8/29/18 Plaque Assay of Enriched Lysate

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 likely that there are phages present (see spot test results below). In this procedure a plaque assay on the enriched lysate will be conducted to confirm the results of the spot test.

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?

Procedures and Protocols:

Materials:

• CiDecon
• 70% Ethanol
• Ethanol Burner
• .5 ml Arthrobacter
• refrigerator
• Pipette
• Test tube stand
• 50 ml tubes
• 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. Divide the bottom of an Agar plate into four sections and label them Groups 1, 2, 5, and 6 in order to create a TA control. Set aside.
2. Label a second agar plate with initials, date, and description (plaque assay will be preformed in this plate).
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 culture 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 *Note:this was a collaborative effort so the agar was prepared for three full agar plates and a control:

1. Prepare the agar according to the following recipe (use 4 plate recipe):
2. Under aseptic conditions, pipette 8.o ml of LB broth into a 50 ml tube. Cap the tube.
3. Under aseptic conditions, pipette 90 µL of 1 M CaCl2 into the same 50 ml tube. Cap the tube.
4. Under aseptic conditions, pipette 10.o ml of 2X TA into the same 50 ml tube
5. 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 culture tube
2. Pipette the mixture several times to mix it *Note: in the process of pipetting the mixture and air bubble caused some of the mixture to spill out of the culture tube, potentially upsetting the ratios in place*
3. Pour the mixture in the culture tube into the agar plate labeled with initials, date, and description
4. Cap the plate and allow the plaque assay agar to solidify for 10 minutes
5. Aseptically Pipette 1 ml of the contents in the 50 ml tube onto the control TA plate in the section labeled group 6
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

Results:

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

Update: The plates appear to be inconclusive based on cloudy appearance and due to a lack of positive or negative controls, one cannot make any assertions about phage presence.

Analysis:

It is hard to analyse the results of this lab because the results themselves are unclear. However, it can be concluded that when the agar plates are redone thicker agar lawns should be used and i would be beneficial to include positive and negative controls as a means of comparison. It is also possible that samples were contaminated, but it is hard to confirm this.

Future:

Due to the inconclusive nature of the plaque assays, I will need to redo my plaque assay likely with positive and negative controls in order to confirm the results of my spot test.