8/27/18 Spot Test

Objective:

The goal of this procedure is to determine weather or not bacteria phages were present in the soil collected last week. This will be determined by using small amounts of lysate on auger plates to look for plaques that would indicate phage particles. By performing this test

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
• Micropipette

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 spot test could be preformed.

1. Divide the bottom of an Agar plate into three sections and label them E for enriched isolation, D for direct isolation, and B for phage buffer *note that this was a collaborative effort to some degree so three agar plates were created*
2. Create a separate Agar plate for a top agar (TA) control, label and set aside
3. Gather previously created enriched isolation and direct isolation lysate
4. Use a syringe to aseptically draw 3 ml of the enriched lysate out of the 50 ml tube
5. Reseal tube
6. Attach a filter to end of syringe and gently push 1.5 ml of lysate through the filter and into a pipette tip
7. Cap the tip and set aside
8. Set aside two 50 ml tubes and label them as TA control and Arthrobacter
9. In the Arthrobacter tube add 13.ml of LB broth *The Arthrobacter tube was originally overfilled so some of the LB broth was pipette into the TA control tube*
10. In the TA control tube add 4.5 ml of LB broth *Note that while adding LB broth to the TA tube the broth was spilled and the tube contents had to be emptied and new broth added*
11. Set aside
12. Add 42.75 μl Calcium Chloride and 5.0 ml of 2X TA to the TA control tube
    1. *The amount of calcium chloride to be added was determined based on the following calculations:
13. Mix the contents of the TA control by shaking the tube vigorously *note that a pipette should have been used*
14. Pipette 10 ml of the contents in the TA control tube into the agar plate labeled TA control
15. cap plate and set aside for agar to solidify
16. In the tube labeled Arthrobacter add 135 μl Calcium Chloride, 1.5 ml Arthrobacter, and 15.0 ml of 2X TA
17. Use a pipette to mix solution by pulling it into the pipette and then expelling it several times
18. Pipette 10 ml of solution from the Arthrobacter tube into each of the Agar plates
19. On the Agar plate pour the remaining contents of the tube onto the plate because slightly less than 10 ml are left
20. Allow Agar to solidify
21. Using a Micropipette pipette 10 μl of filtered enriched lysate onto the section of plate labeled E, then pipette 10 μl of direct isolation lysate onto the section of plate labeled D, finally pipette 10 μl of phage buffer onto the section of plate labeled B
22. Allow the plate to sit for about 10 minutes before being placed into the incubator
23. Leave to incubate until next class (approximately 46 hours)

Results:

The results of this procedure will not be immediately clear until Wednesday’s lab, but once they are available they will be included here. Based on visible observations from lab today, the results of this procedure were four agar plates that appear to have been created correctly.

Update: There are visible plaques!!

Analysis:

Based on the Wednesday update, it can be inferred that there are bacteria phage present in my enriched and direct lysates. I will confirm this will a plaque update.

Future:

My future actions will heavily depend on the results of these spot tests. Regardless of weather or not plaques form though I will be doing plaque assay. So my next lab will detail that.