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.

Scientific Question:

Does the presence of Arthrobacter phage vary in oak species? Is there one species where they are more dominant?

Rationale:

In today’s lab, the goal was to successfully perform a plaque assay assessment to test the presence of phage in soil samples. Although the results of the spot test yielded negative results for the presence of phage, plaque assays have the ability to yield different results.

Materials Used: 

• 10 μL Lysate
• PY Broth
• PY 2X TA
• 40% Dextrose
• 1M Calcium Chloride
• 0.5 mL of Arthrobacter host
• Agar plate
• Pipettes (Micropipettes and serological pipettes)

Procedure: 

1. I began this procedure by setting up an aseptic zone on my workbench.
2. Next, I gathered the necessary materials to create the top agar. Each group member grabbed their own plate for their plaque assay. I labeled mine with a sharpie and began to make the top agar.
3. Because the top agar made was supposed to be split 4 ways, the measurements for the top agar solution varied slightly from the spot test. For this procedure the top agar consisted of:
   • 8 mL of LB Broth
   • 10 mL of PY 2x Top Agar
   • 90 μL of Calcium Chloride
4. I added the 8 mL of LB broth into our 50 mL conical tube by using the serological pipette.
5. After the LB Broth was added, I used a 20-200 μl micropipette to transfer 90 μL of the calcium chloride to the conical tube.
6. Then I took the filtered lysate from the spot test procedure in the micro centrifuge tube and I used a 1-10 μL micropipette to transfer 10μL of the lysate to the vial containing the 0.5 mL of arthrobacter. I allowed this mixture to sit for approximately 15 minutes to let any possible phage present infect the bacterial hosts.
7. After 15 minutes had past, 5 mL of the top agar was extracted from the tube to add to the arthrobacter solution, the vial was mixed gently, and then plated immediately onto my plaque and let it sit for 15 minutes.
8. Unfortunately, while transferring the top agar to my partner’s vial, we noticed the vial had broken and was leaking my partner’s solution all over the table, so before advancing any further, an aseptic zone was reestablished. We then continued with the transfer of top agar to the remaining vials.
9. We then took the leftover agar and added it to the control plate, leaving the agar to solidify and add to the incubator for 48 hours.

Observations/Results/Data:

• The top agar this time looked very similar to the top agar created for the spot test. There was no contamination of the top agar as everything was performed aseptically.
• The color of the top agar stayed relatively dark yellow, very clear once poured and solidified.

Conclusions/Next Steps:

• The top agar has yet to be examined, but if the results for phage are positive there will be empty lawns throughout the plate where the phage has killed its bacterial host.
• If no spots are examined, then the soil sample will be confirmed negative for phage and I will have to collect more soil samples from different plots on campus.

Rationale: Today’s goal was to correctly perform a spot test to determine the presence of phage in the soil samples that we had extracted in the week prior to this one. Hopefully, if done correctly with no contamination, spots will appear in our plaque after 48 hours of incubation.

Materials:

• Lysate
• PY Broth
• PY 2X TA
• 40% Dextrose
• 1M Calcium Chloride
• 0.5 mL of Arthrobacter host
• Agar plate
• Pipettes (Micropipettes and serological pipettes)

Procedure:

1. This procedure started with establishing an aseptic zone to decontaminate the work bench. The bench was sprayed and wiped down with CiDecon, then followed up with 70% Ethanol, which was spread on the table allowed to evaporate. Following this, I grabbed our burner to finish setting up the aseptic zone.
2. Next, I gathered the materials necessary to create the top agar that is needed for the spot test. I grabbed 4 plates (one for each group member and one for control), 2 serological pipette tips,a 50 mL conical tube, and I also set out four micro centrifuge tubes which will be used later in the lab. I then marked my plate with a sharpie to show where I would spot my direct isolation, my enriched isolation, and my phage buffer.
3. Next, I began to create the top agar, I measured out 4.5 milliliters of LB broth to add to the conical tube using a serological pipette. My pipette was having issues, and could not go past 4 milliliters of liquid, so I had to use the serological pipette for the 4 milliliters, then I had to use a micropipette to gather 500 micro-liters to add to my broth to equal 4.5 milliliters to add to my 50 mL conical tube.
4. After the LB broth was added, I needed to add calcium chloride to our top agar. Initially, the quantity of calcium chloride needed was unknown, but what was known was that we needed the molarity of calcium chloride to equate to 4.5 mM or millimolar. To solve for the volume needed, there is a simple equation that I used to get a final value of 45 microliters of calcium chloride
5. 
6. Following the calculation, the calcium chloride was added to our broth mixture using a 20-200 microliter pipette.
7. Instead of adding arthrobacter next, I filtered my enrichment from the previous lab through a 22 micron filter by syringe. I pulled out approximately 2 milliliters and pushed it gently through the filter into a micro-centrifuge tube.
8. After the filtration process, 0.5 ml of arthrobacter was added to the broth mixture.
9. Followed up immediately by pipetting 5.0 milliliters of PY 2x Top Agar with serological pipette and allowed the top agar to mix.
10. Then I very quickly plated my top agar and the group’s control plate, swirled the plate around, and waited 15 minutes for the plate to solidify.
11. After 15 minutes, I added 10 microliters of my direct, enriched, and phage buffer to their designated spots and put the tray to incubate for approximately 45 hours.
12. After 45 hours I removed the tray to analyze my results.

Observations/Results/Data:

• After examining the plate, it was concluded that there was no phage present in the lysate that I had extracted from my soil sample. The plate looked the same as it did when it was put in the incubator, no spotting whatsoever. This indicates a lack of phage as the arthobacter had not been cleared from the plate. There was no contamination as the agar looked exactly like the control.
• Plate had the same color, but had a liquid forming on the top. Unsure what exactly caused that. 

Interpretations/Conclusions:

• The results of the spot test indicate that there is no phage present in the soil sample that I collected the week before. This can be deduced by the lack of any clearing or any indication of bacterial death on the plate. If phage were to be present, there should be minor bacterial clearing in any part of the plate.

Next Steps:

• There may still be hope for the soil sample. The next step is to perform a plaque assay to view the results of that experiment, as plaque assays and spot test have the ability to yield different results. The plaque assay will be performed the next time I am in lab, and if the results are negative that means I have to collect more soil samples to test for phage.