10/3/18 The 50 μl Plaque Assay

Objective:

The goal of this procedure is to do anything possible to try to find a plauqe to confirm that I do in fact have phage. This is necessary as my re-do of passage 2 yielded no plaques, and my spot test did not yield great results. This caused me to wonder whether or not phage were present and I decided doing a final assay with a lot more lysate would help me answer the question.

The overarching question this test seeks to address is: Is the presence of phage determined by species of oak tree from which soil was collected?

In other words, are specific oak tree species more likely to have Arthrobacter bacteria phages in the soil surrounding them?

The question specific to my lab table is: Is the difference in the presence of phage between live oaks and red oaks on Baylor’s campus?

As a group, we hope to expand our question to include more species as we gather data so that we can better address our overarching question and we will look at our metadata to examine whether or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for an Aseptic zone:

• CiDecon
• 70% Ethanol
• Ethanol Burner

Materials for Plaque Assay:

• .5 ml Arthrobacter
• incubator
• Pipette
• Test tube stand
• 50 ml tubes
• Culture tube
• LB Broth
• 2X TA
• 1M Calcium Chloride
• Agar plate
• Serological pipette

In order to complete the procedure, an aseptic zone was created.

1. CiDecon was applied to the lab table with a squeeze bottle and wiped away with a paper towel
2. 70% Ethanol was also applied with a squeeze bottle, spread with a paper towel, and allow to evaporate
3. An ethanol burner was light in order to use the rising heat from the flame to form the aseptic zone
   

Then a plaque assay on the solution in the tube labeled P1 (refred to as PA1 in previous lab and the rest of this lab) was performed

1. Four agar plates were labeled. An agar plate was labeled with initials, date, and description for each group member, and one agar plate was labeled with data and “TA control”
2. The remaining PA1 lysate, stored in a microcentrifuge tube, was gathered (like the tube in the picture below)
3. 50 µL of the remaining PA1 lysate was aseptically transferred into a culture tube containing .5 ml of Arthrobacter using a Serological pipette
4. The culture tube was capped and set aside for 15 minutes. This process was repeated twice more using different lysates for each group member).

While the lysate and bacteria are allowed to sit in the culture tube the agar was prepared.

1. The agar was prepared according to the following recipe (makes four plates):
2. Under aseptic conditions, 8.0 ml of LB broth was transferred into a 50 ml tube.
3. Under aseptic conditions, 90 µL of 1 M CaCl2 was transferred into the same 50 ml tube.
4. Under aseptic conditions,  10.o ml of 2X TA was transferred into the same 50 ml tube
5. The mixture was pipetted several times to mix it (table set up seen below)
6. 4.5 ml of the contents in the 50 ml tube was transferred to the plate labeled “TA control”
7. The plate was swirled and set aside
8. 4.5 ml of the contents in the 50 ml tube was transferred into the culture tube containing lysate and bacteria
9. The mixture was pipetted several times to mix it
10. Then the mixture was poured from the culture tube into the agar plate labeled with initials, date, and description
11. The plate was swirled and then set aside for 10 minutes to allow agar to solidify. This procedure was repeated twice more, once for each group member.
12. The agar failed to solidify after 20 minutes so the plates were placed in the incubator, but not inverted.
13. Plates were left to incubate until nest class

Results:

The results of this lab will be crucial in light of the results of the 9/26 and 10/1 labs. The 9/26 procedure yielded a passage two plaque assay with no plaques and necessitated the 10/1 procedure, which yielded only one possible plaque. While it is impossible to know the results of this procedure immediately, I will assert that if I do not have plaque, I do not have phage. *Note: it is my goal to update this lab before the 5:00 Friday submission deadline, if that does not happen I will update the blog and not the submission on Monday. Becuase I do not have the results on hand right now I will write the next two sections without them and change them if I can.*

Analysis:

The odd results of the 9/26 and 10/1 procedures suggest that there is something potentially odd happening with my plates or that I just don’t have phage (a possibility that I find likely). Of the three possible explanations suggested in the last lab report, all three are all still possible. If it turns out that I do not have phage then it is likely that I have been picking air bubbles or somehow managed to kill my phage. In ether instance, this means I will need to be extremely careful in my next soil enrichment attempt and the following testing. In addition, if my group members don’t have phage, I might try to collect soil from an area that is less sandy with the hopes that the soil is more conducive to phage growth. I will need to change how I am conducting my procedures and what I am testing simply because I cannot do the same thing over and over again and expect different results.

Future:

My next steps will depend on the results of the plaque assay. If I have phage then I will pick the most promising one and passage it (in what will be its second passage) and I will spot test it and three or four other promising plaques just to ensure that they are in fact phage. If I do not have phage I will make an enriched lysate from my soil sample number three and probably collect a forth soil sample.