8/31/18 Plaque Assay of Enriched Lysate Attempt Two

Objective:

The goal of this procedure is to determine weather or not bacteria phages are present in the soil collected last week. Based on the results of the spot test it is possible that there are phages, but the previous plaque test was contaminated (see below) so now this assay will be conducted to recheck.

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.

Procedures and Protocols:

Materials:

• CiDecon
• 70% Ethanol
• Ethanol Burner
• .5 ml Arthrobacter
• incubator
• Pipette
• Test tube stand
• 50 ml tubes
• 15 ml tube
• LB Broth
• 2X TA
• 1M Calcium Chloride
• Agar plate
• Serological pipette

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

1. Clean off the work space (lab table) with CiDecon applied with a squeeze bottle and wiped away with a paper towel
2. Apply 70% Ethanol with a squeeze bottle, spread with a paper towel, and allow to evaporate
3. Light an ethanol burner in order to use the rising heat from the flame to form the aseptic zone

Then the plaque assay on the enriched lysate was preformed.

1. Label two agar plates. Label one with initials, date, and description “TA control”
2. Label a second agar plate with initials, date, and description “Enriched Lysate Assay (HB)”
3. Gather the remaining enriched lysate from last procedure (found in the pipette cap seen below)
4. Using a Serological pipette, aseptically transfer 10 µL of the remaining enriched lysate into a 15 ml tube containing .5 ml of Arthrobacter
5. Reseal tube and recap pipette cap
6. Allow the lysate and bacteria solution to sit for 15 minutes while the agar is prepared

While the lysate and bacteria are allowed to sit in the culture tube prepare the agar

1. Prepare the agar according to the following recipe (makes two plates):
2. Under aseptic conditions, pipette 5.o ml of LB broth into a 50 ml tube. Cap the tube. *Note: This was a mistake that will be addressed later in the procedure*
3. Under aseptic conditions, pipette 45 µL of 1 M CaCl2 into the same 50 ml tube. Cap the tube.
4. Discard 50 ml tube with LB broth and CaCl2 because the measurements were wrong
5. Under aseptic conditions, pipette 4.o ml of LB broth into a 50 ml tube. Cap the tube.
6. Under aseptic conditions, pipette 45 µL of 1 M CaCl2 into the same 50 ml tube. Cap the tube.
7. Under aseptic conditions, pipette 5.o ml of 2X TA into the same 50 ml tube
8. Pipette the mixture several times to mix it

When agar preparations are finished the bacteria and lysate have been allowed to sit for 15 minutes

1. Pipette 4.5 ml of the contents in the 50 ml tube into the lysate and bacteria 15 ml tube
2. Pipette the mixture several times to mix it
3. Pour the mixture from the 15 ml tube into the agar plate labeled with initials, date, and description “Enriched Lysate Assay (HB)”
4. Cap the plate and allow the plaque assay agar to solidify for 10 minutes
5. Aseptically Pipette 5 ml of the contents in the 50 ml tube onto the control TA plate
6. Allow the TA control plate to sit for about 10 minutes before being placed into the incubator
7. Once the labeled plaque assay has solidified, invert the plate and place it in the incubator
8. Leave to incubate until next class (~94 hours)

Results:

The results of this procedure will not be immediately clear until Wednesday’s normal lab, but once they are available they will be included here.

Update: The top agar appears to have not solidified all the way because there is liquid in the plate. There do not appear to be any plaques in the areas where that agar remained intact suggesting a negative assay.

Analysis:

Because the Agar did not stay solidified it is hard to analyse these results with certainty; however, because there did not appear to be plaques on the parts of the agar that remained solid it is likely that the assay was negative. This suggests that there were not phages in the soil collected, or that there are not enough to cause results.

Future:

Due to the two unsuccessful assays, I will be collecting soil next lab in an attempt to find viable phage for isolation and purification.