QUANTITATION OF VIRUS
Introduction: As you saw from the demonstration
plates, many different types of bacteria, along with
different types of bacteriophage can be easily isolated from
waste water. Other environmental sources can also yield a
variety of viruses, depending upon the appropriate target
cells and enrichment methods. Regardless of isolation
method, it is frequently important to quantitate the
concentration or amount of virus obtained. This is fairly
easy to do for lytic viruses. The situation is more
difficult with non-lytic viruses.
The plaque-forming method for bacteriophage used in this
exercise is a general method which can be applied, with
modification, to animal and plant viruses as well. For plant
viruses, samples are applied to leaves instead of agar
plates. For animal viruses, samples are added to cell
cultures. In all cases, holes or plaques form where cells
have lysed due to the activity of a lytic virus. Each plaque
represents one virus particle capable of an infection cycle
which occurs first in one cell, then repeats in neighboring
cells. The titer is determined by counting the number of
plaque-forming units. First, the number of plaques on a
plate are counted. That number is then multiplied by the
reciprocal of the amount of sample added to the plate and
multiplied by the reciprocal of the dilution factor. For
example, 85 plaques on a plate which received 0.1 ml sample
from a 10-4 dilution tube is equal to
85 x 10 x 104 = 8.5 x 106
pfu/ml.
For bacteriophage, a second method is sometimes used
called the broth-clearing assay. A dilution series of phage
in broth is set up, then inoculated with target bacteria. An
endpoint is determined based on the highest dilution
producing lysis of bacteria and clearing the broth culture.
This method can be used in parallel with the plaque-forming
assay for a given phage, then used later alone as a quick
check on concentrations of samples.
Basic methods: Rationale
1. Sterile technique is critical in this type of
work. You must prevent contaminating bacteria and fungi
from getting into your samples and plates. All materials
are initially sterilized, including tubes of diluent,
solid and liquid media, and pipet tips. While working
with these materials, care needs to be taken to avoid
contamination. Tubes and plates are kept covered, except
for brief periods to manipulate them. Pipet tips are
aseptically mounted on the pipettors and care is used in
avoiding touching anything with your pipet tip but your
desired sample.
2. Proper micropipet use is important in order to get
the appropriate quantities consistently. Practice holding
the pipettor and pushing the plunger until you are
comfortable. The plunger can be pushed to two positions.
The first position is to draw up a measured sample. The
second, farthest, position is to dispense the contents.
Be sure to carefully seat the pipet tip on the pipettor.
Otherwise you may have leaks and inconsistent volumes as
a result.
3. Labeling petri plates first is important in this
exercise. Plates are labeled on the bottom, not the top.
Include plate number, initials, and date.
4. Biohazard discard of pipet tips will be in the
provided beakers. Used agar tubes and dilution tubes are
placed in the provided racks. If there is a spill, let me
know. Spills need to be surrounded with bleach, then
covered with paper towels. The contaminated towels need
to be discarded in biohazard waste.
5. Organization in setting up and in doing this
exercise is necessary to get good results. Read the
entire exercise. Working in pairs helps. Decide who does
what, especially when transferring bacteria and diluted
phage to the top agar tubes. Reasonable speed is
necessary to avoid overheating the samples. Time is also
of the essence when pouring the top agar onto a petri
plate to facilitate a smooth and even layer.
EXERCISE: Titration of Bacteriophage: Work in
pairs
Materials: Per pair
1 rack to hold eppendorf tubes
6 eppendorf tubes with 0.9 ml sterile saline
1 P-100 pipettor set to "100" [100 ml
or 0.1 ml]
6 melted top agar tubes [3 ml 0.4% TSA each]:
in water bath
6 TSA plates
1 stock tube of assigned bacteria [in log phase
growth]
Materials: Per four
1 marking pen
1 box sterile pipet tips
1 discard beaker for used pipet tips
1 sample tube of assigned phage
1 heat block, calibrated to 50ºC
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Record assigned bacteria:
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Record assigned phage:
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Procedure:
1. Label the eppendorf dilution tubes and the
petri plates "1" through "6" by writing on the bottom of the
plates. On the plates, also label with date and initials for
later identification.
2. Aseptically mount a pipet tip on the P-100
pipettor, as demonstrated, by pushing the pipettor tip into
the top of the disposable tip in the box and lifting it free
of the box without touching any surface with the tip.
3. Aseptically add 100 ml
[0.1 ml] phage suspension to tube 1 containing 0.9
ml saline. Blow out tip into tube 1, then mix by carefully
aspirating up and down three times with the pipet. Discard
tip and mount a new pipet tip. Transfer 100 ml
from tube 1 to tube 2 and mix well. Transfer 100
ml from tube 2 to tube 3 and mix
well. Change pipet tips.
4. Repeat the transfer steps through tube 5. Add
nothing to tube 6- this is your control tube.
Why are the pipet tips changed between dilution
steps?
What dilution exists in each dilution tube,
relative to your sample tube?
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Tube
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Tube
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Tube
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Tube
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What purpose does the control, tube 6, serve?
5. Transfer 6 top agar tubes from the water bath
to the heat block. Heat block temperature should remain
between 48-50ºC.
6. With a fresh pipet tip, transfer 100
ml bacteria suspensions to each
of the 6 top agar tubes. Roll the tubes briefly between your
palms, as demonstrated, to mix, then return to the heat
block. Discard tip.
7. Starting with tube 6, aseptically transfer 100
ml to a top agar tube. Mix tube
by rolling between your palms. Quickly pour the inoculated
top agar over the surface of petri plate 6, tilting the
plate to spread evenly. Let it sit upright [lid up]
on the counter to solidify.
8. You may use the same pipet tip to continue
transferring 100 ml of each
successive dilution tube to a top agar tube, provided it
remains uncontaminated. [If sterility is broken or if in
doubt, change tips.] After each transfer of dilution
sample, mix and pour top agar over the surface of the
appropriately labeled plate.
9. When all plates have solidified, they may be
stacked and taped as a bundle. Be sure the plates are
labeled clearly as to date, names, etc. Invert your plates
and incubate at 35ºC in D234.
10. Plaques should be visible within a few hours.
You may check your plates in the morning in D234. To count,
select a plate with between 30 and 300 plaques. Count the
number of plaques, then multiply by 10, then multiply by the
reciprocal of the dilution of that plate to give the number
of plaque forming units per milliliter. If you see distinct
differences of plaques on a plate, you may choose to count
the types separately. To do so, use different colors of pens
to facilitate your counting. Please return your plates to
the tray. They will be removed from the incubator about 12
noon and stored for next week's discussion. A sign will be
posted to indicate their location in case you want to check
them later.
Why is the number of plaques multiplied by 10?
Record your results in pfu/ml:
Show your calculations:
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