Viral Plaque Assay


today we’re going to demonstrate the
viral plaque assay we’re gonna begin by talking about how to use the pipettor
when you press the plunger on the pipettor you’ll notice that the pipettor
stops you this is how far you want to press the plunger when you want to bring
liquid into the pipettor when you want to expel liquid from the pipettor
press your thumb past that all the way down and leave your thumb
pressed until after you pull the pipette tip out of the liquid when you want to
get the pipette tip off just press the button by your thumb now we’re gonna demonstrate making
serial dilutions of the t4 phage transfer 0.1ml of the t4 phage
into the tube you labeled 1:100 mix the broth using a sterile transfer
pipette transfer 0.1ml of the 1:100
dilution into the tube you labeled 1: 10,000 mix the broth with a sterile transfer
pipette transfer 1ml of the 1:10,000
dilution into the tube you labeled 1:100,000 mix the broth with the sterile
transfer pipette transfer 1 ml of the broth you labeled
1:100,000 into the broth you labeled 1:1,000,000 mix the broth with a sterile transfer
pipette transfer 1 ml of the broth you labeled
1:1,000,000 into the broth you labeled 1:10,000,000 mix the broth
with a sterile transfer pipette now we’re going to mix the bacteriophage
solutions with E.coli pipette 0.1ml of E.coli into the microcentrifuge tubes that you labeled with the same dilutions using the same pipette tip using a different pipette tip for each
transfer pipette 0.1ml of each of the phage dilutions into the
corresponding microcentrifuge tube of E.coli
so put 0.1ml of 1:100 phage dilution into the tube of
E.coli labeled 1:100 after you’ve pipetted all of the
dilutions into the tubes you’re gonna rotate the tubes in your palms and then
let them incubate at room temperature for five minutes now we’re going to demonstrate plating
the phage E.coli mixture transfer the contents of the phage E.coli mixture
with a sterile transfer pipette into the melted top agar and give it a gentle
mix then pour the contents onto the corresponding luria agar plate quickly
swirl it so that it spreads over the surface of the luria agar plate avoid
producing bubbles because they can affect your results

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