9.17.18 Obtaining and Washing Soil Sample C

Questions posed by Lathan:

1. The reason why Justin’s plate contained phage in the spot test and the plaque assay and the rest of group four only displayed plaque in the plaque assay was due to the titer of their solutions. Justin’s likely had the highest titer, or concentration, of phage in the plated sample, which would have allowed the spot to clear a plaque on the lawn growth. A lower titer would not be able to display a plaque in the same manner, which would have explained why the rest of group four had the results they did.
2. 4.018μL

Rationale: Since previous soil samples (Soil Sample A and Soil Sample B) did not contain a plaque that contained phage, it became necessary to obtain a third sample and prepare it (by washing the soil and collecting metadata) for testing that will occur on Wednesday in the form of a Spot Test and Plaque Assay.

Data from Friday, September 14

The Plaque Assay performed on September 14 needed at least 48 hours to develop results, which were first available to be observed during this lab session. As predicted by the Spot Test, there were no plaques on the plate containing lysate from soil sample B. However, the negative control exhibited slight presence of small Arthrobacter colonies of unknown origin. These colonies could be due to an error in preparation, or they could be from general contamination in the incubator or otherwise. Furthermore, there are uneven areas present on the experimental plate that cause imperfections in the lawn growth. These breaks are likely caused by uneven top agar spreading rather than phage, so they have no bearing on result. Pictures below illustrate these occurrences.

Procedure: (Sections in italics are metadata related)

1. Cleaned lab bench. Lit ethanol burner to create aseptic zone.
2. Observed plates with results and observations listed above.
3. Obtained materials for soil collection and obtained soil from an Earle Hall tree. Placed soil in bag labeled “HMB Soil Sample C Earle Hall”. Conical Tube labeled “HMB Earle Hall Soil Sample C 9/17/18”.
4. 2mL of soil was added to Conical Tube.
5. 12mL LB Broth added to Conical Tube.
6. Mixture of LB Broth and soil was shaken for 15 minutes. During the 15 minute period, the mass was found to be 20.795g. To match Nathan’s sample for centrifuging, water was added to the tube to make it 21.028g.
7. Sample was centrifuged for 10 minutes to pellet soil and waste.
8. Metadata: Weigh boat was massed and found to be 2.43g. 4g of soil was added to weigh boat. After, the weigh boat labeled with a “2” and distinguished by a crack in the plastic was placed under the hood to be reexamined on Wednesday. 
9. 4mL of soil was added to a 50mL Falcon Tube.
10. 8mL of DI water was added to 50mL Falcon Tube.
11. 3 drops of Soil Dispersion Solution were added to Falcon Tube. Tube was shaken for 30 seconds to mix soil. Soil let sit until Wednesday.
12. Syringe and filter used to add 10mL of lysate to 50mL conical tube and about 1.5mL of lysate into microcentrifuge tube as a direct lysate (which was labeled “HMB D 9/17/18” and placed into the fridge for storage).
13. 0.5mL of Arthrobacter was added to 50mL conical tube to create enriched lysate. Labeled “HMB Enriched 9/17/18”.
14. Enriched lysate was stored in shaker to be recovered in 48 hours.
15. Lab station cleaned and materials were returned to places they were found.

Observations

• During the metadata section, the soil seemed to separate more evenly than the first analyzed sample.
• As filter was used more during the filtration step, it became more and more difficult to push the lysate through the syringe. However, the tension never let up, which confirmed that the filter was at no point broken or dysfunctional.

Next Steps: On Wednesday, the enriched lysate will be used to perform both a Spot Test and a Plaque Assay. This will allow the sample to be read on Friday, which will reveal whether or not Soil Sample C had a viable phage that was causing plaques. Furthermore, if the testing process returns with a negative result, a new sample will be able to be collected more efficiently than if the washing process was put off until Wednesday.

Conclusions:
Since the second soil sample did not have a phage, it was necessary to repeat the process to obtain a third. Hopefully, this sample will contain phage and new procedures learned regarding picking the plaques and creating a webbed plate can be practiced.