Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/3.20 Phage Titer


Phage Purification May - June Notebook: Experiments


3.20 Phage Viability Titer

I) Purpose

Determine the viability of phage that we had on hand.
Plate phage to determine pfu's (plaque forming units) or concentration of phage.

II) Expected Outcome

Phage plaques that decrease along with the dilution series on plates.

III) Materials Used

1x LB Top Agar (TA)
LB Broth
LB plates
Eppendorf tubes
Phage ( strains T2, T5, and T3)

IV) Actual Procedure

We filled five eppendorf tubes full of 90 microliters of Liquid Broth each.
We added 10 microliters of our desired phage to the first test tube and then mixed
We took 10 microliters of the first tubes mixed solution and added it to tube 2, and followed the same procedure for each test tube down the line to tube five.
We then labeled 6 culture tubes 0 to -5.
In the first culture tube, we added 20 microL phage to .5 mL bacteria.
In tubes -1 to -5 we took 20 microL from eppendorfs and added to .5 mL bacteria.
We then allowed a 20 minute waiting period for the virus to infect the E. coli.
5 mL top agar was added to each culture tube.
Each tube was then plated and incubated at 37 degrees C.
The following teammates were assigned phage as follows:
Amber - T2
Arick - T5
Darren - T3

V) Results

We ran into several problems while doing the titer. After we had completed the titer, we found out that the pipet tips we had used were contaminated. When preparing the top agar, we had to melt it in the microwave which caused it to boil over. This also caused a lot of condensation in the plates and caused the auger to crack. This could have caused some contamination. While filling our -5 plate with top agar, there was only enough to put in 4mL of agar instead of the 5mL that was called for in our procedure.

None of the plates had any phage. There was just a lawn of bacteria growing. This could either be because of the problems mentioned above or because the source of T3 was bad. Seeing as nobody else was able to grow any phage we believe that the source was either old or we need a new lab technique.