Date: 8/29/2018

Plaque Assay Soil A

Objectives:

• Analyse the results of the spot test from Monday (08/27/2018)
• Make a plaque assay with the enriched lysate of soil sample A that was filtered on Monday

Results from Spot Test:

• No plaques have formed on my Spot test for soil A. According to these results, there are no bacteriophages in my soil sample.
• I have also made a major error. I forgot my filtered enriched sample on the table on Monday and this may affect the results of the plaque test today.
• There was also some moving liquid inside my spot test dish. May be just some of the TA that had no solidifies before incubation.
• 

Adjustments:

Due to a lack of an adequate number of Agar plates, adjustments were made to the experiment for today. Everyone had to do an enriched sample plaque Assay and the following adjustments were made to the measurements of the ingredients required to make the top agar. the top agar was made for the entire group in one conical vial.

2 ml LB broth

2.5 ml 2X  Top Agar

22.5 microliters 1 M CaCl2.

These values were then multiplied by 4 to account for the fact that it was for the entire group. the measurements used for the group:

8 ml LB broth

10 ml 2X Top Agar

90 microliter 1 M Cacl2

5 ml of this mixture for the top agar was poured onto each plate.

Calculations:

conversion factors:

1M= 1000mM

1 ml =1000 microliters

M1V1=M2V2

1M(V1)=(4.5mM)(10ml)

1000mM(V1)=(4.5mM)(10000 microliters)

V1=45 microliters

Materials Required: Filtered Enriched lysate, serological pipettes, micropipettes, micropipette tubes, LB broth, Top Agar 2X, 50 ml conical vials, 15 ml conical vials

Procedure:

1.  First the aseptic zone was set up: pour Cidecon on the desk and wiping it till the desk dry. then pour ethanol and wipe it all over the table and let it evaporate.
2. Light the ethanol lamp to create an air current near which the samples can be opened to prevent things from getting into the samples.
3. Retrieved the LB broth, the same one I used on monday, a 50 ml conical tube and a serological pipette
4. While in the aseptic zone, transfer 8 ml of LB broth to the 50 ml conical vial.
5. now retrive the 1 M CaCl2 stock solution.
6.  Using the micropipette, i transfered 90 microliters of the CaCl2 to the 50 ml conical tube with the LB broth.
7. Set this vial in the rack.
8. retrieve 0.5 ml of arthrobacter.
9. using the micropipette, i transfered 10 microliters of my filtered enriched lysate to the arthrobacter.
10. i let the testtube rest for 10 minutes in its rack.
11. after the 10 minutes are up, add 10 ml of top agar to the conical vial.
12. using a serological pipette, transfer 5 ml of the top agar mixture to the test tube with the arthrobacter and the lysate.
13. My test tube cracked and spilled onto the table. i had to restart the process with the measurements for one sample.
14. i added 10 microliters of the filtered enriched lysate to another 0.5 ml culture of arthrobacter. let it rest for 10 minutes
15. Into a 15 ml conical vial in the aseptic zone, add 2 ml of LB broth, 22.5 microliters of 1M CaCl2 and 2.5 ml of 2X top agar, using the same methods as before.
16. Transfer the contents of the 15 ml conical vial to the test tube of arthrobacter and lysate after the 10 minute rest.
17. pour the contents of the test tube into the agar plate.
18. to let the top agar solidify, i waited for 10 minutes.
19. i put the plate, upside down in the incubator, where it will remain for 48 hours.
20. pour a part of the top agar mixture prepared into the top agar control plate for the 4 lab groups.

Analysis:

there was no event that may have caused contamination to the sample. the aseptic zone was properly maintained. the procedure was properly followed.

Future notes:

take extra care while handling lab equipment to prevent future damage that could lead to more delays. We have also decided on a research question : Does the presence of arthrobacter appear more dominant in the soil of one oak species than the others? Is there a correlation between the presence of Arthrobacterphage and the presence of oak wilt fungus?