Plaque Assay Procedures
DO NOT PUBLISH WITHOUT PERMISSION OF AUTHORS
K. Eric Wommack
Prepare your media AHEAD OF TIME. Use the protocol for making media specific to your bugs. You will need to have your media ready to go in these flavors:
Broth
Agar plates
Top agar
Plating Bacterial Hosts
1. Bacteria are stored in 15% glycerol at –80 C. Locate the stocks you will need (e.g., CB908, E. coli host for phage T4).
2. Have plates of the appropriate media prepared, equilibrated at room temperature, labeled with the date, media type, your initials, and the bacteria it is to receive.
3. Have a cooler ready with dry ice. Place your frozen bacteria stocks in the dry ice immediately upon removing from the –80 freezer.
4. Using aseptic technique, spread bacteria over each plate with a loop. (See separate instructions on streaking plates using aseptic technique.)
5. Allow bacterial colonies to grow at least overnight, until distinct colonies can be picked.
Growing Overnight Host Cultures in Broth (do this in the afternoon)
1. Label a sterile 50 ml tube with the date, media type, your initials, and the bacteria it is to receive.
2. Aseptically transfer about 10 ml of broth into a sterile 50 ml tube. (Do this in a laminar flow hood to help keep things sterile.)
3. Using a sterile loop, touch a colony of bacteria on the agar plate.
4. Insert the loop into the broth, being careful not to insert past the circular end of the loop.
5. Carefully swirl the loop around and then remove it from the tube.
6. Flame the loop and replace the tube cap.
7. Incubate the tube at the appropriate temperature for your bugs overnight on a rotary shaker.
Growing Log-Phase Cultures (do this first thing in the morning)
1. Aseptically transfer about 10 ml of broth into a sterile 50 ml tube. (Do this in a laminar flow hood to help keep things sterile.)
2. Using a P200 pipetter with a sterile 200 ul pipette tip, transfer 100ul of the overnight culture into the fresh media. Make sure the tube is labeled!
3. Incubate the tube at the appropriate temp for about 2-8 hours (depending on host). Keep an eye on the turbidity, you want the cultures at early log phase.
Plaque Assay
Have ready:
Phage stocks
Phage dilution buffer (SM buffer or PBS are fine)
Log-phase host culture
Agar media plates
Top agar
50 C water bath
5 ml pipettes and pipetter
200 ul pipette tips and P200
Sterile Disposable slip-cap tubes (size?)
Set up dilutions of phage stocks (if necessary) by pipeting 2.7 ml of sterile dilution buffer into tubes. Add 0.3 ml phage stock to 1st tube and make serial dilutions as needed.
Set up and label tubes and plates for each dilution of phage X host combination. For example, if you are plating a 10 –2 dilution of T4, label one sterile tube and one agar plate as “E. coli X T4 10 -2.” If you are making replicate plates, label accordingly (e.g., “E.coli X T4 10 –2 R1”).
Pipette 100ul of host into the appropriately labeled slip-cap tubes. Add 100ul of the appropriate dilution of phage.
Add 4-5 ml of molten (50 C) top agar to ONE or TWO tubes at a time. Vortex briefly and gently pour over the appropriately labeled agar plate. Try to avoid pouring bubbles onto the plates. Carefully tilt and rotate the plates to spread the molten agar over the entire surface of the plate.
Allow plates to set up for about 10 min. Then incubate, inverted, overnight at the appropriate temp.
TIPS:
1. When vortexing, the agar will go up as high as your fingers, so hold the tube with thumb and forefinger about 3/4 way up. Do not hold tubes by the lids while vortexing – the agar will slosh out!
2. Tilt and rotate plates to spread agar immediately after pouring. The agar sets up fast, so you will not be able to pour several plates and then go back to spread. Pour, then spread, then move to the next tube
