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- Overview
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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.
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