10/1/18 Spot Test of Five Lysates and Plaque Picking

Objective:

The goal of this procedure is to use lysates from previous passages and old plates to perform a spot test to test for phage presence. This is necessary as my re-do of passage 2 yielded no plaques. This caused me to wonder whether or not phage were present and I decided a spot test would be my best option.

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 a 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 weather 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 Spot test

• .5 ml Arthrobacter
• refrigerator
• Pipette
• Test tube stand
• 50 ml tubes
• LB Broth
• 2X TA
• 1M Calcium Chloride
• Pipette cap
• Phage Buffer
• Agar plate
• Micropipette
• Syringe Filter

Materials for Phage Picking:

• Agar plates with plaques of interest
• Micropipette tip
• Phage buffer
• Microcentrifuge tubes (incorrectly referred to as pipette caps in previous entries)

*Note: I had to erase my phone on Tuesday and I lost all photos from this lab before I could upload them, as a result, the images shown below are either taken after the fact or are examples from previous labs*

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 phages were picked

1. 100 µL of phage buffer was transferred into a microcentrifuge tube labeled with initials, date and the description ST1
2. 100 µL of phage buffer was transferred into a microcentrifuge tube labeled with initials, date and the description ST2
3. 100 µL of phage buffer was transferred into a microcentrifuge tube labeled with initials, date and the description ST3 *Note: this image was taken before the spot tests were labeled* 
   

   

4. A pipette tip was used to stab the center of the chosen plaques on each plate  *Note: My hands shake and it is possible I contaminated my pipette tip with the surrounding agar when I tried to stab my plaque* 
5. The (hopefully) phage-infected tip was swirled in the phage buffer and then the solution was vortexed and set aside.
6. This was repeated for ST2 and ST3

Then the spot test could be performed.

1. The bottom of an Agar plate was divided into six sections and label PA1 for passage one, PA2 for passage 2, ST1 for spot test one, ST2 for spot test two, ST3 for spot test three, and PB for phage buffer
2. A separate Agar plate for a top agar (TA) control was created, labeled and set aside
3. All of the previously created lysates were gathered
4. Agar was prepared according to the following recipe: 
5. 4.0 ml of LB broth was added to a 50 ml tube
6. 45.0 μl 1M CaCl2 was added
7. 5.0 ml of 2X Top Agar was added and pipetted to mix
8. 4.5 ml of the mixture was pipetted into the agar plate labeled TA control, swirled, and set aside for 10 minutes
9. Then the remaining mixture in the 50 ml tube (~4.3 ml) was pipetted into a culture tube and pipetted to mix
10. The contents of the culture tube were poured into the labeled agar plate, swirled, and set aside for 10 minutes
11. Once agar had solidified a Micropipette to pipette was used to transfer 10 μl of each lysate onto the section of the plate that corresponded to the correct label
12. This was repeated with 10 μl of phage buffer
13. The plates were set aside for 15 minutes before being placed in the incubator until next class

Results:

*Note: When I had to erase my phone I also lost my photos of the results of the 9/26 lab. I will be referencing these results here, but as of right now I do not have photos and the plates weren’t saved because they had negative results. If I am able to recover the photos I will update the labs appropriately.*

The results of this lab will be crucial in light of the results of the 9/26 lab. That procedure yielded a passage two plaque assay with no plaques and necessitated this procedure (possible reasons for no plaques will be discussed in the analysis section). While it is impossible to know the results of this procedure immediately, it is reasonable to assert that if all spots are negative then I do not have phage.

Update:

These results are not the most promising. There is one potential plaque in the section that was created using passage one lysate, and this will be confirmed with further testing (it is hard to see the potential plaque on the image below because of the glare and because it is close to the sharpie line). Based on these results I do not feel comfortable asserting that there is phage present definitively, but I cannot rule out phage presence ether. The results require further testing. I am also not sure if the TA control is contaimnated of if the agar just solidifed in an odd way.

Analysis:

Based on the results of this procedure and the 9/26 procedure, there are several possible explanations. The first possibility that would explain my results is that I have no phage and I’ve been picking air bubbles which looked a lot like plaque; however, this explanation seems unlikely as my other two group members appear to have phage, and we collected soil from the same tree. The second possibility is that my phage have all died somehow, which would explain why I suddenly stopped seeing plaques form when they seemed to form before. The third possibility is that I did pick an actual, phage containing plaque when I prepared they lysate for passage one, and I picked an air bubble instead of a plaque when I was picking for passage two. I will be unable to decide which of these possibilities makes the most sense until I complete further testing on the one potential plaque that appeared on my spot test, but once I do I will be able to figure out what likley happened.

Future:

The next step I have to take is to test my passage one lysate with a spot test. If I find plaque on this spot test I will pick one and passage it with the hopes of puriying it, if I do not then I will preform and enrichment on my third soil sample and start over.