Date: Monday, August 27th, 2018

Title: Spot Test

Rationale: The purpose of today’s lab is to set up a spot test on an agar plate with an ATC 21022 lawn in order to find a bacteriophage.

Procedure:

1. We began by creating an aseptic zone spreading CiDecon over the workplace then letting Ethanol (70%) evaporate off the table, dehydrating and therefore killing any organisms that could contaminate the equipment or samples.
2. We lit a burner in the middle of the table in order to keep falling particles from contaminating the equipment or samples from above since the flame creates a circulating air current.
3. I filtered 2 mL of my enriched isolation using a 22 micrometer filter into a 3 mL microcentrifuge tube.
4. My group (Group 5) then received four agar plates. We each used one individually, then used the fourth plate as a top agar control to make sure our top agar wasn’t contaminated, thus invalidating any potential results.
5. I labelled my plate with my initials (BJR) and divided the plate into three equal sections, labelled E, D, and B for Enriched isolation, Direct isolation, and Phage buffer respectively.
6. We calculated how much of each material we would need for our plates: for my personal plate, I would use .5 mL arthrobacter ATC 21022, 4.5 mL LB Broth, 5 mL x2 top agar, and 45 microliters 1M CaCL2. For Group 5’s top agar control, we would use 4.5 mL LB broth, 5 mL x2 top agar, and 42.5 microliters 1M CaCl2.
7. We used a P200 pipette to transfer 42.5 microliters 1M CaCl2 to a new 50 mL conical vial. This was to be used for Group 5’s top agar control.
8. I used the same P200 pipette to transfer 45 microliters  1M CaCl2 to my own 50 mL conical vial.
9. We used another pipette to transfer 4.5 mL LB broth into the group conical and 4.5 mL LB broth into my personal conical.
10. I then added 5 mL of x2 top agar to my conical.
11. I received .5 mL arthrobacter ATC 21022 to add my conical from the TA Lathan.
12. I transferred the contents of my 50 mL conical to my agar plate then let it sit for  15 minutes. We also added the top agar to the control plate and let it sit for 10 minutes.
13. I used a P10 pipette to drop 10 microliters of my enriched sample to the division of my plate marked “E,” 10 microliters of my direct sample to the division of my plate marked “D,” and 10 microliters of a phage buffer to the division of my plate marked “B.”
14. We placed our plates into a room temperature incubator.

Observations:

• Top agar solidifies quickly, so it’s better to add top agar last. I had added the top agar to my conical before adding arthrobacter, leading rushing and possible sloppy work.

Results:

• This experiment yielded me a spot test set up to evaluate at a later date.

Next Steps:

• My next step is to examine the plate in ~48 hours to see if there is a positive result.