GUIDE MANUAL: WATER AND WASTEWATER ANALYSIS

3. MICROBIOLOGICAL ANALYSIS

3.1 PREAMBLE

Water borne pathogens can enter the human body through intact or compromised skin, inhalation,
ingestion, aspiration, or by direct contact with mucosa of the eye, ear, nose, mouth and genitals, and
cause disease. Besides failure of potable water systems, which usually contribute to the greatest
number of outbreaks of waterborne diseases, common outdoor recreational activities such as
swimming, boating, bathing, camping and hiking, all place humans at risk of waterborne diseases
from ingestion or direct contact with contaminated water. A risk analysis approach is required for
overcoming the problems with waterborne diseases and water quality monitoring. While this could be
achieved relatively easily with respect to chemical contaminants, and standards could be set up for
chemical contaminants, setting standards for microbial contaminants has proved more difficult,
because, (a) methods for detection of many pathogens are not yet available; (b) days to weeks are
some times required to obtain results; and (c) expensive and time consuming methods are often
required to detect and quantify the presence of specific pathogens.

To overcome these difficulties, the concept of testing water samples for indicator organisms was
introduced as far back as the turn of the 19th century. The concept was developed for assessing faecal
contamination of water and the concept relies on the fact that certain non-pathogenic bacteria present
in water can be easily isolated and quantified by simple bacteriological methods; detection of these
bacteria would essentially mean that faecal contamination has occurred and also suggest that enteric
pathogens also may be present. The essential criteria for indicator organisms are given in the box
below:

Criteria for indicator micro-organisms in water

• Should be useful for all types of water

• Should be present whenever enteric pathogens are present
• Should have a reasonable longer survival time than the hardest enteric pathogen
• Should not grow in water
• Testing method for the organism should be easy to perform
• Density of the indicator should have some direct relationship to the degree of faecal pollution
• Should be a member of the intestinal micro-flora of humans and warm-blooded animals

It should, however, be appreciated that the choice of monitoring indicator(s) presupposes an

understanding of the parameters to be measured and the relationship of the indicator(s) to the
pathogen(s). No single indicator provides assurance that water is pathogen-free. Pathogens such as
Psedomonas, Aeromonas, Plesidomnas, Yersinia, Vibrio, Legionella and Mycobacterium may not
correlate with the most commonly used coliform indicators, and also, traditional bacteria indicators
may not also correlate with viruses and parasites in pristine waters or ground waters. A brief
consideration of the common indicator organisms that are currently employed in
qualitative/quantitative tests for water would be appropriate here.
Indicator microorganisms of faecal pollution:

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GUIDE MANUAL: WATER AND WASTEWATER ANALYSIS

3.1.1. Coliform bacteria:

Coliform bacteria include all aerobic and facultative anaerobic gram negative, non-sore forming, rod-
shaped bacteria that produce gas upon fermentation in prescribed culture media within 48 hr at 35°C.

In recent years, new criteria have been added to traditional definition of coliform bacteria and E. coli,
using the presence of characteristic enzymatic activities. Definition of coliform or faecal coliform
basically relies on the activity of a single enzyme β- galactosidase. The new enzymatic definition of
TOTAL COLIFORM bacteria is based on the presence of β- galactosidase; and that of E. coli is
based on the enzymatic action of β- glucuronidase. This aspect is well utilized in developing very
sensitive and specific presence-absence (P-A) tests for detection of total coliforms and E coli.

Deficiencies with the use of coliform bacteria as indicator of water quality include:
• Regrowth in aquatic environments
• Regrowth in distribution systems, including biofilm colonization
• Suppression by high background bacterial growth
• Not indicative of health threat
• No relationship with enteric protozoan and viral concentration.

Coliform bacteria had been used historically to assess the microbial quality of drinking water.
However, by the 1980s it had become quite clear that coliform bacteria did not indicate the presence
of pathogenic waterborne Giardia or enteric viruses. Numerous outbreaks had occurred. In which
coliform standards were met, because of greater resistance of viruses and Giardia to chlorination. A
new approach was needed to ensure the microbial safety of drinking water.
Most Probable Number (MPN), the Membrane filter (MF) and presence-absence (P/A) tests are used
to detect the total coliforms.

3.1.2. Faecal coliform bacteria:

Faecal coliform bacteria are differentiated in the laboratory by their ability to ferment lactose, with
production of acid and gas at 44.5ºC within 24 h.
Faecal coliforms pose some of the same limitations as those posed by coliforms (Regrowth in
distribution system, less resistance to water treatment than viruses and protozoa, etc.)
Faecal coliforms are also detected by similar methods (MPN, MF and P/A) used for total coliforms.

3.1.3. Faecal streptococci:

Faecal streptococci include the gram-positive, Lancefield Group-D streptococci, belonging to the
genera Enterococcus and Streptococcus.
The genus Enterococcus is differentiated from other streptococci by ability to grow in 6.5% NaCl, pH
9.6 and 45°C (includes spp. E. avivum, E. faecium, E. faecalis and E. gallinarium). E. faecium and E.
faecalis are more specific to human gut.

In the genus Streptococcus, only S. bovis and S. equines are considered to be true faecal streptococci
have certain advantages over the coliforms, as indicators:

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GUIDE MANUAL: WATER AND WASTEWATER ANALYSIS

• They rarely multiply in water

• More resistant to environmental stress and chlorinated that coliforms
• Persist longer in the Environment
• Suggested as useful indicators for recreational waters

Both MPN and MF methods may be used for isolation and enumeration of faecal streptococci.

3.1.4. Clostridium perfringens:

They are sulphite-reducing anaerobic spore-formers, gram-positive, rod-shaped and exclusively of

faecal origin. The hardly spores (resistant to 75ºC for 15 min.) limit its usefulness as an indicator; it
could be used as an indicator of past pollution and an indicator of removal of protozoan parasites and
viruses during treatment of drinking water and wastewater.

3.1.5. Pseudomonas aeruginosa:

They are gram-negative, non-sporulating, rod-shaped bacteria, producing characteristics pyocyanin

(blue-green) and the green fluorescent pigment fluorescein. One of the most common opportunistic
pathogens. Numerous cases of folliculitis, dermatitis, ear and urinary tract infections are due to P.
aeruginosa, associated with swimming in contaminated waters. Because of this association and also
its consistent presence in high numbers in sewage, this organism has been suggested as an indicator
for water quality in swimming pools, hot tubs and other recreational waters. But its value as an
indicator is very little, because it is ubiquitous in nature and can multiply under natural conditions
(can grow even in distilled water).

3.1.6. Hydrogen sulphide (H 2 S)-producing bacteria:

H 2 S-producing organisms (bacteria) are invariably present in faeces. There is a very strong
correlation between presence of H 2 S-producing bacteria and faecal pollution of water. By detecting
the presence of H 2 S-producing bacteria in water samples, faecal pollution of such waters can be
deduced.

3.1.7. Coliphages and other bacteriophages:

Their use as indicators of faecal pollution is based on the assumption that their presence in water
samples denotes the presence of bacteria capable of supporting replication of the phages. Two groups
of phages have been studied for their suitability as indicator organisms:

• Somatic Coliphages that infect E. coli host strains through cell wall receptors.
• F-specific RNA coliphage-which infect strains of E. coli and related bacteria through the F +
or sex pili

Advantages of using coliphage is that they can be detected by simple and inexpensive techniques, in
8-18 hr. Both plating (agar-overlay method) and MPN method can be used to detect coliphages, in
volumes ranging from 1-100 mL of water.

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Another bacteriophages of interest is the bacteriophages that infect Bacteriodes fragilis. These
bacteria (Bacteriodes spp.) are strict anaerobes and are a major component of the human faecal
microflora. The bacteriophages that infect Bacteriodes fragilis appear to be exclusively of human
origin and can serve as an ideal indicator organism. The main disadvantage, however, lies with the
fact that since the host organism (B. fragilis) is an anaerobic organism, the detection of the indicator
organism would involve tedious and complicated methodologies.
The coliphages and other bacteriophages have also been suggested as indicators of viral pollution.
This is because the structure, morphology and size, as well as their behaviour in the aquatic
environment closely resemble those of enteric viruses. These bacteriophages are quite useful in
evaluation of virus resistance to disinfectants.

An essential goad for the provision of safe drinking water is that it be essentially free of (at low risk
of containing) disease-causing micro-organisms. Since the beginning of the 20th century, the detection
of faecal indicator bacteria in drinking water has been used as the bases of criteria, guidelines and
standards for acceptable limits of faecal contamination and as the basis for judging or predicting the
possible presence or absence or pathogenic (disease-causing) micro-organisms. The WHO Guidelines
for Drinking Water Quality (GDWQ) and many other authorities continue to support the use of
bacterial indicator levels and their measurement as a basis for judging and verifying drinking water
quality. However, such faecal indicator analysis of drinking water as a measure of end-product
quality and determinant of microbial disease risk is only one of many measures and activities in an
overall system for providing safe drinking water.

It is now well established waterborne pathogens enter the drinking water systems through faecal
contamination, and the list of such organisms (which include bacteria, viruses, protozoans and
parasites) is a very long one. It would neither be practicable, nor is it necessary, to test a sample of
drinking water for all the pathogens that might possible be present in it, as that would involve many
different procedures, a multitude of media and other materials, incubation conditions, etc. It would,
therefore, be sufficient if we can determine whether the water sample was contaminated with faces,
such water is not fit for human consumption, as there would be a possibility that such water might
harbour pathogenic organisms. Therefore, by establishing faecal contamination of water, we will be
able to infer upon its potabilility or otherwise.
In the laboratory there are three simple procedures of testing water samples for faecal contamination:

1. H 2 S-Strip method: This is a simple, reliable and easy-to-perform (by even untrained
personnel), ‘Presence / Absence’ test for bacteriological quality, which works on the principle
that there is a close correlation between faecal contamination and the presence of hydrogen
sulphide (H 2 S)-producing bacteria and, the faecal pollution of water can be deduced by
demonstration of H 2 S production. It has been claimed, by various workers, that the H 2 S-strip
method shows >90% agreement with the conventional MPN test.
2. Most Probable Number (MPN) method for coliform bacteria-using the multiple tube
fermentation technique: In this method the MPN of total coliform bacteria, faecal coliform
bacteria, faecal coliform bacteria (or the thermotolerant coliforms) present in the water sample
is determined, along with the presence / absence of Escherichia coli.

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GUIDE MANUAL: WATER AND WASTEWATER ANALYSIS

3.2 COMPOSITION OF COMMON BACTERIOLOGICAL CULTURE MEDIA

3.2.1. MacConkey broth

Double-strength medium:
• Dissolve the peptone, sodium chloride and bile salts in the water by heating and store at 4ºC
overnight.
• Filter while still cold, add the lactose and dissolve.
• Adjust to pH 7.4 ± 0.2 and add the Neutral Red.

Single-strength medium:
• Prepare single-strength medium by dilution of the double-strength medium with an equal
volume of distilled water or make separatory using half the concentration of ingredients.
• Distribute single-strength medium in 5 mL volumes and double-strength medium in 10 mL
and 50 mL volumes. Each tube or bottle used should contain an inverted fermentation
(Durham) tube.
• Autoclave at 115ºC for 10 min.

3.2.2. Brilliant Green Lactose Bile Broth:

• Dissolve peptone and lactose in 500 mL distilled water
• Add 20 g dehydrated oxgall dissolved in 200 mL distilled water. The pH of this solution
should be 7.0-7.5
• Mix and add water to make 975 brilliant green in distilled water
• Add distilled water to make 1 litre
• Dispense into fermentation tubes, making certain that fluid level covers inverted vials.
Autoclave 15 min at 121ºC. Final pH, 7.2 ± 0.1.

3.2.3. EC Broth:
• Distribute 8 mL portions to 16 x 150 mm test tubes containing inverted fermentation
(Durham) tubes
• Autoclave 15 min at 121ºC. Final pH, 6.9 ± 0.2.

3.2.4. EC-MUG medium:

• Prepare as for EC Broth above, but add 50 mg 4-methylumbelliferyl-β-D-glucuronide (MUG)
per litre before autoclaving (15 min, 121ºC)
• Do not use Durham tubes, as gas is not relevant to the test and could interfere with the
interpretation of the UV light results.

3.2.5. Indole medium:

• Adjust pH to 7.3 ± 0.2.
• Dispense 4 mL portions to
• Autoclave 15 min at
• Final pH, 7.2 ± 0.2.

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GUIDE MANUAL: WATER AND WASTEWATER ANALYSIS

3.2.6. MacConkey agar: (w/o crystal vilet and NaCl, w/Sodium taurocholate)
• Suspended ingredients and hear with agitation to dissolve
• Boil 1-2 min
• Autoclave 15 min at 121ºC
• Cool to 45-50ºC, pour 20 mL portions into sterile 15 x 100 mm Petri dishes
• Dry at room temperature with lids closed
• Final pH, 7.1 ± 0.2.

3.3 TEST PROCEDURES

A. Multiple tube fermentation technique for coliform bacteria (MPN test):

In the multiple-tube method, a series of tubes containing a suitable selective broth culture medium
(lactose-containing broth, such as MacConkey broth) is inoculated with test portions of a water
sample. After a specified incubation time at a given temperature, each tube showing gas formation is
regarded as “presumptive positive” since the gas indicates the possible presence of coliforms.
However, gas may also be produced by other organisms, and so a subsequent confirmatory test is
essential. The two tests are known respectively as the presumptive test and the confirmatory test.
For the confirmatory test, a more selective culture medium (brilliant green bile broth) is inoculated
with material taken from the positive tubes. After an appropriate incubation time, the tubes are
examined for gas formation as before. 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 specially devised statistical tables. This technique is known as the MPN
method

Equipment and supplies:

• Culture tubes containing inverted vials (Durham tubes): each tube should be large enough for
a vial, completely filled with medium, to be submerged in it.
• Gas burner: a Bunsen or similar burner is adequate
• Inoculation loop and holder: lengths of 2 or 26 gauge wire (7.5-10 cm) should be used.
Nichrome wire is acceptable, but platinum-iridium is better. The wire is set in a handle made
of metal or glass, of diameter similar to that of a pencil. To make the inoculation loop, the
wire is bent to form a circle 3-4 mm in diameter.
• Clearing and maintenance equipment: items such as brushes for cleaning tubes, bottles, etc., a
waste bin, and a tool kit are required.
• Safety equipment: there should be an adequate first-aid and a fire extinguisher or other means
of fire control in every laboratory.

The following consumable items are required:

• Culture media / Reagents: the following culture media and reagents are required

- MacConkey Broth with neutral red (double strength)

- MacConkey Broth with neutral red (single strength)

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GUIDE MANUAL: WATER AND WASTEWATER ANALYSIS

- Brilliant Green Blue broth (BGB)

- Tryptone water/Peptone water (for indole test)
- Kovac’s Reagent

• Laboratory disinfectant: for cleaning laboratory surfaces and the pipette discard bin.
• Detergent: for washing glassware, etc
• Sodium thiosulphate solution: required when chlorinated supplies are tested. Sodium
thiosulphate neutralizes any residual chlorine in samples at the time of collection, preventing
it from acting on any micro-organisms present in water samples.
• Autoclave tape

Culture media preparation

• Commercially available dehydrated media simplify the preparation of culture broths and are
therefore recommended for laboratory work. Various manufacturers produce these media as
powders, which can then be easily weighed, dissolved in distilled water, and dispensed into
culture tubes before sterilization.

Preparation of media: Media should be prepared in accordance with the manufacturer’s instruction, as
follows:

a. Dissolve the stated amount of the dehydrated medium in distilled water to obtain the double-
strength or single-strength presumptive medium (MacConkey broth). The confirmatory
medium (BGB) is required only in single-strength.
b. Dispense the requisite volume into culture tubes containing an inverted Durham tube, and cap
the culture tubes; simple cotton plugs may be used or more ideally metal slip-on caps may be
used to cap the tubes.
c. Sterilise in an autoclave at 115ºC for 10 minutes (or in accordance with the manufacturer’s
specifications). It is particularly important that media containing disaccharides, e.g. lactose,
are not autoclave at higher temperatures.
d. The sterilized medium may be stored at room temperature (approximately 25ºC) or, ideally, at
2-8ºC. Media should in any case be warmed to room temperature before use to ensure that all
components have re-dissolved. In addition, since several dyes are light sensitive, the solution
should be protected form exposure to light.

Procedure for inoculation of samples: The procedure to be used for testing relatively unpolluted water,
such as treated water from waterworks or distribution system or end user’s tap.

Note down the details of the sample collected (from the label on the bottle) in the register.

a. With the stopper in position, shake the bottle vigorously to achieve a homogeneous dispersion
of bacteria. (If the bottle is completely full, remove the stopper and discard about 20-30 mL of
water; then replace the stopper and shake. This ensures thorough mixing).
b. Add 50 mL of sample to a tube / flask containing 50 mL of presumptive broth (double
strength). With a sterile 10 mL pipette, inoculate 10 mL of the sample into each the five tubes
containing 5 mL presumptive broth (single strength). It is advisable to shake the tubes gently

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to distribute the sample uniformly throughout the medium. Be careful as to not shake so hard
that air is introduced into the inverted tubes.
c. Incubate the tubes at 35º C ± 5ºC for 24 hours.
d. At the end of the 24-hour incubation period, examine each tube for the presence of gas. If
present, gas can be seen in the Durham tube. If none is visible, gently shake the tube; if any
effervescence (streams of tine bubbles) is observed, the tubes should be considered positive.
e. Record the number of positive tubes after 24 hours.
f. Re-incubate negative tubes for a further 24-hour period. At the end of this period, check the
tubes again for gas production as in 5 above. Gas production at the end of either 24 or 48
hours’ incuba-tion is presumed to be due to the presence of coliforms in the sample.
g. Record the number of positive tubes after 48 hours.
h. The confirmatory test should be carried out at the end of both the 24-hour and the 48-hour
incubation. Using a sterile loop, transfer one or two loops-full from each presumptive positive
tube into tow tubes containing respectively confirmatory broth (BGB) and tryptone water.
(Sterilise the inoculation loop before each transfer by flaming and allow cooling). To confirm
the presence of thermotolerant coliforms, incubate the subculture tubes from each
presumptive positive tube for 24 hours at 44.5 ± 0.5ºC.

[Alternatively, transfer a loopful of a positive MacConkey broth tube into BGB medium and
incubate at 35ºC for 24-48 hours. This will be a better confirmation of Total Coliforms.]

i. At the end of 24 hours’ incubation, examine each broth tube for growth and the presence of
gas in the Durham tube. Record the results, as done previously.
j. To each tube of tryptone water, add approximately 0.1 mL of Kovacs reagent and mix gently.
The presence of indole is indicated by a red colour in the Kovacs reagent, forming a film over
the aqueous phase of the medium.
k. Confirmatory tests positive for indole, growth, and gas production show the presence of E.
coli. Growth and gas production in the presence of indole confirms thermotolerant coliforms.

Determination of MPN: For treated water, where one 50 mL, five 10 mL and five 1 mL portions are
inoculated, the MPN can be found from the test results by referring to the MPN Table (also called
McCrady’s Table) given in Annexure-G. It would be best to use the numbers from the confirmatory
tests for the determination of water quality (Table 3.3: I).

Table 3.3: I: Water Samples are classified based on the presumptive count in the following way:
Class Grading Coliform Count / E. coli cont per
100 mL (MPN) 100 mL*
Class-1 Excellent 10 0
Class-2 Satisfactory 1-3 0
Class-3 Suspicious 4-10 0
Class-4 Unsatisfactory >10 0 or more

*Presence of E. coli immediately places the sample in Class-4, irrespective of coliform count

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B. Membrane filtration method for total coliform and thermotolerant (faecal) coliforms

Principle: The method is based on the filtration of a known volume of water through a membrane
filter consisting of a cellulose compound with a uniform pore diameter of 0.45 or 0.2 µm; the bacteria
are retained on the surface of the membrane filter. When the membrane containing the bacteria is
incubated in a sterile container at an appropriate temperature with a selective differential culture
medium, characteristic colonies of coliforms/ thermotolerant coliforms develop, which can be
counted directly. In contrast to the multiple-tube method, the membrane-filtration method gives as a
direct count of total coliforms and thermotolerant coliforms present in a given sample of water. A
comparison of the two methods, namely the multiple tube method and the membrane filtration
method, depicting the relative advantages and disadvantages, is given in the table below (Table 3.3:
II).

Table 3.3: II: Comparison of multiple-tube technique vs membrane filter technique

Multiple-tube method Membrane filter method

Principle Principle
Fermentation tubes (other suitable vessels) Measured volume of water if filtered
containing lactose broths (MacConkey) are through a membrane (pore size 0.45 or 0.2
inoculated with measured volumes of water µm, which retains the bacteria on its
samples; the coliform bacteria present in surface; the membrane is then incubated on
the water sample multiple and are detected a suitable selective medium for coliform
by formation of acid and gas. From the bacteria), allowing the bacteria to multiply
number of tubes inoculated and the number and form colonies. The number of colonies
with a positive reaction, the most probable counted is directly related to the
number (MPN) of bacteria present in the bacteriological content of the water sample
original water sample can be determined being analysed.
statistically.
Applicable to all kinds of water samples: • Not suitable for turbid water
clean, coloured or turbid, containing • Clay, algae, etc. prevents filtration
sewage or sewage or mud/soil particle, of a sufficient volume for analysis;
provided the bacteria are homogeneously may produce a deposit on the
distributed in the prepared test sample. membrane that could interfere with
bacterial growth
• Presence of high counts of non-
coliform bacteria in the sample may
interfere with detection of coliforms
• Sufficiently sensitive to measure • Results are direct and obtained
low levels of bacteria in water more quickly; coliform counts can
samples. (but a minimum time of 48 be made in less than 24 hr.
hr is required either for negative
result or for a presumptive positive
coliform count)
• Not suitable for field use. • Portable equipment, suitable for
field use.

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Volume of water sample for filtration: Since the filtration area is relatively small, it can support the
growth of only a limited number of colonies: the optimum number is between 20 and 80, with a
maximum of 200. If this figure is exceeded, very small atypical colonies or superimposed colonies
may develop, or there may be growth inhibition due to overpopulation. The choice of the volume of
sample to be filtered will depend on the type of water. Examples of typical volumes for membrane in
Table 3.3: III.

Table 3.3: III: Typical volumes for membrane-

Sample type Sample volume (mL)

Treated drinking-water 100
Partially treated drinking-water 10-100
Protected source water or groundwater 10-100
Surface water and water from open walls 0.1-100a
a
Volumes less than 10 mL should be added to the filtration apparatus after addition of at least 10 mL
of sterile diluent to ensure adequate dispersal across the surface of the membrane filter.
Equipment and glassware: In addition to the basic equipment and glassware used in the multiple-tube
method, the following items are needed for the membrane-filtration technique.
• Membrane-filtration apparatus: including an electric or hand-powdered vacuum pump, a
vacuum flask (e.g. an Erlenmeyer side-arm flask), and a filter support. One such commercially
available filter assemble is shown below to illustrate the various components.
• Residual Petri dishes: made from glass or metal (disposable plastic Petri dishes may also be
used).
• Blunt-ended forceps: For safe handling of filter membranes handle membrane filters without
damage using highly polished stainless steel forcep blades with beveled, unserrated tips.
Sterilise by autoclaving or by flaming.
• Reusable (autoclavable) bottles: for culture media (e.g. / 25 mL polypropylene bottles).
• A magnifying lens: with 34 or 35 magnification for examining and counting the colonies on
the membrane filters. Alternatively, a colony counter (such as Quebec Colony Counter) may
also be used.
• A boiling bath / pan: if filtration apparatus is to be disinfected in boiling water between
analyses.
• Sterile pipettes: 1 mL and 10 mL
• A graduated cylinder: 100 mL
• In addition to the consumables needed for the MPN, the following are required:
• Membrane filters: 47 mm in diameter, with a pore diameter of 0.45 µm. Singly packed,
presterilised membrane filters are very convenient. Unsterilised membrane filters can also be
used, however, and should bee wrapped in paper packets in convenient numbers (depending
on the number of water samples to be tested). These can then be sterilized in the autoclave and
dried by rapid exhaustion of the steam.
• Nutrient absorbent pads: These are essentially filter-paper discs about 1 mm thick, with the
same diameter as the membrane filters. They are available with suitable dispensers which help
in rapidly dispensing large numbers of pads without individual forceps-handling and hence
reduce contamination risks.
• Culture media: different types are available
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• Wax pencils: for labeling Petri dishes

• Polythene bags: for wrapping Petri dishes if a dry incubator is used, to prevent drying of the
sample and media

Culture media and dilution water: Various media can be used for the examination of coliform
organisms by the membrane-filtration method. Of these, lactose Tergitol agar, lactose TTC Tergitol
agar and membrane lauryl sulfate lactose broth may be used for coliform organisms at 35 or 37ºC and
for thermotolerant coliform organisms at 44ºC or 44.5ºC. Membrane faecal coliform (MFC) broth
should be used only at 44 or 44.5ºC for thermotolerant coliform counts. Although the use of all these
media for the detection of presumptive coliform organisms is based on the fermentation of lactose,
the characteristic reaction varies with each medium (Table 3.3: IV and 3.3: V). It is common to
confirm sheen colonies from mENDO by inoculating confirmatory broth media with colonies. Your
might require technician to swab the filter surface with a sterile cotton swab and use this to inoculate
MacConkey, BGB and EC broths.

Table 3.3: IV: Comparison of media for the examination of Coliforms

Medium Uses Incubation Remarks

temperature
Lactose TTC Total or 18-24 hours at 35 ± Adjust pH before
Agar with Tergitol thermotolerant 0.5ºC or 37 ± 0.5ºC for sterilization. Filter
7 coliforms total coliforms and 18- TTC supplement to
24 hours at 44 ± 0.25ºC sterilise. Tergitol
for thermotolerant supplement sterilised
coliforms by autoclaving.
Supplements of
Tergitol and TTC to
be added aseptically.

Lactose agar with Total or 18-24 hours at 35 ± Prepared plates have

Tergitol 7 thermotolerant 0.5ºC for total coliforms max. shelf-life of 10
coliforms and 18-24 hours at 44 ± days. Store in dark.
0.25ºC or 44.5 ± 0.25ºC
for thermotolerant
coliforms

Membrane Total or 18-24 hours at 35 ± Check pH before

enrichment with thermotolerant 0.5ºC or 37 ± 0.5ºC for sterilization
Teepol broth coliforms total coliforms and 18-
24 hours at 44 ± .25ºC
or 44.5 ± 0.25ºC for
thermotolerant
coliforms

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Membrane lauryl Total or 18-24 hours at 35 ± Check pH before

sulphate broth thermotolerant 0.5ºC or 37 ± 0.5ºC for sterilization
coliforms total coliforms and 18-
24 hours at 44 ± .25ºC
or 44.5 ± 0.25ºC for
thermotolerant
coliforms

Endo medium Total coliforms only 35-37ºC Basic fuchsin may be

a carcinogen. Also
requires ethanol. Do
not autoclave.
Prepared medium has
a shelf-life of 4 days.
Store prepared
medium at 4ºC in the
dark.

LES Endo medium Total coliforms only 35-37ºC Basic fuchsin may be
a carcinogen. Also
requires ethanol. Do
not autoclave.
Prepared medium has
a shelf-life of 2 days.
Store prepared
medium at 4ºC in the
dark.

MFC Thermotolerant 44ºC Do not autoclave.

coliforms Discard unused
medium after 96
hours. Rosalic acid
stock has a maximum
shelf-life of 2 weeks.
Check pH before
sterilisation. Store
prepared medium at
2-10ºC.

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Table 3.3: V: Colony characteristics of total coliforms and thermotolerant coliforms, following
analysis by the membrane-filtration methoda

Medium Colony characteristics

Total coliforms at 35/37ºC Thermotolerant coliforms at
44/45.5ºC

Lactose TTC* agar with Yellow, orange or brick-red As for total coliforms at
Tergitol 7 colouration with yellow 35/37ºC
central halo in the medium
under the membrane

Lactose agar with Tergitol 7 Yellow central halo in the As for total coliforms at
medium under the membrane 35/37ºC

Membrane-enriched Teepol Yellow colour extending on to As for total coliforms at

broth the membrane 35/37ºC

Membrane lauryl sulfate broth Yellow colour extending on to As for total coliforms at
the membrane 35/37ºC

Endo agar or broth Dark red colour with golden- -

green metallic sheen

LES-Endo agar Dark red colour with golden- -

green metallic sheen

Membrane faecal coliform - Blue colonies

(MFC) broth
a
Adapted from ISO 9308-1: 1990, Detection and enumeration of coliform organisms, thermotolerant
coliform organisms and presumptive Escherichia coli- part 1: Membrane filtration method.
* 2, 3, 5-Triphenyltertrazolium chloride.

Although it is possible to presence the media from the basic ingredients, this may be impractical in a
small laboratory. The use of dehydrated media is therefore recommended. The media can be prepared
as a broth and used together with nutrient absorption pads, or as solid agar plates. The broths may be
solidified by the addition of 1.2-1.5% agar before boiling.

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GUIDE MANUAL: WATER AND WASTEWATER ANALYSIS

Procedure:

The procedure generally used is described here, but different types of filtration units and equipment
exist.
a. Connect the Erlenmeyer (side-arm) flask to the vacuum source (turned off) and place the
porous support in position. If an electric pump is used, it is advisable to put a second flask
between the Erlenmeyer flask and the vacuum source; this second flask acts as a water trap,
and thus protects the electric pump.
b. Open a sterile Petri dish and place a sterile absorbent pad in it.
c. Add broth medium to saturate the pad; remove excess broth.
d. Place a sterile 47 mm dia. membrane filter on the porous support surface or screen, using
blunt forceps sterilised by flaming. The gridded surface should be if you are using gridded
membrane filter. Assemble the filter unit in the order shown in.
e. Place the upper container in position and secure it. The type of clamp used will depend on the
type of equipment.
f. Pour the volume of sample chosen as optimal for the types of water into the upper container.
If the test sample is less than 10 mL, at least 20 mL of sterile dilution water should be added
to the top container before filtration. Apply the vacuum.
g. Take the filtration unit apart and, using the sterile forceps, place the membrane filter in the
Petri dish on the pad with the grid side up. Make sure that no air bubbles are trapped between
the pad and the filter.
h. Leave the Petri dish at room temperature or at 35 or 37ºC for 2-4 hours, for resuscitation of
stressed microbes.
i. Place the dishes in an incubator at 44 ± 0.5ºC for 18-24 hours with 100% humidity.
Alternatively, tight-fitting or sealed petri dishes may be placed in waterproof plastic bags for
incubation.
j. Place the dishes in an incubator at 44 ± 0.5ºC for 18-24 hours with 100% humidity.
Alternatively, tight-fitting or sealed petri dishes may be placed in waterproof plastic bags for
incubation.
k. Submerge the bags in a water-bath maintained at 44 ± 0.5ºC for 18-24 hours. The plastic bags
must be below the surface of the water throughout the incubation period. They can be held
down by means of a suitable weight, e.g. a metal rack.

The colonies of coliform/thermotolerant coliform bacteria should be identified from their

characteristics on the medium used. The number of coliforms/thermotolerant coliforms per 100 mL is
then given by:

Coliform / thermotolerant coliforms per 100 mL

= [(No. of coliforms/thermotolerant coliform colonies counted) / (no. of mL of sample filtered) x 100]

Cleaning:

a. Immediately after use, disassemble the filter holder and clean its components with a sponge,
hot water, and a nonabrasive cleanser. If you are cleaning the Stainless Screen Glass 47 mm
Filter-Holder, the screen drops out easily when the base is inverted. Take care not to mislay or
damage the Teflon gasket that lies under the screen.

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GUIDE MANUAL: WATER AND WASTEWATER ANALYSIS

b. Use a stiff bristled brush (if available) to remove all traces of stubborn residue from recesses
and orifices, but do not use the brush on the filter support screen: it can break the screen mesh
and cause a ruptured filter. A test tube brush with detergent solution is helpful in cleaning the
inside of the holder outlet tube. Never use steel wool or abrasive materials on any part of the
holder.
c. Clean the porous glass frit of the filter support by back-flushing with warm tap water and then
soaking overnight in a chromic-acid cleaning solution. Follow the soaking with another back-
flushing.
d. After cleaning thoroughly, rinse the components with clean water and air dry. Do not wipe
with paper or cloth, which may leave traces of fibres and lint. Autoclave the funnel and bases
(if desired), wrap separately, and store for later use.
e. If filtering funnels and bases are needed for multiple analyses in a single day, they may be
sanitized in boiling water for 2-3 minutes in-between samples.

C. EC-MUG Test for confirmation of E. coli

This is a simple test for confirmation of presence of E. coli in water samples and may be knitted into
the Multiple Tube Fermentation (MTF) procedure, as a confirmatory test. If the lab chooses to use
EC-MUG test, it would replace using BGLB and tryptone broth (indole test) at 44.5ºC.
Background information: ECJ_MUG method test for the presence of E. coli bacteria. MUG stands for
4-methlumbelliferyl-β-D-glucoronide. It is the substrate for the enzyme β-glucoronidase. This
enzyme is primarily found only in E. coli. The enzyme cleaves the MUG molecule,

Media/Equipment needed:

• EC-MUG medium in tubes (for preparation, see under the section on culture media)
• Water bath capable of maintaining temperature at 44.5ºC
• Long-wave UV lamp, preferable 6-walt bulb.

Procedure: Submit all presumptive fermentation tubes/bottles showing gas/growth/or acidity within
48±3 hr of incubation, to this confirmatory test for E. coli. EC-MUG broth tubes are inoculated from
positive presumptive MacConkey broth tubes/bottles. The inoculated tubes are incubated at 44.5ºC
for 24 hours. A positive reaction is observance of a bright blue fluorescence when the tune is
subjected to long-wave (366 nm) ultraviolet (UV) light.

The use of positive (a known E. coli MUG positive culture) and negative (thermotolerant Klebsiella
pneumoniae, MUG negative) control reactions is highly recommended. An uninoculated medium
control may also be necessary to interpret the results and to avoid confusion of weak auto-
fluorescence of the medium as a positive response.

Interferences: Certain brands of glass test tubes fluorece under UV light. Tubes should be examined
before use. Do not use Durham tubes in the EC-MUG tubes, as gas is not relevant to the test and
could interfere with the interpretation of the UV light results.

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Bibliography

1. Assessing microbial safety of drinking water-improving approaches and methods, (2003).

OECD, WHO.
2. Manja, K.S., R. Sambasiva, [Link], [Link], [Link], [Link], [Link],
[Link] and [Link], (2001). Report of study of H 2 S test for drinking water. UNICEF,
New Delhi.
3. Chandrashekara, K.V., (2002). Microbiological load analysis methods-qualitative and
quantitative (focusing on emerging waterborne pathogens and consequences)., In Water
Quality Management: South Asian Perspective, Vision 2025. ILSI-India & UNICEF.
4. Guidelines for drinking - water quality, 3rd Edition - vol 1: Recommendations. WHO; 2004.
5. Standard methods for the examination of water and wastewater; 20th Edition, Ed. Pub. APHA,
AWWA and WEF, 1998.

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