9.14.18 Spot Test Results and Plaque Assay

Rationale: Since the Spot Test returned negative results (indicating that the soil sample did not possess any bacteriophage), it was necessary to confirm the results by performing a Plaque Assay that would ensure that the results of the Spot Test were not due to an anomaly or a mistake.

Results from Wednesday (9.12.18):

• After observing the plate containing results from the Spot Test performed on Wednesday 9/12, it was clear that there was no presence of phage in the sample. There was perfect lawn growth with no interruption in the negative control or in any of the experimental sections. This is visualized in the image located below.

Procedure:

1. Lab surface was cleaned with Cidecon and 70% Ethanol by spraying on surface and then wiping dry. An ethanol burner was lit to establish an aseptic zone that was used for each step of the following procedure except for the steps where materials were obtained.
2. Obtained Filter Sterilized Enriched Lysate (labeled “HMB Filtered”) that was created on 9/12 from the fridge.
3. Obtained 0.5mL of Arthrobacter in tube.
4. Added 10µL of Filter Sterilized Enriched Lysate to tube containing 0.5mL Arthrobacter. Let sit for 15 minutes and allow Filter Sterilized Enriched Lysate to mix with Arthrobacter.
5. Obtained 50mL conical tube. Labeled “HMB Plaque Assay Top Agar 9.14.18”. Obtained separate 50mL conical tube for control labeled “NMN HMB 9.14.18 Control TA for Plaque Assay”
6. 2mL of LB Broth was added to both conical tubes.
7. 22.5µL of CaCl2 was added to both tubes.
8. 2 plates obtained. One labeled “HMB Plaque Assay 9.14.18” and the other was labelled “NMN HMB Control Top Agar for Plaque Assay Test 9.14.18”
9. 0.5mL Arthrobacter and 10µL Filter Sterilized Enriched Lysate added to tube labeled “HMB Plaque Assay Test 9.14.18”
10. Added 2.5mL of 2X Top Agar to both tubes, then swished to mix. Promptly poured on the respective plates, spread to all areas, then allowed to sit for 15 minutes.
11. After waiting 15 minutes, the plates were placed into the incubator to be examined on Monday (9/17).
12. Lab surface cleaned with Cidecon and 70% ethanol.

Observations

• Some particles appeared to be in Top Agar overlay that could have been caused by already-hardened agar. Their effect will be determined if the results are adverse on Monday (9/17).

No data was obtained from this experiment, as it will all be collected on Monday and updated in that entry.

Next Steps: If the results are negative yet again on Monday (9/17), it will be necessary to obtain new soil samples and possibly reevaluate the mini-question that our group examined. We will also converse with Group four about their results, as they also were examining the same mini-question as us. However, if the plaque assay reveals a positive result, then the plaque will need to be picked and held for later examination while further studying/sampling may need to occur to allow for trends in observations to be observed.

Conclusions: Based on the fact that Group four all had plaques and that Group 3 (Henry, Nathan, and Shepard) did not, there may be relevance or significance to the scientific question posed by table 2. This would need further data collection and repetition to be supported, but it would be a positive step in determining a question that the entire class could ponder and research.