Bacteriological Analysis of Water

Written by Nisha Rijal ● in Bacteriology  ● Last Updated October 23, 2022

Bacteriological analysis of water is one component of drinking water quality analysis. Water is
screened for the presence of fecal contamination by testing for the presence of an indicator
microorganism.

Indicator microorganisms are ones that have the following

properties:

1. The microorganism is not found in water and will

be present in the water only when a
contamination event has occurred; and

2. The density of the microorganisms present should

be proportional to the degree of
contamination. 

E.coli which is a normal flora present in the intestine of humans and animals meets the above
requirements so detection of E.coli and/or related bacteria termed “coliforms” is done using many
different techniques as a part of a bacteriological analysis of water. One of the techniques is the
membrane filtration method.
Membrane filtration for determining the coliform count (Image source:
https://www.sciencedirect.com )

Membrane filtration method

In this method, water from different samples is passed through a membrane filter with a pore size
of 0.45 µm, and the membrane is incubated on an agar plate. Bacterial cells trapped on the
membrane will grow into colonies that can be counted, and the bacterial density of the water
samples can be calculated. Depending on the source of the water different filtration volumes of
water samples are used.

Table 1: Typical sample volumes for membrane filtration analysis

100 10 1 0.1 0.01 0.001

Sample type
ml ml ml ml ml ml

Treated drinking water X          

Partially treated drinking water X X        

Protected source water   X X      

Recreational water     X X    

Surface water     X X    

Wastewater     X X X  

Discharge from sewage treatment

    X X X  
plant

Ponds, rivers, stormwater runoff       X X X

Raw sewage       X X X
Note:

Small volumes should be added to the filtration apparatus together with a minimum of 9 ml of
sterile diluent to ensure adequate dispersal across the surface of the filter membrane

1.0, 0.1, 0.01, and 0.001-ml volumes are filtered after first preparing serial dilutions of the sample.

Since large volumes of the sample can be filtered, the membrane filtration technique can detect
the presence of a very low number of bacteria. However, the turbidity of the water may limit the
volume of samples that is practical to filter. High numbers of background bacteria or toxic
substances if present may interfere with the test and result in underestimation of the density of
coliforms.

Table of Contents
1. Total coliforms and fecal coliforms:
1.1. Detection of total coliforms and fecal coliforms:
1.1.1. Detection of total coliforms using mEndo agar LES
1.1.2. Detection of fecal coliforms using mFC agar
1.2. Materials required
1.2.1. Apparatus
1.2.2. Consumables
1.3. Water sample collection Precautions
1.4. Procedure of membrane filtration technique
1.4.1. References and further reading
Total coliforms and fecal coliforms:
They are the members of the family Enterobacteriaceae. Coliforms may include bacteria of the
following genera: Escherichia, Enterobacter, Klebsiella, Citrobacter, and Serratia. Fecal coliforms are
the subset of total coliforms which are found within the digestive tract and shed through feces.
Fecal coliforms can ferment lactose with the production of acid and gas at 44.5°C within 24 hours.
They are also said to be thermotolerant coliforms as they can grow at a higher temperature.
Bacteria belonging to the genera Escherichia and Enterobacter are considered fecal coliforms.

Detection of total coliforms and fecal coliforms:

Total coliforms and fecal coliforms are detected using selective and differential culture medium.
For the detection of total coliforms mEndo agar LES is used and for fecal coliforms, mFC agar is
used. On mEndo agar, coliforms will form red colonies with a metallic sheen and on mFC agar,
fecal coliforms will form dark blue colonies. 

Detection of total coliforms using mEndo agar LES

This medium contains lactose and a pH indicator (that changes
color when acid is produced due to fermentation of lactose).
 Membrane filter is incubated at 35°C for 22-24 hours for the
detection of total coliforms.

Coliforms typically will produce a metallic (golden) sheen, which

is due to the extensive production of aldehydes and acid from the
fermentation of lactose.

Atypical total coliforms colonies appear as dark red, mucoid, or have a dark center but without a
metallic sheen. E.coli will form colonies with a metallic sheen.
Detection of fecal coliforms using mFC agar
This medium contains bile salts which inhibit other bacteria
except enteric. It also contains rosolic acid which inhibits
bacteria other than fecal coliforms. Aniline blue is used as a
pH indicator which gives blue color in acidic pH.

 Membrane filter is incubated at 44.5°C for 22-26 hours in mFC

agar for the detection of fecal coliforms.

Fecal coliforms form blue colonies in this medium (acid is

produced due to the fermentation of lactose).  E.coli will form
flat dark blue colonies.

Materials required

Apparatus
Incubator(s) or water-bath(s)

Membrane filtration apparatus, complete with a vacuum source (electrically operated pump,
hand pump, or aspirator) and suction flask

Autoclave for sterilizing prepared culture media

Boiling pan or bath (if filtration apparatus is to be disinfected in boiling water between uses)

Laboratory balance
Racks for bottles of prepared culture media and dilution water. These must fit into the autoclave

Distilling apparatus with storage capacity for at least 5 liters of distilled water

Refrigerator for storage of prepared culture media

Hot-air sterilizer for sterilizing pipettes and glass or metal Petri dishes

Thermometer for checking the calibration of incubator or water bath

Pipette cans for sterilizing pipettes

Boxes for Petri dishes for use in a hot-air sterilizer

Reusable bottles for culture media

Measuring cylinders, capacity 100 ml, and 250 ml

Reusable pipettes, glass, capacity 1 ml, and 10 ml

Bottles to contain 9-ml volumes of buffered dilution water

Flasks for the preparation of culture media

Wash-bottle

Blunt-edged forceps

Pipette bulbs

Spatula

Container for used pipettes

Brushes for cleaning glassware (several sizes)

Fire extinguisher and first-aid kit

 Miscellaneous tools

Waste bin

Consumables
Methanol for disinfecting filtration apparatus using formaldehyde gas (unnecessary in the
laboratory, but essential if analyses are done in the field). It is essential to use methanol.
Ethanol or methylated spirits cannot be substituted.

Membrane filters, 0.45 µm pore size, and diameter appropriate for the filtration apparatus being
used and complete with absorbent pads.

Disinfectant for cleaning laboratory surfaces and a container for discarded pipettes.

Culture media (mEndo Agar LES and mFC Agar).

Phosphate-buffered dilution water.

Petri dishes, glass or aluminum (reusable) or plastic (disposable).

Polyethylene bags for wrapping Petri dishes if a dry incubator is used.

Magnifying lens (as an aid to counting colonies after filters are incubated).

Wax pencils for labeling Petri dishes.

Autoclave tape.

Detergent for cleaning glassware and laboratory equipment.

Positive control sample (100 ml of water with a 5 ml of a 1:100 dilution of an overnight culture
of E.coli)

Negative control sample (sterile water)

 Water samples to be tested: Volume of water needed for testing. 

Water sample collection Precautions

Water sample must be collected in a sterile bottle

Water sample must be representative of the supply from which it is taken.

Contamination of the sample must be avoided during and after sampling.

The sample should be tested as promptly as possible after collection.

If there is a delay in the examination of the sample, it should be stored at a temperature

between 0 and 10°C.

Procedure of membrane filtration technique

1. Sterilise the tips of the blunt-ended forceps in a flame and allow them to cool.

2. Carefully remove a sterile membrane filter from its package, holding it only by its edge.

3. Place the membrane filter in the filter apparatus, and clamp it in place. (If the apparatus has been
disinfected by boiling, ensure that it has cooled down before inserting the membrane filter.)

4. Mix the sample by inverting its container several times. Pour or pipette the
desired volume of sample into the filter funnel. This volume should normally be chosen in the
light of previous experience, but suggested volumes are given in Table 1. If the volume to be
 filtered is less than 10 ml, it should be made up to at least 10 ml with sterile diluent so that the
sample will be distributed evenly across the filter during filtration.

5. Apply a vacuum to the suction flask and draw the sample through the filter; disconnect the
vacuum.

6. Dismantle the filtration apparatus and remove the membrane filter using
the sterile forceps, taking care to touch only the edge of the filter.

7. Remove the lid of a previously prepared mENDO agar LES plate and place
the membrane, grid side uppermost, onto the agar. Lower the membrane, starting at one edge in
order to avoid trapping air bubbles between membrane and agar. Mark the petri dish with the
sample number or other identification. The sample volume should also be recorded. Use a wax
pencil or waterproof pen when writing on Petri dishes.

8. Repeat procedure (1 to 8) with the same volume, but place membranes on mFC plates.

9. Incubate mEndo agar LES plates at 35 ± 0.5°C for 22 – 24 hours and mFC plates at 44.5 ± 0.2°C
for 22 to 26 hours, all lid side down. In order to maintain the temperature within such a narrow
 range, a water bath is typically used for incubation of the mFC agar plates. These plates are
placed in watertight plastic bags and then submerged in the water bath.

Day 2 (24 hours later):

1. After 22 – 24 hours, remove the mEndo agar LES plates from the 35°C
incubator and count the colonies that are dark red, mucoid, have a dark center or (more
typically) produce a metallic sheen. These are considered to be total coliform colonies.

2. From the mEndo agar LES plates, choose two total coliform colonies that are isolated on the
membrane and confirm that they are Gram-negative rods and non-spore formers.

3. After 22 to 26 hours, remove the mFC agar plates from the 44.5°C
incubator and count the colonies that have any blue color. These are considered to be fecal
coliform colonies.
4. From the mFC agar plates, choose two fecal coliform colonies that are isolated on the
membrane. Confirm that they are Gram-negative rods and non-spore formers.

5. Calculate the total and fecal coliform CFU per 100 ml for each sample as described below:

The original density is estimated from the volume of sample filtered (or the volume of dilution
and the dilution factor), and the number of colonies counted on the membrane.

As counts are reported per 100 ml of sample (not per ml), the per ml values must be multiplied
by a factor of 100.

No. of CFU per 100 ml = [(No. of colonies on the membrane)/(volume filtered)] × 100

If the sample is diluted and the volume of the dilution was filtered, the denominator will be
(volume (ml) filtered x dilution)

References and further reading

Water Quality Monitoring – A Practical Guide to the Design and Implementation of Freshwater
Quality Studies and Monitoring Programmes (WHO)
 Brian Forster, Catalina Arango Pinedo. 2015. Bacteriological examination of waters: membrane
filtration protocol. American Society for Microbiology (ASM)

Manual for Bacteriological Analysis of Natural Water Supply Sources in Disaster Situations. Pan
American Health Organization (PAHO)

Pelczar MJ, Chan ECS, Krieg NR (2007). Microbiology. 5th edn. Tata McGraw-Hill.

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Nisha Rijal
I am working as Microbiologist in National Public Health Laboratory (NPHL), government national
reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work
lies in Bacteriology, especially in Antimicrobial resistance.
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