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.

  1. Obtain 10 microcentrifuge tubes and place in a rack. (Remember: These tubes are sterile and we want to keep them sterile.)
  2. Label the tubes on their tops, -1, -2, -3, …. -10
  3. Place 90 µl of buffer in each of the tubes, using a new sterile tips for each tube.
  4. Obtain a sample of phage. Pipette 10 µl of that sample into the tube labeled -1.
  5. Mix well, using the vortex.
  6. Pipette 10 µl of the -1 mixture into the tube labeled -2.
  7. Mix well, using the vortex.
  8. Pipette 10 µl of the -2 mixture into the tube labeled -3.
  9. Mix well, using the vortex.
  10. Pipette 10 µl of the -3 mixture into the tube labeled -4.
  11. Mix well, using the vortex.
  12. Continue until you have pipetted 10 µl of the -9 mixture into the tube labeled -10.
  13. Mix well,using the vortex.

Tube # / Amt. of buffer (µl) / Amt. of phage (µl) / Total volume (µl) / Concentration / Concentration Scientific
notation / Number of Plaques counted
“Neat” / 0 / 100 / 100 / 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!)
  1. Label a Petri dish with agar (on the agar side of the dish, aka the bottom) with your initials, the date, and SPOT Test.
  2. 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.
  3. Obtain a tube of smeg (0.5 ml of smeg).
  4. Pipet 4.5 ml of molten top agar into the tube with the smeg.
  5. Pipet up and down to mix
  6. 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.)
  1. In each segment, place 5 µl of each appropriate dilution.
  1. 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 20- 200 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