West Bengal University of Animal and Fishery Sciences

To isolate bacteriophages from

sewage sample.
Submitted by
Diptomoy Bhattacharya
M/D/2020/03
 Principle
 Bacteriophages are viruses of bacteria which require
bacterial hosts for their growth, developments and
multiplication. These are similar to enteric viruses and found
in higher numbers than enteric viruses in waste water and
other environment. Bacteriophages can be detected easily
and rapidly in environmental samples as compared to
enteric viruses. Thus bacteriophages are potential indicator
of environmental pollution which indicates the presence of
host bacteria such as coliforms and other enteric
pathogens.
 Coliphage is one type of bacteroiphage attack the
coliform group of bacteria. Thus coliphage is a potential
indicator of quality of water in estuaries, sea water,
potable water, and assessment of the efficiency of water
and waste water treatrnent plants, performance of water
treatments process and there is a good correlation
between coliphage and enteric viruses and pathogens,
though it does not indicate the fecal pollution
 Bacteriophages are bacteria eaters and they have a
complex structure. The protein coat consists of a
polyhedral head and a helical tail to which other structures
are attached. The head contains the nucleic acid. To initiate
an infection, the bacteriophage absorbs onto the surface of
a bacterial cell by means of its tail fibers and base plate.
After adsorption the tail sheath contracts, bringing the core
through the cell wall and injecting the DNA into the
bacterium.
1. Bacteriophages can be grown in liquid or solid cultures
of bacteria. The use of solid media makes location of
the bacteriophage possible by the plaques·detection
method.
2. Host bacteria and bacteriophages are mixed together
in melted agar.
3. The agar is then poured into a Petri plate containing
hardened nutrient agar.
4. Each bacteriophage infecting bacteria produces new
progeny of viruses.
5. All of the bacteria in the area surrounding the original
virus are destroyed leaving a clear area or plaque
against a confluent lawn of bacterial growth.
 Inthis method of bacteriophages from sewage sample
are enriched and isolated by centrifugation and
membrane filtration. Membrane filtration of viruses is
done using membrane filters of pore size 0.45 micron
that physically exclude bacteria from filtrate.
 Materials required:
1. Sewage sample
2. E. coli culture
3. Nutrient broth -10 ml of 5x NB(5 tubes)
4. CaCl2 - 3.5% (10 ml)
5. Sterile 250 ml flask - (3)
6. Nutrient agar
7. Petri plates
8. Spreader
9. Centrifuge tube
10. Membrane filter (0.45 micron in dia)
Procedure:
1. Mix properly 40 ml of sewage sample, 10 ml (5x)
nutrient broth. 5 ml E.coli culture and 1 ml CaCl2
solution were mixed and incubated at 37 degree
centigrade for 24hrs.
2. After incubation centrifuge the culture at 10,000 rpm
for 10 min to exclude the bacterial cells.
3. Filter the supernatant through a membrane filter.
4. Prepare the nutrient agar plates and a smooth lawn of
E. coli culture on it by using a bent glass rod .

CONTINUED…
5. Inoculate the bacteriophage by either method.
a)Overlay method: Prepare soft agar by mixing
0.75% of agar to nutrient broth. To this add few ml of
CaCl2. Supernatant containing virus and bacterial
culture and this was pour on the hardened nutrient
agar plates and spread it uniformly. Then incubate
these plates at 37 degree centigrade for 24 hrs in
upright position.
b) In other method, inoculate a loopful of filtrate and
CaCl2 as drops on four zones in plate. Incubate the
plates at 37 degree centigrade for 24 hrs.
6. Observe the plates for plaque formation
Thank
You