Titering phage

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ACCESS Mini Research Campl
Titering activity
January, 21-22, 2010
Determining the titer of plaque forming units (pfu/ml) in an
unknown sample
The number of plaques that appear on each plate can be manipulated by how much sample you
put on each plate. I took one of those plates that looked like this,
and flooded it with buffer. The phages floated into the liquid and that liquid was removed from
the plates by pipette. It was the filtered (to remove any bacteria). How many pfu are in your
sample? We have to be able to count them. However, there are too many to count. One of the
ways to do this is to dilute your sample by factors of 10. When you get to a plate you can count,
you multiply the number of PFUs by the dilution factor. The best plate to count is one that has
20 - 200 plaques. Can you guess why that is best?
An example: You plate 1 ml of a sample that was diluted by a factor of 1/10 and counted 6
plaques on it. In the original sample there are 60 PFUs.
6 plaques X 10 (dilution factor) = 60 PFUs/ml
We make dilutions that are more dilute by factors of 10. We use the powers of 10 because the
number of pfu in our sample is large.
Materials:
Sample with unknown number of pfu
Micro-centrifuge tubes & rack
Pipetteman Pipettors (P20 and P200)
Sterile pipet tips
Pipettor for 5ml plastic pipets
Sterile 5 ml pipettes
Test tubes & racks
PB buffer
agar plates (with growth factors and antibiotics
added)
Top agar
Mycobacterium smegmatis
Vortex
Incubator
Markers
Making a serial dilution
1. Make ten-fold serial dilutions of your phage: 10-1 to 10-10.
a. Obtain 10 microcentrifuge tubes and place in a rack. (Remember: These tubes are
sterile and we want to keep them sterile.)
b. Label the tubes on their tops, -1, -2, -3, …. -10
c. Place 90 µl of buffer in each of the tubes, using a new sterile tips for each tube.
d. Obtain a sample of phage. Pipette 10 µl of that sample into the tube labeled -1.
e. Mix well, using the vortex.
f. Pipette 10 µl of the -1 mixture into the tube labeled -2.
g. Mix well, using the vortex.
h. Pipette 10 µl of the -2 mixture into the tube labeled -3.
i. Mix well, using the vortex.
j. Pipette 10 µl of the -3 mixture into the tube labeled -4.
k. Mix well, using the vortex.
l. Continue until you have pipetted 10 µl of the -9 mixture into the tube labeled -10.
m. Mix well,using the vortex.
Tube
#
Amt. of
buffer
(µl)
“Neat”
0
Amt. of
phage (µl)
Total
volume (µl)
100
100
Concentration Concentration Number of
Scientific
Plaques
notation
counted
100/100
1
-1
90
10 of Neat
100
1 x 10/100
10-1
-2
90
10 of -1
100
10-1 x 10/100
10-2
-3
90
10 of -2
100
10-2x 10/100
10-3
-4
90
10 of -3
100
10-3 x 10/100
10-4
-5
90
10 of -4
100
10-4x 10/100
10-5
-6
90
10 of -5
100
10-5 x 10/100
10-6
-7
90
10 of -6
100
10-6 x 10/100
10-7
-8
90
10 of -7
100
10-7 x 10/100
10-8
-9
90
10 of -8
100
10-8 x 10/100
10-9
-10
90
10 of -9
100
10-9 x 10/100
10-10
These serial dilutions can be assayed by two different methods, the spot test, or by
plating the phage.
Making a spot test
1. Prepare a Petri dish with M.smegmatis (smeg for short!)
a. Label a Petri dish with agar (on the agar side of the dish, aka the bottom) with your
initials, the date, and SPOT Test.
b. Divide the plate into 10 sections. Label each segment -1, -2, -3, …. -10. (make the label
as small as possible. Check out these two examples shown above. Turn it over.
c. Obtain a tube of smeg (0.5 ml of smeg).
d. Pipet 4.5 ml of molten top agar into the tube with the smeg.
e. Pipet up and down to mix
f. Pipette the entire contents (molten agar + smeg) onto the agar surface. Swirl in both
directions for 2 seconds. Allow to stand STILL until cool. (~5 minutes. Do not disturb
during this time.)
2. In each segment, place 5 µl of each appropriate dilution.
3. Allow the plate to stand until we are done with the next steps. Do not disturb.
Pouring Dilution Plates
1. Obtain 11 0.5 ml tubes of smeg. Label each tube: control, -1, -2, -3,. -10.
2. Pipette 10 µl of buffer into the control tube of smeg.
3. Pipette 10 µl of the appropriate dilution into each of the tubes of smeg. (Place 10 µl of -1
sample tube in the red cap tube labeled -1, 10 µl of the -2 sample tube in the red cap tube
labeled -2, etc.)
4. Pipette 4.5ml of molten top agar into the -1 red cap tube. Pipette the entire contents of the
tube onto the agar of the plate labeled -1. Swirl into both directions for 2 seconds. Allow to
stand STILL until cool. (Do not disturb.)
5. Repeat step 3 & 4 for all of the dilutions.
6. Allow the plates to stand until they are solid. Do not disturb.
7. Once the agar is solid, incubate the plates at 37˚ C for 24-48 hours. Find the plates with 20200 plaques on them. Count and record your results
Record Results in your notebook.
Dilution of plate
Number of Plaques counted
Determining the Titer
8 plaques/ 10 µl x 106 x 1000 µl/ml = .8 x 106 x 103 µl/ml = .8 x 109 = 8 x 108 plaques/ ml
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