Plaque Assay 8.29.18
Plaque Assay 8.29.18
Rationale)
To conduct a plaque assay using an enriched lysate derived from Soil Sample A in the effort to find and isolate a phage via the presence of a plaque, and if no plaque is formed the plaque assay will determine the necessity of collecting another soil sample to try again.
Results from 8.27.18)
I was able to check my Spot Test from 8.27.18 which based upon careful visual inspection did not have any plaques that had formed in the top agar. Therefore my results were negative, however, there was no evidence of contamination present in the dish. Subsequently, chances are high that another soil sample will have to be collected in order to isolate a lysate to be tested.
Procedures)
1. Setup an aseptic zone using CiDecon, 70% ethanol, and an ethanol flame.
2. Retrieve a 50mL conical vial and label “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay”
3. Retrieve micro test tube labeled “Enriched Lysate for Spot Test NMN 8.27.18”, a red-capped vial of arthrobacter, which is then labeled as “Top Agar for Plaque Assay 8.29.18 NMN”, and a plate with base agar, labeled as “NMN 8.29.18 Plaque Assay”.
4. Add 10μL of “Enriched Lysate for Spot Test NMN 8.27.18” to the red-capped vial labeled “Top Agar for Plaque Assay 8.29.18 NMN” and let the arthrobacter and lysate interact for 13 minutes.
5. Add 8mL of LB Broth to “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” under aseptic conditions using a 10mL serological pipette.
6. Add 90μL of 1M CaCl2 to “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” using a 20-200 micropipette under aseptic conditions.
7. Add 10mL of 2xTop Agar to “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” using a 10ml serological pipette under aseptic conditions.
8. Mix, by swirling for 10 seconds, “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” then using a 1000μL micropipette add about 1mL of “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” to the group 3 section of the Top Agar control plate, put the remaining top agar in the micropipette back into “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” all under aseptic conditions.
9. Distribute 5mL of the remaining top agar in “EAG, SS, NMN 8.29.18 Top Agar for Plaque Assay” into the 3 separate vials for group 3, including “Top Agar for Plaque Assay 8.29.18 NMN” all under aseptic conditions using a 10mL serological pipette.
10. Mix carefully, by swirling for 15 seconds, “Top Agar for Plaque Assay 8.29.18 NMN” then add the vial’s top agar solution, under aseptic conditions, to the plate “NMN 8.29.18 Plaque Assay” under aseptic conditions, shake the plate gently to evenly distribute the top agar across the surface of the plate.
11. Let the plate solidify for 5 minutes then invert the plate and place it in the incubator for a total of 48 hours with the intent to check on Friday for the development of plaques.
Observations)
I observed minor bubbles when pouring the top agar into my plate which could potentially be mistaken to be plaques if care is not taken when looking for them. I also observed that there were string-like structures forming in the top agar as I was mixing it right before I was about to pour it into the plate. Ultimately we perceived little to no error in the performing of these procedures.
Conclusion)
From the negative result from the spot test on 8.27.18 I can conclude that if the plaque assay produces negative results as well another soil sample will need to be acquired. As Soil Sample A will be shown to not contain a phage that can affect arthrobacteria, however, if the plaque assay produces positive results it can be seen that Soil Sample A may contain arthrobacter phage and the spot test or the manner in which it was conducted was ineffective in allowing the formation of plaques.
Next Steps)
The next step in conducting this plaque would be to wait about 48 hours or until Friday to check and see if the plaque assay produces positive or negative results. This would be determined, respectively, by the presence or absence of a plaque in the plaque assay that I conducted. If a plaque was formed then the plaque could be picked and a phage could potentially be isolated from that, however, if the results are negative another soil sample will have to be acquired.
Update from 8.31.18)
As of checking Friday, August 8th, 2018 the plaque assay, “NMN 8.29.18 Plaque Assay”, produced negative results in terms of the formation of plaques, however, the bacteria growth was very grainy in appearance potentially illustrating an issue with the top agar mixing or setting. Either way, based upon the two negative results I have observed in both the previous spot test and this plaque assay it can be concluded that lysate from Soil Sample A did not contain any phage that infected Arthrobacteria. Subsequently, I will have to collect another soil sample, Soil Sample LH, in the hopes of isolating a phage.
