09/26/18

Objective:

• Make another plaque assay from the extracted phages from the plate from the first serial dilution

Pre-Lab Observations:

• After checking the positive control made by the lab instructors, it was discovered that the arthrobacter culture used on 09/24/18 was not actually arthrobacter.
• So, new plaque assays must be made from the same phage extract used on 09/24/18
• The control plate from the plaque assays prepared on 09/24/18 was not contaminated.

Procedure:

1. Cidecon was poured on the desk and wiped till the desk was dry. Then, 70% ethanol was poured and wiped until it was all over the table and then it was allowed to evaporate. After the ethanol had evaporated, an ethanol lamp was lit, setting up the aseptic zone.
2. 400 μl of arthrobacter was retrieved from the lab instructor
3. Using the micropipette, 10 microliters of the 10^0 bacteriophage mixture was transferred to the arthrobacter vial.
4. The vial was then allowed to rest on the test tube rack for 15 minutes
5. While the vial was resting, one Top Agar mixture was made for the group.
6. The LB broth was retrieved from its storage bath, along with a 50 ml conical tube and a serological pipette
7. While in the aseptic zone, 8.4 ml of LB broth was transferred to the 50 ml conical vial.
8. Then, 1 M CaCl2 stock solution was retrieved from the lab instructor.
9.  Using the micropipette, 90 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
10. The vial was then set on the rack.
11. 10 ml of the 2X TA was added to the LB broth and Cacl2 after the sample was allowed to enrich the arthrobacter for 15 minutes.
12. Using another serological pipette, 4.5 ml of the top agar mixture was transferred to the test tube with the arthrobacter and the phage extract.
13. The contents of the test tube were then poured onto the agar plate.
14.  Part of the top agar mixture was poured into the top agar control plate for the group.
15. To let the top agar solidify, the plates were allowed to rest for 15 minutes.
16. The plates were placed upside down in the incubator, where they will remain for 48 hours

Analysis and Conclusion:

The procedures were properly performed in the aseptic zone and the chances on contamination were minimized. the control plate from 09/24/18 was not contaminated, which was a surprising outcome due to the repeated contamination of the control plates in previous spot tests and plaque assays. it may have been the plates that were contaminated when contaminated control plates were a result because more caution was used while picking plates for assays on Monday.