Objectives:
To
demonstrate the ability of bacteriophage to replicate inside a susceptible host
cell.
To
determine the concentration of phage particles in a suspension.
Principle:
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 cells. 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.
Materials Required:
Cultures:
24- hour nutrient broth cultures of Escheria coli B and T2 coli
phage.
Media:
Tryptone agar plates, Tryptone soft agar tubes (2ml/tube) and
tryptone broth tubes (9ml/tube)
Equipments:
·
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.
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 .
Label all dilution tubes and media as follows. 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
Label all dilution tubes and media as follows. 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
·
Put on gloves, fill 9 ml of tryptone broth to ten culture tubes
labeled as 10-1 through 10-10. These tubes will be
used for viral serial dilutions.
·
Take 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 Escheria 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
Counting
and Calculating Titers
The viral titer is a quantitative measurement of the biological
activity of your virus and is expressed as plaque forming units (pfu) per
ml.
To calculate the viral titer,
To calculate the viral titer,
·
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.
Where, d = dilution
v = volume of diluted virus added to the plate
v = volume of diluted virus added to the plate
Sample
calculation:
• An average of 50 plaques formed in the 1:10,000 dilution wells
• Volume of diluted virus added: 0.2 ml
Result:
The titer value of Phage in the given sample is
___________pfu/ml
Courtesy : http://vlab.amrita.edu/?sub=3&brch=76&sim=719&cnt=2
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