28 November 2018 ✷ Pick a Different Plaque

Rationale: Members of group 6 adopted phage from Soil 4 from Melissa (collected 10/5/18) in order to help amplify the phage sample. A plaque was picked from Melissa’s original plate because the prior plates run by members of group 6 came up negative.

Procedure

• The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
• Plaque “Y” on the plate from the prior lab session was picked and swirled into 50 µL of phage buffer. 30 µL of this mixture was added to 0.5 mL of arthrobacter and allowed to sit for 15 minutes while the plates were made.
• A plate for 2 plaque assays and a control plate were made with the following concentrations and volumes:

• component	volume	final concentration
  LB Broth	6 mL	–
  2X Top Agar	7.5 mL	1X
  1M Calcium Chloride	68 µL	4.5 µM

• The arthro/lysate mix was combined with 5 mL of the above plate mix and the plate was poured, allowed to harden, and then inverted and incubated at 37 degrees celsius. The remaining lysate was refrigerated.

Observation, Results, Data

The originally positive plaque assay was picked and plated but ended up with negative plaque assays that had weak spots that mimicked plaques, but weren’t actually plaques.

 The spots, when looked at under the microscope, weren’t circular nor clear, as true plaques should be. Thus, when Melissa’s initial plate was looked at again under the microscope, the plaques present were more easily identifiable and were able to be picked and plated.

Interpretations, Conclusion, Next Steps

In the following lab periods, the presence of a webbed plate will mean that the lysate is a high titer and can be studied more closely using TEM. The current titer is very low, which may indicate a weaker phage or phage presence.