated from the number of tubes inoculated and the number of positive
tubes obtained in the confirmatory test using statistical reference table.
Culture media:
  MacConkey broth
 Eosin Methylene Blue Agar (EMB)

Media preparation: Media should be prepared as per the manufacturer's instructions, as

follows:
 To prepare the double- or single-strength presumptive media, dissolve the specified
quantity of the dehydrated medium in distilled water (MacConkey broth). BGB
medium is only required in single-strength used for the confirmation test.
 Fill culture tubes with an inverted Durham tube to the necessary volume, then cap the
culture tubes with simple cotton plugs.
 Sterilize in an autoclave for 10 minutes at 115 °C (or as directed by the
manufacturer).
 Keep the sterilised medium at room temperature.
Procedure for inoculation of samples:
 For each water sample that will be examined, use 6 tubes of single strength and
3 tubes of double strength.
 Add 10 mL of water with a sterile pipette to 3 tubes holding 10 mL of double-strength
medium.
 Then, add 0.1 mL water to the remaining 3 tubes of 10 mL single strength media and
1 mL water to the 3 tubes that had 10 mL of single strength medium.
 For 24 hours, incubate all the tubes at 37°C. Re-incubate the tubes for up to 48 hours
if no tubes appear positive.
 Record the quantity of bacteria present in each tube and compare the number of tubes
that react positively to a reference chart included in ISO 7218.
 Incubate the prepared EMB plates from sample taken from positive tubes of
MacConkey broth.
For instance, if a water sample is tested and the results are 3-2-1 (3 positives in 10 mL, 2
positives in 1 mL, and 1 positive in 0.1 mL), this indicates that the MPN value for the
water sample is 17, meaning that it is thought to contain 17 coliforms per 100 ml.
Observation: E.coli colonies are small with metallic sheen, Whereas E.aerogenes colonies
are large without metallic sheen.
References:
 IS 5401-2 : 2012
 ISO 7218 : 2007
 ISO 4832, Microbiology of food and animal feeding stuffs — Horizontal method for
the enumeration of coliforms — Colony-count technique.
 EDWARD, P.R. and EWING, W.H., Identification of Enterobacteriaceae, 3rd edn.,
Burgess Publishing Company, Minneapolis, Minnesota, USA, 1972.
Detection of Faecal coliform bacteria:
In the lab, faecal coliform bacteria are distinguished by their capacity to ferment lactose,
producing acid and gas at 44.5 °C in less than 24 hours.  Some of the restrictions placed by
coliforms are also placed by faecal coliforms (Regrowth in distribution system, reduced
protozoal and viral resistance to water treatment, etc.)  Similar techniques (MPN, MF, and
P/A) used for total coliform detection can also detect faecal coliforms.
Coliform detection
Coliform bacteria are gram-negative, non-spore forming rods that belong to the
Enterobacteriaceae family. They can ferment lactose and produce acid and gas within 48
hours at 30-37°C.
Principle:
A membrane filter is used to filter a test portion of water before it is placed on a plate of
violet red bile lactose agar for incubation. The presence of the inhibitors, bile salts and crystal
violet, makes the medium selective. Gram-positive microbes, particularly Staphylococci, are
inhibited by crystal violet. Lactose-fermenting organisms create red colonies with a red-
purple halo around them. Pale colonies are produced by late and early lactose fermenters.
Culture media:
 Violet Red Bile Lactose Agar (VRBL)
Procedure:
 Use 70% ethanol to sanitise the surface of the bottle, holding the sample. Shake the
sample vigorously to thoroughly mix it and produce a uniform dispersion.
 Place a sterile membrane filter with a 0.45-m pore size over the appropriate volume of
sample, place the filter on a plate of VRBL agar, and incubate the plate at 36°C for
24–2 hours.
Observation:
 Coliform bacteria develop as reddish-purple colonies that are encircled by a crimson
zone of precipitated bile.
 Consider all reddish-purple colonies to be coliform.
Results:
Presence/Absence of Coliform are detected by the confirmation of presumptive colonies in
the sample examined.
References:
 IS15188:2012

BIOCHEMICAL TESTS:
Principle:
IMViC reactions are a collection of four significant reactions that are frequently used to
identify members of the Enterobacteriaceae family. It is a set of analyses used to research the
physiological traits of bacteria from the Enterobacteriaceae family, specifically Enterobacter
and Escherichia. They are made to distinguish between Gram-negative intestinal bacilli from
the Enterobacteriaceae family, encompasses numerous genera that are connected to one
another genetically and biochemically.
IMViC tests are divided into four categories. One of these tests is represented by each letter
in the acronym "IMViC."
Indole - I , methyl red - (M) , V- Voges-Proskauer, Citrate – C, For rhyming purposes only,
the letter I is used.
Culture media:
 Peptone water
 Tryptophan broth
 Glucose-phosphate broth (MR-VP broth)
 Simmon’s Citrate agar
INDOLE TEST:
Principle:
Using the enzyme tryptophanase, some bacteria can synthesise the indole molecule from the
amino acid tryptophan. Using Kovac's or Ehrlich's reagent, indole production is observed.
Red colour is produced when indole and the reagent's aldehyde react. The red colour is
concentrated as a ring at the top by an alcoholic layer.
Procedure:
 Bacterium to be tested is inoculated in peptone water, which contains amino acid
tryptophan and incubated overnight at 37oC.
 Prepare 1% tryptophan broth.
 Incubate one set of test tube with test organism and maintain one set as negative
control without inoculation. Inoculate one set of test tube with E. coli use as positive
control.
 Following incubation few drops of Kovac’s reagent are added. Shake gently. Kovac’s
reagent consists of para-dimethyl amino benzaldehyde 10 gm, isoamyl alcohol 150gm
and con. HCl 50 ml.
 Allow the tubes to stand for 2 min. so that the reagent comes to the top and then
compare test culture with the control tubes.
Ehrlich’s reagent is more sensitive in detecting indole production in anaerobes and non-
fermenters.
Observation: - Formation of a red or pink coloured ring at the top is taken as positive.
Example: Escherichia coli: Positive; Klebsiella pneumoniae: Negative
METHYL RED (MR) TEST:
Principle:
The purpose of the METHYL RED (MR) TEST is to determine an organism's capacity to
develop and maintain stable acid end products from fermentation of glucose. Some bacteria
produce significant quantities of acids from the fermentation of glucose that they overcome
the system's buffering effect. A pH indicator called Methyl Red maintains its red colour at a
pH 4.4 or lower.
Procedure:
The bacterium to be tested in inoculated into glucose phosphate broth, which contains
glucose and a phosphate buffer and incubated at 37 oC for 48 hours. Over the 48 hours the
mixed-acid producing organism must produce sufficient acid to overcome the phosphate
buffer and remain acid.
The pH of the medium is tested by the addition of 5 drops of MR reagent.
Observation: Development of red colour is taken as positive. MR negative organism produce
yellow colour.
Example: Escherichia coli: Positive; Klebsiella pneumoniae: Negative
VOGES PROSKAUER (VP) TEST:
Principle:
TEST FOR VOGES PROSKAUER (VP): Butylene glycol producers can be found using the
VP test, whereas mixed acid producers can be found using the MR test. Butylene glycol is
produced using the intermediate acetyl-methyl carbinol (acetoin). After incubation, two
reagents—40 % KOH and alpha-naphthol—are added to the test broth. If acetoin is present, it
is converted to diacetyl in the presence of air and KOH. In the presence of alpha-naphthol,
diacetyl interacts with the guanidine components of peptone to give red colour. Alpha-
naphthol serves as a catalyst and a colour enhancer.
Procedure:
 Bacterium to be tested is inoculated into glucose phosphate broth and incubated for at
least 48 hours.
 0.6 ml of alpha-naphthol is added to the test broth and shaken.
 0.2 ml of 40% KOH is added to the broth and shaken. The tube is allowed to stand for
15 minutes.
 The negative tubes must be held for one hour, since maximum colour development
occurs within one hour after addition of reagents.
Obseravtion: Appearance of red colour is taken as a positive test.
Examples: Escherichia coli: Negative; Klebsiella pneumoniae: Positive
CITRATE UTILIZATION TEST:
Principle:
It measures an organism's capability to use citrate as its sole source of carbon and energy. On
a medium containing sodium citrate and the pH indicator bromothymol blue, bacteria are
inoculated. Inorganic ammonium salts, which serve as the only source of nitrogen in the
media, are also present. Citrate is used by the enzyme citritase, which converts it to
oxaloacetate and acetate. The subsequent breakdown of oxaloacetate yields pyruvate and
CO2. Alkaline pH is the outcome of the production of Na2CO3 and NH3 from the use of
sodium citrate and ammonium salt, respectively. As a result, the medium turns blue instead of
green.
Procedure:
 Bacterial colonies are picked up using sterile loop and inoculated into slope of
Simmon’s citrate agar and incubated overnight at 37oC.
 If the organism has the ability to utilize citrate, the medium changes its color from
green to blue.
Observation- If colour of the medium change to blue it is citrate Positive E.coli is citrate
Positive. Examples: Escherichia coli: Negative; Klebsiella pneumoniae: Positive
Reference:
 Baird, R., & Bridgewater, L. (2017). Standard methods for the examination of water
and wastewater. 23rd edition. Washington, D.C.: American Public Health
Association.