Team:BYU Provo/Notebook/PhagePurification/Winterexp/Period1/Exp/3.20 Phage Viability Test


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Phage Purification March - April Notebook: Experiments

Phage Purification

3.20 Phage Viability Test

I) Purpose

Test the viability (concentration if possible) of the phage we currently have
Become familiar with virus tittering techniques

II) Expected outcome

Should see plaque forming on a lawn of E coli (confluent growth)with decreasing amounts of plaques on less concentrated plates.

III) Reagent record

E coli
T3, T5, and T7 Phage Stock

IV) Actual procedures / observations

1. Dilution series of bacteriophage and its incubation with E coli

i) 90μL of broth were added to added to each dilution vial, labeled 1-5.
ii)20μL stock phage solution was added to vial 1.
Note: Do not disturb original phage solution; the precipitate at the bottom is most likely bacterial lysate.
iii)Perform a serial dilution (1:10) with vial 1-5, decreasing phage concentration to 10% of the previous vial with each.
iv)Approximately 0.5mL of E coli solution was added to each of the 6 test tubes, previously autoclaved and labeled 0-5.
v) 20μL of phage solution were transferred from vial to test tube. Test tube 0 received 20μL of the stock phage solution; otherwise, each test tube received 20μL of phage solution from the vial with corresponding number
vi)Phage solution was allowed to sit and mix with E coli solution for 20mL. This will let phage attach to bacteria

2. Addition of agar and plating

i)Top agar was warmed/liquefied by microwaving.
Microwaving doesn’t see to work so well, a better alternative would be to use a warm water bath.
ii) 5mL of liquid top agar was added to test tube; mixed well with phage + bacteria solution
iii)5mL of the mixed solution was transferred to the LB plate. The top agar was spread to from a uniform layer.
iv)The same process were repeated the test tube 0-6.

3. Check up on phage + bacteria viability in 24-48 hours

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 could have caused some contamination. While filling my -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 phage were bad. Seeing as nobody was able to grow any phage we believe that the source was old.