Objectives:

• To analyze the plaque assays from 10/01/18
• To make a plaque assay with 50 μL of phage extract from 09/26/18
• To acquire a webbed plate for flooding

Pre Lab Observations:

The control plate for the plaque assays from 10/01/18 was not contaminated. There was one possible plaque on the plaque assay for the third passage of purification. To confirm the strength or lack there of due to the presence or absence of phages in the sample, a plaque assay will be made with 50 μl of phage extract from 09/26/18.

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. 0.5 ml arthrobacter was retrieved from the lab instructor
3. Using the micropipette, 50 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 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 20 minutes.
16. The plates were placed upside down in the incubator, where they will remain for 48 hours

Analysis and Conclusion:

The top agar did not properly solidify and therefore the plates where not inverted to prevent movement of top agar. It seems that the through cleaning and bleaching of lab equipment may have resulted in positive results, implied from the lack of contamination of the control plate from 10/01/18. the procedure was properly performed in the aseptic zone. there were no apparent sources of contamination.