Date: Wednesday, August 29th, 2018

Title: Spot Test Results and Plaque Assay Preparations

Rationale: The purpose of today’s lab is to examine my spot test for positive or negative results and test my remaining lysate with a plaque assay to check for possible missed phages.

Preliminary Class Question: Is there a correlation between certain concentrations/species of bacteriophages and different species of oak trees?

Procedure:

1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
2. We lit a burner in the middle of the table in order to keep falling particles from contaminating the equipment or samples from above since the flame creates a circulating air current.
3. We checked our plates for results. My plate was negative, with a few spots being attributed to bubbles. My plate sat in the incubator for ~46 hours.
4. Group 5 received 3 plates to set up a plaque assay on. In addition, my side of the room (four groups, 1, 2, 5, and 6) received one more plate to share as a control.
5. We used a P200 micropipette to transfer 22.5 microliters 1M CaCl2 to a 50 mL conical vial to be used in the top agar control.
6. We added 2 mL LB broth to our control vial before realizing that we had miscalculated how much material we needed, forgetting to multiply the values by four to have enough solution for each plate.
7. We added 67.5 microliters 1M CaCl2 to the group conical using a P200 micropipette.
8. We then added 6 mL LB broth to our group conical vial.
9. I used a P10 micropipette to transfer 10 microliters of my enriched lysate to to a vial containing .5 mL arthrobacter ATC 21022 and let this solution sit for 21 minutes in order to let any phages infect the arthrobacter.
10. We added 10 mL x2 top agar to the group vial then quickly transferred 1 mL of the group vial solution to the group control plate so the agar didn’t harden early.
11. I transferred 5 mL  of x2 top agar to my personal vial with arthrobacter and enriched  lysate before adding the mixture to my agar plate in the aseptic zone.
12. I moved the agar plate side-to-side in order to cover the plate completely. I let this sit for 10 minutes to solidify.
13. I set my plate in the incubator at room temperature.

Observations:

• The spot test and plaque assay seem very similar on the surface, but have important differences. In the spot test, the plate is divided into separate sections to see if certain isolations or samples yield results. In a plaque assay, the whole plate has the same solution spread all over it and is less organized.

Results:

• Our group spot test control was contaminated, and my personal spot test yielded negative results.
• This experiment yielded me a plaque assay set up which will hopefully be uncontaminated and yield something positive.

Next Step:

• My next step is to evaluate my plaque assay for positive results next week.