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

Overview

March-April

May-June

July-August

September-October

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.