9/24/18 Passage #2 of Enriched Lysate from Soil Sample #2

Objective:

The goal of this procedure is to passage our phage a second time as part of the phage purification process. Last lab we picked a potential plaque, preformed dilutions and the did multiple assays. This could be called the “first passage” (out of three). This lab we will pick the most promising plaque and do another assay (passage two). By passaging our phage we seek to isolate and purify 1 specific strand of phage. Once we do this (following the process found in the image below), we can move on to experimenting with our specific phage strain.

We are also seeking to avoid contamination even further, as can be seen in the analysis section of this report our previous TA control was contaminated.

We are also seeking to address the following questions every lab:

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 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

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)

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 phage was picked *Note: Each group member picked the most promising plaque from their respective plates for a total of 3 picked plaques*

1. 100 µL of phage buffer was transferred into a microcentrifuge tube labeled with initials, date and the description “P2” (P2 meaning passage 2)
2. Four plaques were deemed to be promising, one was selected for a plaque assay, and the other three were selected for later spot test (image below) 
3. A pipette tip was used to stab the center of the chosen plaque on each plate (the chosen plaque is indicated my the red arrow on the image below) *Note: My hands shake and it is possible I contaminated my pipette tip with the surrounding agar when I tried to stab my plaque; however, it does not appear that I contaminated my last picked plaque so I will assume I did not contaminate this one ether* 
4. The phage-infected tip was swirled in the phage buffer and then the solution was vortexed and set aside.

Then a plaque assay on solution in the tube labeled “P2” was preformed

1. Four agar plates were gathered and set aside.
2. The agar was prepared according to the following recipe (makes four plates):
3. Under aseptic conditions, 8.o ml of LB broth was transfered into a 50 ml tube.
4. Under aseptic conditions, 90 µL of 1 M CaCl2 was transferred into the same 50 ml tube.
5. 10 µL of the P2 lysate was aseptically transferred into a culture tube containing .5 ml of Arthrobacter using a Serological pipette
6. The culture tube was capped and set aside for 15 minutes. This process was repeated twice more (once for each group member). *Note: this was a mistake from the usual process because we forgot to put the lysate into the culture tubes before we started prepping the agar, so after the first two steps of agar prep we had to stop and wait the 15 minute incubation period*

The lysate and bacteria were allowed to sit in the culture tube for 15 minutes

1. Under aseptic conditions,  5.o ml of 2X TA was transferred into the same 50 ml tube
2. The mixture was pipetted several times to mix it
3. Three agar plates were labeled with initials, date, and “P2” while a forth was labeled with the date and description “TA control”
4. 4.5 ml of the contents in the 50 ml tube was transferred to the plate labeled “TA control”
5. The plate was swirled and set aside
6. 4.5 ml of the contents in the 50 ml tube was transferred into the culture tube containing lysate and bacteria
7. The mixture was pipetted several times to mix it
8. This process was repeated twice more, once for each group memeber
9. Then the mixture was poured from the culture tube into the agar plate labeled with initials, date, and description
10. 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. *Note: when swirling Aman’s plate some of the liquid agar spilled out of his plate, potentially disrupting his results* 
11. Once the labeled plaque assay had solidified, the plate was inverted and placed in the incubator
12. Plates were left to incubate until nest class

Results:

The results of the plaque assay on the P2 lysate will be recorder here when available. It is reasonable to assume though, that there will be plaques as there have been plaques in the two previous assays.

Update:

There were no identifiable plaques on our plates on Wednesday (see plate below), but after our TA’s ran a control plate they discovered that during this lab we did not plate with artho. This makes all of our results from this lab invalid/unusable, and it explains why there were no plaques to be found. We will infer (and confirm with later testing) that there is still viable phage in our P2 tube, and as we continue to passage, it should be one strain and only one strain.

Analysis:

The results of this lab are invalid because we did not use arthro to plate our phage; however, there are still several valuable things that can be inferred from this. The first thing that this lab demonstrated is that there are a lot of ways for things to become contaminated. The image above shows the control plate from the lab conducted on 9/19/18. The plaques from that assay were picked to run the procedures detailed above, so it is important to understand forms of contamination. In this case we knew that we did not contaminate our sample and that our broth was not contaminated because we took extra precautions on 9/19. Based on how the contaminated appear to spread, we inferred that the contamination came from the plate itself, teaching us to more fully examine our agar plates before using them. In addition, based on the mistake with the arthro, we learned that mistakes can happen in every section of the scientific process and that we should always be careful.

Future:

Had our procedure gone to plan, we would have preformed passage #3 on Wednesday, but because we didn’t plate with arthro, we will redo our passage #2 on Wednesday, following roughly the same procedure, being careful to make the same mistakes.