Objective: 

• Acquire a concentrated sample of bacteriophages
• Pick a plaque from the plaque assays formed after serial dilutions
• Make a plaque assay from the picked plaque

Pre-Lab Observations:

• Plaques were formed on all the plates ( 10^0, 10^-1, and 10^-2 were the dilutions)
• The control plate was 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. Phage buffer was acquired from the lab instructor and microcentrifuge tips.
3. Using the micropipette, 100μl of phage buffer was transferred to a microcentrifuge tube.
4. In the aseptic zone, at a 90° angle, a plaque on the 10^-1 plaque assay was stabbed using a micropipette tip ( attached to the micropipette) and the tip was then put into the microcentrifuge tube with 100μl of phage buffer and stirred to properly remove bacteriophages on the tip.
5. This microcentrifuge tube was then vortexed for 30 seconds and was labelled 10^0.
6. One Top Agar mixture was made for the group.
7. The LB broth was retrieved from its storage bath, along with a 50 ml conical tube and a serological pipette
8. While in the aseptic zone, 8 ml of LB broth was transferred to the 50 ml conical vial.
9. Then, 1 M CaCl2 stock solution was retrieved from the lab instructor.
10.  Using the micropipette, 90 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
11. The vial was then set on the rack.
12. 0.5 ml of arthrobacter was retrieved from the lab instructor
13. Using the micropipette, 10 microliters of the 10^0 bacteriophage mixture was transferred to the arthrobacter vial.
14. The vial was then allowed to rest on the test tube rack for 15 minutes
15. After the 10 minutes had ended, 25 ml of the 2X TA was added to the LB broth and Cacl2.
16. Using another serological pipette, 4.5 ml of the top agar mixture was transferred to the test tubes with the arthrobacter and the lysate.
17. The contents of the test tube were then poured onto the agar plate.
18.  Part of the top agar mixture was poured into the top agar control plate for the group.
19. To let the top agar solidify, the plates were allowed to rest for 12 minutes.
20. The plates were placed upside down in the incubator, where they will remain for 48 hours

Analysis and Interpretations:

The repeated contamination of the plate is perplexing. there may be another factor causing the contamination of the plates is that the agar plate could be contaminated plates before top agar was poured onto the plates. the purification process will not be repeated a few more times to acquire more concentrated and similar phages.

Future Notes 

Check plates properly before using them and test the broth and 2x agar for potential contamination.