Exp. No. 10 Isolation and titration of Coliphages
Isolation and titration of Coliphages
Introduction
Bacteriophages (phage)
are obligate intracellular parasites. They multiply inside a bacterium by
making use of some or all of the host (ie., bacteria) biosynthetic
machinery (viruses that infect bacteria are known as bacteriophage). They enter
the bacterial cell by ‘landing’ on the cell wall and injecting their DNA into
the bacterial cytoplasm. After entry, the phage DNA acts as a template for
production of phage proteins. These proteins replicate the phage and subjugate
the cell, eventually causing lysis and death of the host cell. A
bacteriophage particle is even harder to see than a bacterium. Viruses are
beyond the limits of resolution of the light microscope and can be seen only
with electron microscopes. Fortunately, we can use a technique very similar to
the colony-counting technique used to measure the number of bacteria to count
phage particles, known as the plaque assay. Lytic phages are enumerated by this
method.
The plaque assay is
originally a virological assay employed to count and measure the infectivity
level of the bacteriophages. But later, it was applied to measure and count the
mammalian viruses as well. This assay is the most widely used technique for the
isolation of virus and its purification, and to optimize the viral titers. The
basis of plaque assay is to measure the ability of a single infectious virus to
form a “plaque” on a concurrent monolayer culture cell. A plaque is developed
as a part of infection of one cell by a single virus particle that is followed
by the replication of that virus, and finally, the death of the cell. The newly
replicated virus particles will later infect and then kill surrounding cells.
Aim
To demonstrate the ability of
bacteriophage to replicate inside a susceptible host cell.
To determine the concentration of phage
particles in a suspension.
Materials and Methods
Cultures:
24- hour nutrient broth cultures of Escherichia coli B and
T2 coli phage.
Media:
Tryptone agar plates, Tryptone soft agar tubes (2ml/tube) and
tryptone broth tubes (9ml/tube)
Equipment's:
·
Bunsen burner
·
Water bath
·
Thermometer
·
1-ml sterile pipettes
·
Sterile Pasteur pipettes
·
Mechanical pipetting devices
·
Test tube rack
·
Glassware marking pencil
Media Preparation:
a) Tryptone agar: - Add 10g Tryptone,
0.01-0.03M Calcium chloride (reagent), 5g Sodium chloride and 11g agar in 1L of
water. Heat with frequent agitation and boil for 1 minute to completely
dissolve the powder. Autoclave at 121°C for 15 minutes.
b) Tryptone Broth: -Prepared as above without
the addition of agar in the medium.
c) Tryptone soft agar: -Add 10g Tryptone, 5ml Potassium chloride and 9g Agar in
1L of water. Heat with frequent agitation and boil for 1 minute to completely
dissolve the powder. Autoclave at 121°C for 15 minutes.
Procedure
Since viruses can grow to incredibly high concentrations, we need
to dilute them in order to count them effectively. Perform dilution of the
bacteriophage culture.
Each tube represents a ten-fold dilution of the virus.
a)
Five tryptone soft agar tubes: 10-5, 10-6,
10-7, 10-8, 10-9
b)
Five tryptone hard agar plates: 10-5, 10-6, 10-7,
10-8, 10-9
c) Ten
tryptone broth tubes : 10-1 through
10-10
Serial Dilution
·
9
ml of tryptone broth was filled to ten culture tubes labeled as 10-1 through
10-10. These tubes were used for viral serial dilutions.
·
Add
1 ml of the phage culture stock that you want to titer and transfer it to the
tube titled 10-1 with a pipette. Mix the tube well. This is
your first ten-fold dilution. (ie; a 1 in 10 dilution)
·
Take
1 ml of the mixed culture from your tube labeled 10-1 and
transfer it with a new pipette to the next tube, labeled 10-2. Mix
this tube as well.
·
Continue
this pattern to create a serial dilution series. You will end up with 9 tubes
of 9 ml and 1 tube of 10 ml. The viral loads in your tubes will be diluted
anywhere from 10 times (your first tube) or 100 times (your second tube) to ten
billion times (your final tube).
Preparing Plates
·
Take
five tubes of tryptone soft agar and five Petri plates labeled as 10-5 through
10-9
·
Place
the five labeled soft tryptone agar tubes into a water bath. Water should be of
a depth just slightly above that of the agar in the tubes. Bring the water bath
to 100˚C to melt the agar. Cool and maintain the melted agar at 45˚C. This will
ensure that your agar does not solidify in the tubes before you have a chance
to pour it into the petri dishes.
·
Aseptically
transfer two drops of Escherichia coli B culture with a Pasteur pipette
to the agar and mix it gently. These are the bacteria that will be killed,
allowing you to count the number of virus particles in a particular solution.
·
Add
0.1 ml of each serial dilution to its corresponding soft agar tube while the
tubes are still in the hot water bath. For example, 0.1 ml of your 10-5 serial
dilution should go into the soft agar tube labeled 10-5.
·
Using
separate Pasteur pipettes and sterile pipette tips, repeat the previous step
for the tryptone broth phage dilution tubes labeled 10-6through 10-9.
·
Mix
the tubes well and then pour each tube into the Petri plate with the
corresponding label. This will create a thin layer of agar that has been
inoculated with bacteria and viruses in each plate. Incubate all plate cultures
in an inverted position for 24 hours at 37 ˚C
The viral titer is a quantitative
measurement of the biological activity of your virus and is expressed as
plaque forming units (pfu) per ml.
·
Take
your plates out of the incubator and examine them. You should see cloudy areas
throughout the plate where bacteria have grown, except for small clear spots
called plaques. These plaques are patches of dead bacteria, and each plaque
represents one virus.
·
Find
a plate that has between 30 and 300 plaques and count the
exact number of plaques on that plate.
·
Then
use the following formula to determine the titer (pfu/ml) of your viral stock.
The titer value of Phage in the given
sample is ___________pfu/ml
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