CE 219 : Civil Engineering Laboratory I E 17 Batch

Reg. No.: …………….. Name : ……………………………………….

Environmental Engineering Laboratory
Date : ……………

Title : MEMBRANE FILTER TECHNIQUE CODE: ENV 421

Objective : To verify and estimate the presence of the Total Coliform and Fecal Coliform levels in
given water samples to determine the bacteriological quality of water.

Introduction :
Contaminated water is always been a significant agent in the transmission of various diseases
such as typhoid fever, cholera, infectious hepatitis, bacillary and amoebic dysenteries and varieties of
gastrointestinal illnesses to human beings. The introduction of water treatment together with
disinfection has markedly reduced the incidence of many diseases. But the occasional occurrence of
waterborne disease outbreaks highlighted the continuing importance of strict supervision and control
over the quality of public and private water supplies and enforcement of standards.
The fecal contamination of drinking water sources presents the greatest danger to human health
and thus the examination of bacteriological quality provides the most sensitive means for the detection
of such pollution. Even though, techniques are available for the detection of pathogenic bacteria,
viruses or protozoa in sewage or sewage effluents, it is not practical (time consuming, dangerous and
extremely expensive) to monitor them in drinking water samples. However, as pathogens present in
water are usually greatly outnumbered by normal intestinal organisms such as coliform bacteria, their
isolation and identification which are much easier is used as indicator organisms to evaluate the
bacteriological quality of drinking water today. Therefore as a standard, the presence of such
indicative organism in a water sample is considered a positive identification of the presence of
pathogenic organisms that can lead to a possible health hazard. However even their absence cannot
guarantee total water safety, is taken as a measure to evaluate the bacteriological quality of a water.
Coliform are a gram-negative, aerobic to facultative anaerobic, non-spore-forming, rod-shaped
bacteria that develop red colonies with a metallic (golden) sheen within 24 hrs at 35 0C on an Endo-
type medium containing lactose. They are widely distributed in nature and native to the gut of warm-
blooded animals and human but are not generally harmful themselves. However, presence of Coliform
in water indicates potential health problems. Generally, Coliform are divided into two groups as Total
Coliform (TC) and Fecal Coliform (FC). TC consists all of the coliform bacteria while FC consists
only a very few portion of the TC but are considered much more serious from the hygiene point of
view. FCs are dominated by Escherichia coli, also known as E.coli which passes out in high numbers
with the fecal material of human as well as some warm blooded animals. Counting of FC is done by
growing the bacteria under very specialized conditions at higher temperatures (44.5 0C) which
discourages the growth of TC. In addition, Fecal Streptococci (FS) also are normally found in the
intestines of man and animals. But, as human feces tend to have much more FC than FS and FS tend to
be more common in animal feces, comparing the numbers of FC to FS (FC:FS ratio) is helpful for
getting an idea of the real course of pollution, whether the water has been polluted with human fecal
wastes (>2:1) or animal wastes (1:1).

Principal of Membrane Filter Technique for Counting Bacteria

Membrane Filter (MF) method gives a direct count of the total Coliform and fecal Coliform
present in a given sample of water. This method is based on the filtration of a known volume of water
through a membrane filter consisting of a cellulose compound with a pore size of 0.45 μm. The
bacteria are retained on the filter, which is then placed on a suitable nutrient medium. Nutrients that
diffuse through the filter metabolized by bacteria trapped on the filter. Each bacterium that is trapped
on the filter will develop into a colony. Bacterial colonies growing on the medium can be counted.

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When a selective or differential medium is used at appropriate temperatures, desired colonies will have
a distinctive appearance.

Volume of Sample for Filtration

Due to relatively small area of membrane filter, it can support about 20 – 80 numbers of colonies
(optimum), with a maximum of 200. Thus the choice of the volume of sample to be filtered will
depend on the expected concentration of Coliform in the water.

Type of sample Volume of sample to be filtered (mL)

Good quality treated water 50 – 100
Untreated drinking water 10 – 50
Surface water 1 – 10
Highly polluted water <1

Apparatus :
- Membrane Filters, 47 – 50 mm diameter with a pore size of 0.45 μm
- Absorbent pads
- Broth Media
- Incubator
- Petri dishes
- Filter units
- Autoclave
- pH meter
- Balance
- Dilution bottles
- Pipettes
- Gas burner
- Water aspirator (electric vacuum pump)
- Colony counter or a magnifying lens
- Forceps

Procedure :

Media Preparation
a) M- Endo broth (for total coliform counts at 350 C – 370C)
i) Dissolve 0.96 g of dehydrated culture medium in 20 mL of distilled water and add
0.4 mL of ethanol.
ii) Sterilize by heating gently just to the boiling point
The medium can be stored for up to 4 days in the refrigerator

b) M-FC broth (for fecal coliform counts at 440C)

i) Dissolve 0.74 g of dehydrated medium in 20 mL of distilled water. Add 1 mL of
1% solution of Bacto Rosalic Acid in 0.2N NaOH
ii) Sterilize by heating gently just to the boiling point

Preparation of Dilution water

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 Special buffered, sterilized water is used to prepare the sample dilutions. The water is
prepared from a concentrated stock solution of phosphate buffer. To make the stock solution,
dissolve 34.0 g of Potassium Di-hydrogen Phosphate (KH2PO4) in 500 mL distilled water. This
solution should have pH of 7.2. If not adjust the pH of the solution to 7.2 using 1 N NaOH solution
and bring volume to 1000 mL with sufficient distilled water. Sterilize by filtration of autoclave for
15 minutes at 1210C (15-lb pressure). To prepare working dilution water solution add 1.25 mL of
stock solution to each 1 liter of distilled water and dispense in appropriate amounts into screw-cap
bottles and sterilize in the autoclave for 15 minutes at 121 0C.(15-lb pressure). After sterilization,
tighten the stoppers and store the dilution water in a clean place until needed.

Determination of Total Coliform count

1. Open a petri dish and place an absorbent pad in it using a flamed forceps.
2. Using a sterile pipette add 2 mL of M-Endo broth medium to saturate the pad, and cover
the petri dish and set aside
3. Connect the filtration unit to the vacuum source (turned off) and place the porous support
in position.
4. Assemble the filtration unit by placing a sterile membrane filter on the porous support,
using forceps sterilized by a flame.
5. Place the upper container in position and tighten it carefully
6. Pour the volume of sample chosen as optimal in accordance with the type of water into the
upper container. If the test sample is less than 10 mL, it should be diluted using sterile
dilution solution at least to 20 mL.
7. Apply the vacuum.
8. After the sample has passed through the filter, disconnect the vacuum and rinse the
container with 20 – 30 mL of sterile dilution water. Repeat the rinsing after all the water
from the first rinse has passed through the filter
9. Take the filtration unit apart and using a flamed forcep place the membrane filter with the
grid side up in petri dish on the pad. Make sure that no air bubbles are trapped between the
pad and the filter.
10. Invert the petri dish and keep in the incubator at 35 0C – 370C for 18 – 24 hours with 100%
humidity (to ensure this a piece of wet cotton wool in the incubator). If petri dishes with
tight-fitting lids are used, humidification is not necessary.
Colonies of total coliform bacteria will be formed at 18 – 24 hours to medium red or dark red in
color, with a greenish gold or metallic surface sheen. This sheen may cover the entire colony, or
appear only in the center of the colony. Colonies of other types should not be counted. The
colonies can be counted using the aid of a magnifying lens or a colony counter. Total coliform
count is presented as number of colonies per 100 mL

Determination of Fecal Coliform count

The procedure for the preparation for the determination of the fecal coliform is similar to that used
for the total Coliform. Filter the sample as described and place the membrane filter on the pad
saturated with M-FC broth medium.

Place the dishes in an incubator at 440C ± 0.50C for 24 hours with 100% humidity.

The colonies of fecal Coliform bacteria are blue in color. The colonies can be counted with aid of a
magnifying lens or a colony counter. Fecal coliform count is presented as number of colonies per
100 mL

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Observations:

Water Filtered Volume Number of colonies counted

Sample No.
source (mL) Total Coliform Fecal Coliform

Specimen Calculations :

For Sample ……………………………..

Number of Total Coliform colonies counted
Total Coliform per 100 mL = x 100
Filtered sample volume (mL)

Total Coliform per 100 mL = x 100

= …………………..colonies per 100 mL

Number of Fecal Coliform colonies counted

Fecal Coliform per 100 mL = x 100
Filtered sample volume (mL)

Fecal Coliform per 100 mL = x 100

= …………………..colonies per 100 mL

Results :

Number of colonies/100 mL
Water source
Total Coliform Fecal Coliform

References :

Metcalf & Eddy, Inc., “Wastewater Engineering – Treatment, Disposal and Reuse”, 3 rd Edition,
McGraw-Hill Inc., 1991.

EPA ‘Membrane Filter Method for the Simultaneous Detection of Total Coliforms and
Escherechia coli in Drinking Water’, February 2000

Discussion :

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1. What are Total Coliform, Fecal Coliform and Fecal Streptococci?
2. How will you identify whether a source of pollution is from humans and or animals?
3. What does indicator microorganism mean?
4. Why is it necessary to filter different volume of samples for different type of samples?
5. Why is it necessary to invert the petri dish when incubation, and maintain 100% humidity in
the incubator?
6. Discuss the significance of this test in controlling water borne diseases.
7. What steps would you recommend to be adopted for the step 6.
8. State the possible means of fecal contamination that can happen in a piped water supply
scheme and a domestic well water supply