October
15
10.15.18 Retry of Plaque Assay
10.15.18 Retry of Plaque Assay
Rationale: Since there was no plaque present on the plaque assay completed on Friday (10/12), but there was contamination on the control plate, it was necessary to retry the test to obtain a sample without contamination. The contamination could have been causing a false negative result, therefore it would be beneficial to ensure that the result was accurate with a repeated test.
Procedure:
- Aseptic zone established.
- 2mL of LB Broth was added to two tubes (control and experimental).
- 10µL of possible phage/phage buffer solution to 0.5mL arthrobacter. Let sit for 15 minutes.
- 22.5µL of CaCl2 was added to both tubes.
- 2.5mL 2X top agar was added to control tube and was plated on control plate (“HMB Plaque Assay Control 10/15/18”).
- 0.5mL arthrobacter/phage buffer/ possible phage solutoin was added to experimental tube
- 2.5mL 2X top agar was added to experimental tube and plated on “HMB Plaque Assay 10/15/18”.
Results:
- The plaque assay from Friday (10/12) showed no sign of plaques or clearings. However, the control plate showed significant signs of contamination. The contamination was in isolated colonies, not lawn growth, but nevertheless it was still very present.
Observations:
- There was a small area of no growth on the control plate. This could have been due to uneven top agar spreading or an alternate source.
- There was a small amount of top agar overlay on the control plate created today that went up the side of the plate due to spreading. Important to note for further examination.
- Both top agar and LB Broth used appeared to be the correct color and clear, signifying that they are free of contamination.
Conclusions and Next Steps:
- Since there was contamination, there could be no official conclusions drawn about the contents of soil sample E. Further conclusions will be able to be made on Wednesday when it is apparent whether or not there are clearings if there is a control plate free of contamination to accompany the plaque assay. The next step is to reanalyze on Wednesday (10/17), and a soil sample will be obtained in the event that the negative result from today is confirmed.