Date: Wednesday, October 31st, 2018

Title: Soil Sample D Metadata Continued and Plaque Assay with Positive Lysate

Rationale: The purpose of today’s lab is to make a plaque assay with a lab partner’s positive lysate.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. An aseptic zone was set up
2. The phage-positive enriched lysate from a lab partner was retrieved and 1 mL was filtered to a microcentrifuge tube.
3. 40 microliters of the phage-positive lysate was added to .5 mL of arthrobacter and left to infect for 15 minutes.
4. Agar was made for two plates with the following formula:
   1. 4 mL LB broth
   2. 5 mL 2x TA
   3. 45 microliters 1M CaCl2
5. 4.5 mL of the TA solution was added to each culture tube and the TA control plate.
6. The plates were left for 20 minutes to harden before being inverted and incubated for the next 5 days.
7. The weigh boat from last lab was taken out from under the fume hood and weighed:
   1. Weigh boat with dry soil: 5.420 g
   2. Weigh boat alone: 2.435 g
   3. Dry soil: 2.985 g
8. The weight of the lost water was divided over the weight of the wet soil and multiplied by 100 to deetermine that the soil was 9.33% water.

Observations: The lysate used was from another student who had plaque assays yielding positive results. 40 microliters of lysate was used since previous plaque assays have shown that 30-50 microliters lysate yield enough plaques to cover the plate without webbing it.

Results: This experiment yielded a new plaque assay that can be evaluated at a later date for plaques.

Next Steps: The next step is to evaluate the plaque assay. If it yields positive results, the next step is to calculate the concentration needed to web a plate and work towards getting a high titer plate.