Rationale

Due to  possible miscalculations from previous titer and webbing, I had to redo the same serial dilution and plate only 10^-3 through 10^-5. From result from previous lab day, no plaque present. From the lab day before that, phage was present in the 10^4 plate. I chose to redo it because new and correct calculations are needed for a new webbed plate.

Detailed Procedure

1. 10 µL of “SJ 11/12/18 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1 dilution which was vortexed.
2. 10 µL of 10^-1 was added with 90 µL of phage buffer to create 10^-2 dilution which was vortexed.
3. 10 µL of 10^-2 was added with 90 µL of phage buffer to create 10^-3 dilution which was vortexed.
4. 10 µL of 10^-3 was added with 90 µL of phage buffer to create 10^-4 dilution which was vortexed.
5. 10 µL of 10^-4 was added with 90 µL of phage buffer to create 10^-5 dilution which was vortexed.
6. Took 50 µL of 10^-3 into 0.5 mL of Arthro. Sat for 15 mins to infect.
7. Took 50 µL of 10^-4 into 0.5 mL of Arthro. Sat for 15 mins to infect.
8. Took 50 µL of 10^-5 into 0.5 mL of Arthro. Sat for 15 mins to infect.
9. Took 8 mL of LB Broth, 10 mL of 2X TA, and 90 µL of 1M CaCl2 into a 50 mL vial.
10. Took 4.5 mL of top agar mixture onto control plate. Allowed to solidify for 15 mins.
11. Took another 4.5 mL of top agar mixture into separate 15 mL vial. Combined lysate and 10^-3 mixture into same 15 mL vial. Swirled gently to mix and poured onto 10^-3 plate.
12. Took another 4.5 mL of top agar mixture into separate 15 mL vial. Combined lysate and 10^-4 mixture into same 15 mL vial. Swirled gently to mix and poured onto 10^-4 plate.
13. Took remiaing 4.5 mL of top agar mixture and combined lysate and 10^-5 mixture into same vial. Swirled gently to mix and poured onto 10^-5 plate.
14. Allowed all plates to solidify for 15 mins.
15. Inverted all plates into incubator after 15 mins.

Conclusion

Current titer= 7.4 x 10^-6

Webbed calculations= 97.635 microliters of 10^-5 to web

Only 10^-3 through 10^-5 plates were made because the results from lab day 24 had plaque shown in the 10^-4 spot titer and made calculations based off from there. Since there might have been a miscalculation from that spot titer, individual plates were made from 10^-3 through 10^-5 to insure a more precise calculation. 10^-4 plate was used to calculate the web and current titer since 10^-5 only had 3 plaque present. In the next lab day,  I will be able to make a new webbed plate for it to be flooded.

contamination of control plate, and no phage present on 10^-4 plate from lab day 24

no contamination on control plate, plaque present in 10^-3, 10^-4, and 10^-5 plates.