09/17/2018

Plaque Assay and Soil Collection

Pre- lab Observations:

The spot test made for sample soil B on 09/12/18 was retrieved from the incubator and analyzed. The plate had no plaques. The Top Agar control plate did have peculiar features that seemed to indicate contamination, which was not reflected in the spot test plates. According to the Teaching Assistant, they seemed to be arthrobacter, which may have entered the plate due to contact of the bacteria with an instrument used during the procedure.

Objectives:

• For verification, make a plaque assay from the filtered enriched lysate to test the presence of arthrobacter
• Collect soil sample C

Procedure:

1. Cidecon was poured on the desk and wiped till the desk was dry. Then, 70% ethanol was poured and wiped until it was all over the table and then it was allowed to evaporate. After the ethanol had evaporated, an ethanol lamp was lit, setting up the aseptic zone.
2. The group decided to make 1 top agar for the group.
3. The LB broth was retrieved from its storage bath, along with a 50 ml conical tube and a serological pipette
4. While in the aseptic zone, 8 ml of LB broth was transferred to the 50 ml conical vial.
5. Then, 1 M CaCl2 stock solution was retrieved from the lab instructor.
6.  Using the micropipette, 90 microliters of the CaCl2  was transferred to the 50 ml conical tube with the LB broth.
7. The vial was then set on the rack.
8. 0.5 ml of arthrobacter was retrieved from the lab instructor ( 0.5 ml for each group member)
9. Using the micropipette, 10 microliters of filtered enriched lysate was transferred to the arthrobacter vial.
10. The vial was then allowed to rest on the test tube rack for 10 minutes
11. After the 10 minutes had ended, 10 ml of 2X TA was added to the conical vial.
12. Using another serological pipette, 4.5 ml of the top agar mixture was transferred to the test tube with the arthrobacter and the lysate.
13. The contents of the tube were then poured onto the agar.plate
14. a part of the top agar mixture was into the top agar control plate.
15. To let the top agar solidify, the plate was allowed to rest for 10 minutes.
16. The plates were placed up side down in the incubator, where they will remain for 48 hours.

Soil Collection

1. Due to the low probability of finding plaques, a new soil was acquired in case a new lysate was to be extracted for testing for phages again
2. The lab group went into the field ( went outside ) to find a live oak tree.
3. The group picked a live oak and metadata was collected as the survey required.
4. First, numerous pictures of the tree were taken.
5. The diameter of the tree trunk was measured 137 cm from the ground.
6. Average diameter of the canopy was measured by taking the average of the longest and shortest diameter of the tree canopy.
7. Using the known height of an individual, the length of the individuals shadow and trigonometry, the angle of elevation of the sun was calculated, which was later used with the length of the shadow of the tree and trigonometry to find the height of the tree.
8. A soil sample was collected a certain distance from the tree trunk, one sample per group member, and the sample was put into a Ziploc bag. A leaf sample was also collected.
9. Store the sample in the fridge.

Conclusion and Interpretation:

The analysis of the control plates indicates that there was contamination. It indicates that more caution is required to maintain the aseptic zone and the methods used at the time of this procedure require more care and attention. There is also a chance that the LB broth or Top Agar. Currently it is not clear which is more probable.

Other Notes:

1. Group 4 had plaques on their Plaque Assays. They group also did  spot tests in addition to plaque assays, but only one group member (Justin) had plaques on the spot test whereas the entire group had plaques on their plaque assays. The same group member also had the most well defined plaque on the plaque assay. One possible reason could be that the soil sample collected by Justin was more concentrated with bacteriophages than the samples of the rest of the group, yielding him the best results.
2. Calculations for Lathan’s (lab instructor) webbing plate

no. of plaques= 14

dilution of sample= 10^-3

titer of sample = (14/10 μl)x(1000ul/1μl)(10^3)= 1.4×10^6 pfu/ml

Diameter of plate= 8 cm

Diameter of the plaque= 1mm

Area plate=πr^2=6400π

Area plaque=πr^2=π

area plate/ area plaque=6400π/π=6400

volume of lysate required= 6400/1.4×10^6=4.57×10^-3