11.12.18 Plaque Assay for Lysate from Claire

Clarification: The lysate used for experiments on Monday (11/12) was generated from plates that were flooded on Thursday (11/8). The lysate originally came from Claire’s positive lysate, and the original lysate was diluted to 10^-2. Plaque assays were made with this dilution on Wednesday (11/7), and the plates were flooded on Thursday (11/8) to make the lysate that was used for today’s (11/12) experiment.

Rationale: Since the plates that contained Claire’s lysate had been flooded by Emily on November 8, a lysate that was ready to plate was obtained. Therefore, the next step in obtaining a high titer was using plaque assays to visualize the results that would indicate whether or not the new lysate had become a high titer through the processing steps.

Procedure:

1. Aseptic zone was established.
2. Claire diluted the original lysate that was obtained from Thursday (11/8) by placing 90µL of phage buffer in 3 separate tubes and adding 10µL of sample to the first tube. 10µL of this new sample was added to the next tube, etc.
3. **For the rest of this procedure, each member of Claire’s team completed these steps independently**
4. 10µL of each of the 4 dilutions (10^0, 10^-1, 10^-2, 10^-3) was added to 0.5mL arthrobacter and let sit for 15 minutes
5. 10mL of LB Broth was added to a conical tube
6. 90µL of CaCl2 was added to the conical tube
7. 2mL of the LB Broth and CaCl2 solution was added to each tube with the arthrobacter and sample.
8. 2.5mL 2X Top Agar was added to each of the arthrobacter tubes
9. Each plate was labeled with the date, dilution of the sample, my initials, and Claire’s initials to indicate where the lysate came from.
10. Plates were let sit for 10 minutes, then placed in the incubator overnight.
11. Station was cleaned

Observations:

• No top agar overlay solutions resulted in any slipping or tearing. Measurements were more precise than in the past, so this contributed to preventing the slippage.
• Samples spent slightly more time infecting arthrobacter than normal. This could contribute to a more lysed plate than normal, but will not be confirmed until Wednesday (11/14).

Results:

• Since no plates were made after the lysate was flooded, I had no results to report. Results from today’s (11/12) plates will be able to be visualized during Wednesday’s (11/14) lab session.

Conclusions and Next Steps:

• After flooding the plates on Thursday in an attempt to increase the concentration of phage, it can be hypothesized that after another cycle of phage buffer flooding that the concentration will have gone up. The next step after creating a plaque assay to support or reject this hypothesis will be to examine the plates on Wednesday and determine the titer of the lysate used. This will allow for specific results and conclusions to be obtained from the procedures completed today (11/12).