Rationale

Try to create a plate that is webbed enough for future flooding.

Detailed Procedures

1. Took 0.5 mL Arthro and added 524 microliters of 10^0 lysate from donor. Sat for 15 mins.
2. Took another 0.5 mL Arthro and added 550 microliters of 10^0 lysate from donor. Sat for 15 mins.
3. Took 6 mL of LB Broth, 7.5 mL 2X TA, and 69 microliters of 1 M CaCl2 into a 50 mL vial.
4. Took 4.5 mL of mixture from step 3 and poured onto a plate labeled as “control.”
5. Took another 4.5 mL of mixture from step 3 and poured into a 15 mL vial labeled as “524 microliters.”
6. Took another 4.5 mL of mixture from step 3 and poured into a different 15 mL vial labeled as “550 microliters.”
7. Took mixture from step 1 and added to 15 mL vial labeled as “524 microliters.”
8. Took mixture from step 2 and added to 15 mL vial labeled as “550 microliters.”
9. Took “524 microliter” vial and poured onto “524 microliters” plate.
10. Took “550 microliter” vial and poured onto “550 microliters” plate.
11. Let all three plate solidify for around 15 mins and inverted into incubator.

Conclusion/Results/Next Steps

Control plate was contaminated again. The plaque assay plate from previous lab day had no phage on the plate, so I had to redo 550 microliters again. Another member who had the same donated lysate also had no phage on plate as well as contamination. There was no problems in making the plates (no bubbles, good aseptic technique, no sliding.) If the plate seems to be webbed enough by next lab day, then flooding will take place. If not, then webbing will continue and will have to re-calculate.