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


Small Phage March - April Notebook: Experiments

Small Phage

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)

III) Reagent record

LB; E coli overnight prepared by Dr. Grose; phiX174 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)10μL 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

V) Results

March 21, 2013 at 1:00pm – E coli have formed a uniform lawn. No plague is seen in any of the plates

VI) Conclusion

E coli is viable, but the phiX174 phage is not.
Obvious contamination seen in 2 out of the 6 plates

VI) Limitations and Questions

We did not have all the equipment ready at the start of lab. With the waiting periods in between, contamination might be a huge problem.