10/08/18

Objectives:

• To pick a plaque from the first purification run ( from the 10 ^ -1 dilution plaque assay)
• To make a plaque assay to check for the presence of phages in apparent plaques.

Pre-Lab Observations:

The control plate was not contaminated in this plaque assay run from 10/03/18. There were no apparent plaques in the plaque assay. therefore, another plaque will be picked in the hopes to acquire phages. There may have been an error when this plate was picked the first time, possibly an air bubble was picked instead of a plaque. The plaque assay that is to be created will verify whether the plaques are plaques or they are air bubbles that seem to look like a plaque.

Procedure:

1. The aseptic zone was set up
2.  100μl of phage buffer was transferred to a microcentrifuge tube.
3. a plaque on the 10^-1 plaque assay was picked using a micropipette tip ( attached to the micropipette) and then put into the microcentrifuge tube with 100μl of phage buffer and stirred.
4. This microcentrifuge tube was then vortexed for 30 seconds and was labelled 10^0.
5. One Top Agar mixture was made for the group.
6. While in the aseptic zone, 8 ml of LB broth was transferred to a conical vial.
7.  90 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
8. The vial was then set on the rack.
9. 0.5 ml of arthrobacter was retrieved from the lab instructor
10. 10 microliters of the 10^0 bacteriophage mixture was transferred to the arthrobacter vial.
11. The vial was then allowed to rest on the test tube rack for 15 minutes
12. After the 10 minutes had ended, 25 ml of the 2X TA was added to the LB broth and Cacl2.
13. 4.5 ml of the top agar mixture was transferred to the test tubes with the arthrobacter and the lysate.
14. The contents of the test tube were then poured onto the agar plate.
15.  Part of the top agar mixture was poured into the top agar control plate for the group.
16. To let the top agar solidify, the plates were allowed to rest for 12 minutes.
17. The plates were placed upside down in the incubator, where they will remain for 48 hours

Analysis And Conclusion:

Analyzing the past two plaque assays, it seems that there were no phages in the lysate prepared from the plaque that was picked on 09/24/18.  The apparent plaques seemed to look more like air bubbles and therefore are a possible error in judgement. the plaque chosen on this day to be picked was upon consultation with the lab instructor. there were no obvious events that occurred which may have caused contamination.