Date: Wednesday, November 14th, 2018

Title: Serial Dilutions and Spot Test with Positive Lysate

Rationale: The purpose of today’s lab is to make serial dilutions and then spot test them in order to evaluate which dilution will yield a plate that has a high titer and isn’t lysed fully.

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. Plates from last lab were evaluated and yielded negative results.
3. Flooded lysate from positive plates with high titer was borrowed from a lab partner (Gabriel Andino) and 5 mL was transferred to a new conical vial.
4. Nine microcentrifuge tubes were labelled PB for Phage Buffer and then 10^-1 through 10^-8.
5. 1 mL of phage buffer was added to the tube labelled PB.
6. 90 microliters of phage buffer was added to each other tube.
7. 10 microliters of the flooded lysate were added to the 10^-1 tube and vortexed.
8. 10 microliters from the 10^-1 tube were added to the 10^-2 tube and vortexed.
9. Step 8 was repeated until 10 microliters from the 10^-7 tube were transferred to the 10^-8 tube and vortexed.
10. Two plates were taken. One was labelled as TA control and the other was divided into nine sections, with each section being labelled after each of the microcentrifuge tubes.
11. Top agar was made using the following recipe:
    1. 4 mL LB broth
    2. 45 microliters 1M CaCl2
    3. 5 mL 2x Top Agar
12. 4.5 mL of the solution was added directly to the TA control plate.
13. 4.5 mL of the solution was added to a culture tube containing .5 mL arthrobacter and pipetted to mix the solution.
14. The culture tube was emptied into the spot test plate and left to harden for 15 minutes.
15. 10 microliters from each microcentrifuge tube were added to the corresponding sections on the spot test plate and left for another 15 minutes to allow the samples to absorb into the agar.
16. The plates were left in the incubator.

Observations: Some of the top agar and LB broth jars still seem to have contamination. From Gabriel’s experiments, the first few serial dilutions still lyse a plate fully. It could be beneficial to come in earlier after the plates have only sat in an incubator for a day so that a plate can be flooded before the plate is fully lysed.

Results: This experiment yielded a spot test with 8 different serial dilution samples as well as a top agar control.

Next Step: The next step is to choose a dilution based off the results of the spot test that has a high titer but will not lyse a plate fully. Then, a plaque assay will be made and flooded if it is webbed.