ISOLATION
AND TITRATION OF COLI PHAGES
AIM: To isolate
and titrate of coli phages from the given sample
INTRODUCTION:
Bacteriophages, they are “bacteria eaters” and are
infectious agents that replicate as obligate intracellular parasites in
bacteria. A typical phage contains head, neck and a protein tail. Bacteriophages
are classified into two major groups on the basis of their mode of propagation:
1. Virulent (Lytic
phage): Growth of virulent phage in
susceptible bacteria destroys the host cells and produces many copies of
themselves. e.g. T2 and T4 phages of E. coli.
2. Temperate Phage: phages which are followed in lysogenic cycle.
Plaque assay is one of the widely used approaches for
determining the quantity of infectious virus in a sample. Only viruses
that cause visible damage of cells can be assayed in this
way. Plaque assay was first developed to calculate the titers of
bacteriophage stocks. Currently, its modified procedure is being used for the
determination of titer of many different animal viruses too.
PRINCIPLE:
When a suspension of an infective phage (e.g. T4 phage) is
spread over the lawn of susceptible bacterial cells (e.g. Escherichia
coli), the phage attaches the bacterial
cell, replicate inside it, and kills it during its lytic release. Lysis of the
bacteriophage is indicated by the formation of a zone of clearing or plaque
within the lawn of bacteria. In the absence of lytic phage, the bacteria form a
confluent lawn of growth.
Each plaque corresponds to the site where a single
bacteriophage acted as an infectious unit and initiated its lytic cycle. The
spread of infectious phage from the initially infected bacterial cell to the
surrounding cells results in the lysis of the bacteria in the vicinity,
eventually forming the plaque that is large enough to be visible to the naked
eye. Plaques do not continue to spread indefinitely. The size of the plaque
formed depends on the virus, the host, and conditions of culture. The
number of plaques that develop and the appropriate dilution factors can be used
to calculate the number of bacteriophages i.e. plaque forming units (PFU) in a
sample.
Isolation of a Bacteriophage from Sewage
Sludge
1. Bacteriophages were isolated from sewage samples by using enrichment
cultures. A total of five sewage samples were collected from sewage near our
college campus.
2. 35.0 ml of a filtered (Prefiltered to remove debris) sample was mixed
with 35ml of 10× Nutrient broth and with
5.0 ml of pure cultures of isolated E.coli.
After proper mixing the enrichment cultures were incubated for 24 h at 37°C to
allow amplification of lytic Coliphages.
3. 10.0 ml of sewage bacteriophage culture was transferred into a
centrifuged tube and the sample was centrifuged at 2000 rpm for 5 minutes.
4. Most of the remaining cells were pelleted. The supernatant was
transferred to a 10.0 ml syringe barrel fitted with a 0.45 micron filter.
5. The supernatant was filtered to remove bacteria from the phage sample.
The filtrate (lysate) was stored at 4oC.
Procedure for Bacteriophage Plaque Assay:
Preparation of
Stock Solution by serial dilution
1.
Place six sterile saline tubes (4.5
ml each) in your test-tube rack.
2.
Label one tube “control” and label
the remaining five tubes consecutively from 10-1 through 10-5.
3.
Label six nutrient agar plates the
same as the tubes.
4.
Using a sterile 1 ml pipette,
aseptically transfer 0.5 ml of the bacteriophage suspension provided to the
saline tube labelled 10-1.
5.
Mix the tube well by rolling it
between the palms of your hands.
6.
With another 1 ml pipette, transfer
0.5 ml from the 10-1 tube to 10-2 tube.
Mix the tube as in step 5.
7.
Using a fresh pipette for each
transfer, transfer 0.5 ml of the suspension from the 10-2 tube to the 10-3 tube,
and continue this diluting procedure consecutively for the remaining saline
tubes. Do not forget to mix each tube well before and after diluting.
Overlaying Plate with
Phage-Agar Mixture
1. Obtain six tubes of melted soft overlay agar from
the waterbath. Pipette 0.3 ml of a broth culture of E.coli into each of the soft agar tubes. Mix each tube well
by rolling between your palms.
2. Remove one inoculated tube of soft agar from
the waterbath. Using a 1 ml pipette, aseptically transfer 0.1 ml of the 10-1 saline
phage dilution into the soft agar tube. Mix the agar tube by rolling it between
your hands.
3. Immediately, aseptically pour the soft agar onto the surface
of the nutrient agar plate correspondingly labelled as 10-1. Replace the lid and without picking up the
plate, rotate it gently in a 6-to 8-inch circle on the surface of the table to
evenly distribute the agar.
4.
Using a fresh 1 ml pipette each time
and working quickly, repeat steps 1 and 2 for the remaining saline phage
dilution tubes and for the saline control tube.
5. For each dilution tube, use its correspondingly labelled
nutrient agar plate.
6.
Allow the soft agar to solidify.
7.
Invert and incubate plates at 35°C
to 37°C for 24 hours.
Results
1.
After incubation, each plate was
examined and the number of plaques was counted on each plate that has clearly
differentiated plaques.
2.
The plaques were counted and
recorded.
3.
The number of lytic phages was
calculated per millilitre that was in the original bacteriophage suspension
using formula mentioned above.
Results of Bacteriophage Plaque Assay
If
48 plaques are observed in 10-5 dilution
factor, as the 0.1 ml virus is added, Plaque forming units/ml will be 4.8 X 107. In your practical you can count the plaque
forming units, calculate and tabulate is as follows:
|
Dilution of phage
|
10-1
|
10-2
|
10-3
|
10-4
|
10-5
|
|
Number of plaques
|
|
|
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|
|
|
Calculations of plaque units/ml
|
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