Enrichment Day 2 - Plaque Assay
Name: Estefany Gutierrez
Date: 1/30/24 - 2/1/24
Lab Session (A or B): A and B
Experiment: Enrichment Day 2 - Plaque Assay
Purpose: Get new phages
Results from previous experiment: I centrifuged my soil sample from the last lab, and I got the phage lysate. I did it 2 times because the first one didn't work, so I did the same thing on Tuesday and Thursday.
Protocol
1. Obtain the phage lysate that you would like to test with a plaque assay.
2. Use sterile technique to transfer 250µl of M. smeg bacteria into a sterile glass test tube for each plaque assay. Label the culture tubes with a sharpie. Add the proper amount of phage lysate to the bacteria based on your experiment. In general, if your lysate is from a direct isolation, you should add 400-500µL, and if your sample is from an enrichment or a serial dilution, you should add 10µL.
3. Let the tube sit undisturbed for 5 minutes.
4. Label the L-agar plate(s) properly for your experiment. Include your initials, lab section, and experiment information.
Note: when plating your samples (steps 5 and 6), it is best to work quickly and plate samples one at a time to avoid the top agar solidifying in the culture tube.
5. After the 5 minutes, get your top agar bottle from the water bath and use a 5mL pipette and sterile technique to transfer 3mL of warm top agar to each glass test tube containing bacteria and phage. Tap the bottom of the test tube with your fingers to mix.
6. Gently pour the top agar/bacteria mixture onto the labeled L-agar plate. Swirl the plate to spread the agar over the surface of the plate. Make sure the agar completely covers the bottom of the plate.
7. Allow the top agar to solidify (approximately 10 minutes).
8. After the top agar has hardened, invert the plate and place it in a 37°C incubator for 48 hours. If the top agar does not harden after 10 minutes, carefully put the plate in the incubator, but do not invert the plate. Discard all phage lysate tubes (direct isolation or serial dilutions) in the milk cartons on top of the benches; these samples cannot be used again.
Lab Notes
Labeled: 1-30-24, EG, section 11, Plaque assay
Future Directions: Get a new conical tube, label it with 8 spaces and a negative sign to make the spot test.