Objectives:

• Extract bacteriophages from plaques on plaque assay from 09/17/18
• make serial dilutions of extracted plaque to 10^0, 10^-1, 10^-2
• make plaque assays for each dilution

Pre Lab observations:

• The plaque assays prepared on 09/17/18 had plaques on them.
• Therefore, soil sample will not be washed and enriched at the time
• Bacteriophages must now be extracted from a plaque and serial dilutions must begin to acquire a more purified sample.
• The control plate was yet again contaminated, possibly by arthrobacter in a similar pattern as the control plate that was contaminated from the previous spot test.

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 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. Using the micropipette, 90μl of phage buffer was transferred to a microcentrifuge tube.
7. 10μl of the 10^0 bacteriophage and phage buffer mixture was transferred to the microcentrifuge tube with 90μl of phage buffer and the tube was labelled 10^-1.
8. Using the micropipette, 90μl of phage buffer was transferred to a microcentrifuge tube.
9. 10μl of the 10^-1 bacteriophage and phage buffer mixture was transferred to the microcentrifuge tube with 90μl of phage buffer and the tube was labelled 10^-2.
10. One Top Agar mixture was made for the group.
11. The LB broth was retrieved from its storage bath, along with a 50 ml conical tube and a serological pipette
12. While in the aseptic zone, 20 ml of LB broth was transferred to the 50 ml conical vial.
13. Then, 1 M CaCl2 stock solution was retrieved from the lab instructor.
14.  Using the micropipette, 225 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
15. The vial was then set on the rack.
16. 3 of the 0.5 ml of arthrobacter test tubes were retrieved from the lab instructor
17. Using the micropipette, 10 microliters of the 10^0 bacteriophage mixture was transferred to a arthrobacter vial.
18. the same was done using the other 2 test tubes with arthrobacter and the 10^-1 and 10^-2 bacteriophage mixtures
19. The vials were then allowed to rest on the test tube rack for 10 minutes
20. After the 10 minutes had ended, 25 ml of the 2X TA was added to the LB broth and Cacl2.
21. Using another serological pipette, 4.5 ml of the top agar mixture was transferred to each of the test tubes with the arthrobacter and the lysate.
22. the contents of each of the test tube were then poured into the 3 separate agar plates.
23.  Part of the top agar mixture was poured into the top agar control plate for the group after each member had followed step 22.
24. To let the top agar solidify, the plates were allowed to rest for 10 minutes.
25. The plates were placed upside down in the incubator, where they will remain for 48 hours.

Analysis and Conclusion

Proper methods and measurements were used. All procedures were performed properly in the aseptic zone. The contaminated plate from the plaque assay control from 09/17/18 was similar to the contamination on the spot test control 09/12/18. Many of the other lab groups have also had contaminated control plates. It seems that there is a greater chance that the LB broth and 2X TA were contaminated. It is also possible that everyone is making the same mistakes in protocol, but the previous is more likely.

Future Notes:

to keep track, the LB broths and 2X TA used will now labelled so as to locate possible contaminated LB broths and TA