Lab Day 27: Serial Dilutions, Plating, Calculations
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
- 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.
- 10 µL of 10-1 was added with 90 µL of phage buffer to create 10-2 dilution which was vortexed.
- 10 µL of 10-2 was added with 90 µL of phage buffer to create 10-3 dilution which was vortexed.
- 10 µL of 10-3 was added with 90 µL of phage buffer to create 10-4 dilution which was vortexed.
- 10 µL of 10-4 was added with 90 µL of phage buffer to create 10-5 dilution which was vortexed.
- Took 50 µL of 10-3 into 0.5 mL of Arthro. Sat for 15 mins to infect.
- Took 50 µL of 10-4 into 0.5 mL of Arthro. Sat for 15 mins to infect.
- Took 50 µL of 10-5 into 0.5 mL of Arthro. Sat for 15 mins to infect.
- Took 8 mL of LB Broth, 10 mL of 2X TA, and 90 µL of 1M CaCl2 into a 50 mL vial.
- Took 4.5 mL of top agar mixture onto control plate. Allowed to solidify for 15 mins.
- 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.
- 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.
- 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.
- Allowed all plates to solidify for 15 mins.
- 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.