10.17.2018 “Flooding” A Plate

Rationale: Since the plate that was produced through Monday’s tests showed many small clearings, it was found necessary to use a procedure to create a 10^0, 10^-1, and 10^-2 lysate solution to begin the process of purifying phages.

Procedure:

1. Aseptic zone was created
2. Top agar was scraped off of the experimental plate completed on Monday (10/15) and placed in a conical tube.
3. 10mL of phage buffer was placed in the conical tube.
4. Tube was shaken for one hour.
5. Centrifuged for 5 minutes at 4000rpm.
6. 1mL lysate obtained from centrifuged tube was added to a microcentrifuge tube (10^0 tube)
7. 10µL chloroform was added to the 10^0 tube.
8. 90µL phage buffer was added to two additional tubes (10^-1 and 10^-2), and the proper dilutions were made from the 10^0 tube.
9. 10µL of each tube was added to 0.5mL Arthrobacter and let sit for 15 minutes.
10. 8mL LB Broth placed in conical tube
11. 90µL CaCl2 was added to the same tube
12. 2mL LB Broth and CaCl2 solution was added to each of the three dilution tubes that contained 0.5mL arthrobacter
13. 2.5mL top agar was added to all four of the tubes. Tubes were swished, then plated on separate tubes with one being the control tube.
14. Plates let sit, then placed in incubator
15. Cleaned table.

Results:

The results from Monday show several small clearings on the plate that contained the retry of Soil Sample E. The control plate also displayed similar levels of contamination to the first time it was completed. No spot test was attempted because the top agar for only the plaque assay was contaminated, not the spot test. The small clearings appeared to be mostly uniform circles.

Observations:

• The top agar created today did not set well and appeared to have tears in it. This resulted in the plates being left right side up in the incubator.
• The control plate persisted to have contamination, so extra care was taken to ensure that the top agar used today was different and was very clear. Same protocol was used for the LB Broth.
• Lysate from after the top agar shaking process had some impurities in the lysate after centrifugation. This would have caused problems in a filter, but since none were able to be used an alternate procedure made the impurities not a problem.

Conclusion and Next Steps:

• Since the clearings on the plate appeared to be roughly uniform, the plate was flooded to obtain as much phage as possible as part of amplification. The next step will be to examine the plates the were prepared today to confirm firstly that phage is present and secondly to begin to determine the best method to create a high titer lysate.