Question on Board:

Lathan checked a purified lysate by doing a plaque assay with 10-μl of lysate at a 10^-3. He counted 14 plaques. How many μl of Lathan’s 10^0 lysate should he add to web a plate (75-mm in diameter) if his average plaque diameter is 1-mm?

Answer: 4.0178-μl

Rationale:

Today’s rationale was to analyze the plaque assay performed the week prior for the presence of phage. If there were any plaques, the next step would have been to create a serial dilution using the lysate. If there were no plaques, a second plaque assay was to be performed and new soil was to be gathered as well.

Materials:

• Serological Pipette
• Micropipette
• 2.0 mL LB Broth
• 2.5 mL 2X Top Agar
• 0.5 mL Arthrobacter
• 22.5 μl Calcium Chloride
• 50 mL Conical Vial
• Lysate

Procedure for Analyzing Plaque Assay and Spot Test:

• Plates were removed from the incubator to analyze for spots.
• Plate was held up to the light to check for any spots and plaques were empty with nothing to signify the presence of phage.
• After plate was looked at, the control plaque was examined and contamination was present in the plate. After determining possible causes for contamination, a second plaque assay was begun for certainty.

Procedure for Plaque Assay:

• An aseptic zone was established with CiDecon, 70% Ethanol, and a burner.
• Initially began with creating a top agar for the whole group. Added 8-mL of LB broth and 90 μl of calcium chloride to the conical vial before realizing the concentration of the calcium chloride would be incorrect in the plates. It was decided to do a top agar independently with original measurements.
• 2.0-mL of LB broth was added to a new 50-mL conical vials. Repeated for the control group as well.
• 22.5-μl of calcium chloride was added to the LB broth by micropipette in both vials.
• 0.5-mL of Arthobacter was then introduced to 10-μl of the lysate created for the previous plaque assay and left in a micro centrifuge tube for 15 minutes to infect.
• After the 15 minutes had passed, 2.5-mL of the 2x TA solution was added to the 50-ml conical vials.
• Immediately after the 2x TA was added, the lysate and broth were both mixed together in the 50-mL conical vial, swirled, and immediately poured onto the plate to solidify. Control agar was plated as well
• After waiting 15 minutes, plates were left in the incubator for 48 hours.

Procedure for Soil Gathering:

• A white oak (species unknown, possibly Burr Oak) was found near the Baylor Sciences Building.
• Soil was extracted with a scoop by digging  and placed into a plastic bag.
• Stored in a cold environment for the enrichment process during the next lab.

Data:

• Plaque assay from previous lab was clearly contaminated. It was unsure if the contaminant was Arthobacter or external contamination from failure to aseptically perform procedures.
• Plate was empty with no signs of phage present in neither spot test or plaque assay.
• Tree seemed very healthy with no clear signs of disease. Soil was a very dark color with moderate amounts of moisture in it.
• 

  Control Group

  

  Plaque Assay

  

  Spot Test

Analysis/Conclusion:

• The same LB broth and 2x TA were used from the last plaque assay and spot test to determine if they were the source of the contamination. If the plaque assay result comes out contaminated again, they will be examined for contamination.
• Very high possibility that soil may just be negative for phage. This could be due to the fact that the soil that the tree was planted in is gardeners soil and not naturally occurring. The differences of minerals present in the soil could very well influence the presence of phage in the soil.

Next Steps:

• The next steps are to enrich the new soil and examine the plaque assay for any contamination and presence of phage. Each member gathered soil from 3 different white oaks, so each sample will be tested for any phage concentrations present in the soil.