8.29.18- Plaque Assay

Results from Monday: After 48 hours in the incubator, the results from the Spot Test were ready to be analyzed. Upon analysis, the plate had no trace of plaque. The negative control plate properly displayed a bacterial lawn with no disruption, but neither enriched lysate nor direct isolate caused any formation of plaque on the experimental plate. The negative control section of the experimental plate also showed no plaque formation or disruption of bacterial lawn, which was to be expected because any other result would have been indicative of contamination.

Rationale: Plaque Assay test needed to be done to determine whether or not results from the Spot Test on Soil Sample A could be confirmed by presence or lack of a plaque (since the description above shows that Monday’s Spot Test showed negative results for presence of a bacteriophage, a result to confirm this would be a lack of plaque on the Plaque Assay). In conjunction, the Plaque Assay was performed to gain further comprehension of the procedure and how to perform each step.

Procedure:

1. Lab space cleaned with CiDecon (sprayed on surface and wiped until dry) and 70% ethanol (sprayed on surface and wiped until 75% dry).
2. Ethanol burner lit to establish aseptic zone. Each addition to tubes was done in this aseptic zone in order to prevent particles from falling into the samples and causing contamination.
3. (In aseptic zone) Tube with 0.5mL of Arthro was obtained and labelled HMB Arthro 8/29/18.
4. (In aseptic zone) 10μL of lysate added to tube with 0.5mL of Arthro. This mixture was set to the side to give the Arthro and lysate to interact while the rest of the experiment was set up.
5. 50mL Conical tube was obtained.
6. (In aseptic zone) 8mL LB Broth was added to the Conical Tube
7. (In aseptic zone) 90μL CaCl2 added to the Conical Tube
8. Plate with agar was obtained and labelled “HMB Plaque Assay 8/29/18”.
9. (In aseptic zone) 10mL of 2X Top Agar was added to the Conical Tube with LB Broth and CaCl2
10. 2X Top Agar, LB Broth, CaCl2 was mixed in Conical Tube (components of overlay solution) by gently shaking the tube back and forth.
11. (In aseptic zone) 4.5mL of overlay solution was added into tube containing 0.5mL of Arthro and lysate. Mixture was swirled to mix.
12. (In aseptic zone) Overlay solution was poured onto plate labelled “HMB Plaque Assay 8/29/18” and let settle.
13. 1mL of overlay solution from Conical Tube added to Control plate shared by groups 3, 4, 7, and 8. This was done due to the shortage of plates in the lab because of contamination.
14. Overlay on experimental plate was allowed to solidify and was stored in the incubator to be checked on Friday.

Observation:

• Plaque Assay plate had many bubbles and the surface appeared to be slightly bumpy. This could be a point to examine if problems occur.
• Excess overlay solution was observed in Conical Tube. The mathematics seemed to excuse this observation (3 individuals used 4.5mL out of 18mL in tube resulting in 4.5mL leftover. 1 mL was used for the control plate, therefore allowing 3.5mL to be excess in the Conical Tube), but the discrepancy should be reported.
• Overlay solution was a golden yellow, as seen before in the Spot Test.

Results from the Plaque Assay cannot be obtained until plate has had time for bacteria to grow and be influenced by any present phages. Therefore, results from plaque assay will be found on Friday during open lab session.

Conclusions drawn: Based on the results of the Spot Test, it is more than likely that the original sample obtained does not have bacteriophages. The lack of plaque shows that there is no death of cells, which is the trademark of a bacteriophage as they reproduce. However, the Plaque Assay may be able to overturn this conclusion if it does contain plaques.

Next Steps: Control and experimental plate will be observed on Friday to determine whether or not the results of the Spot Test are supported or not. If there are no plaques present, it is confirmed that the sample did not contain bacteriophage and another sample will be needed. If there are plaques present, they will be picked and processed further.

Update: Plate was observed and confirmed the negative result for presence of a plaque or bacteriophage. The bacterial lawn was not formed particularly well (as seen in picture below), but there was still no presence of a plaque. The next steps to take from this result will be obtaining a new Soil Sample that ties in with the overarching research question to aid in developing data to answer the question.