WEBVTT

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Welcome to the laboratory.

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This video is a demonstration

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of the standard
plate count technique,

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also known as serial dilutions,

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and a calculation
of the colony forming units per mil.

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The aim
of the standard plate count is

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to determine the number
of living,

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that is viable,
cells in a culture.

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So, in theory,
one bacterial or yeast cell

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should give rise to one colony,

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and it's the same for fungi.

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One spore or one mycelium unit

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should be equivalent
to one colony forming

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on your plate.

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So when performing
serial dilutions,

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you need to use
aseptic technique.

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So that means you need
to make sure

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that your bench has been wiped down
with disinfectant

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and a Bunsen turned on.

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You've been provided
with five McCartney bottles,

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each containing nine mils
of diluent.

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Diluent is just another word
for dilution solution.

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So that is the solution

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that you're going to perform
your serial dilutions in.

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On your bench,
you should also have a culture

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of Micrococcus luteus.

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So you can see
this McCartney bottle

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contains a bacterial culture,

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it's cloudy,
and Micrococcus is also yellow in color.

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So this is our stock solution.

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And this is the solution
we're going to calculate

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the number of viable cells for

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by completing
the serial dilution.

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So we're going to perform ten
to the minus one dilution.

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So one in ten dilutions,

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from ten to the minus one

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to ten to the minus five.

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So the first thing you should do
is label each

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of the McCartney bottles
with the correct dilution factor.

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So to do that,
just grab your texter

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and label each of the bottles
with the dilution factor.

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So, ten to the minus one,

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ten to the minus two,

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ten to the minus three,

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ten to the minus four,

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and finally,

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ten to the minus five.

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So if we're performing one
in ten dilutions,

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that means we need
to add 1000 microliters,

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or one mil, of the stock culture

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to our first tube to collect--

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or to create a one
in ten dilution.

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And then serially dilute that

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into the other bottles
using one mil.

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So you need your pipette,

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which measures
1000 microliters,

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so make sure it's set
to the correct volume.

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Make sure
that your Bunsen flame is turned

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to the blue flame
because we do need to work aseptically.

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And you also need your blue tips.

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So the blue tips go
with the pipette

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that measures 1000 microliters.

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So pop a tip on to your pipette,

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and then working aseptically,
we need to transfer one mil

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of the stock culture
to our first bottle

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to create the one
in ten dilution.

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So using the crook
of your finger,

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loosen the lid of the bottle.

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Flame the neck of the bottle,

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collect one mil.

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Flame the neck of the bottle.

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Replace the lid.

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Grab your ten
to the minus one bottle.

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Remove the lid.

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Flame the neck of the bottle.

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Transfer the one mil
of stock culture.

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Flame the neck of the bottle
and replace the lid.

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Once you've done that,
you can eject your pipette tip

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into the pipette bucket.

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Before we do the next dilution,

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we need to make sure that our ten

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to the minus one dilution
is mixed properly.

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So to do that, we use the vortex.

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So turn the vortex on,

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and then hold the bottle
in the vortex

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until the solution swirls

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around for a couple of seconds.

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And then it should be
thoroughly mixed.

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So to perform the ten
to the minus two dilution,

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we need to grab a new tip
on our pipette,

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and aseptically transfer one mil

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from the ten
to the minus one bottle.

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So using the crook
of your finger,

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remove the lid of the bottle,

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flame the neck of the bottle.

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Collect one mil.

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Flame the neck of the bottle.

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Replace the lid.

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Loosen the lid of the ten
to the minus two bottle,

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flame the neck of the bottle,
transfer the one mil,

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flame the neck of the bottle,

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replace the lid,

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and then eject your pipette tube.

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Again, we need to make sure

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this solution is mixed properly.

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So we use the vortex,

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give it a quick mix

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for a few seconds,

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and then it's ready to perform

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the ten
to the minus three dilution.

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So we collect a fresh tip.

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Using the crook of our finger,

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remove the lid of the bottle,

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flame the neck of the bottle.

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Collect one mil.

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Flame the neck of the bottle,

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replace the lid,

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and transfer it
by removing the lid

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of our ten
to the minus three bottle,

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flaming the neck of the bottle,

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transferring one mil,

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flaming the neck of the bottle,
and replacing the lid.

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Eject your pipette tip.

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Give it a mix on the vortex.

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And then keep performing

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the dilutions

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from ten to the three
right up to ten to the minus five.

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So it's really important
when you are performing

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a serial dilution

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that it is
a one-person operation.

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So one person in your group

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should be
serially diluting the solution,

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just to ensure that it stays nice
and sterile and clean,

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and that nothing
becomes contaminated,

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and you don't get mixed up

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with what solution
has been transferred

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to which tube.

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[no monologue]

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When you've completed
the dilutions,

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make sure
that you do give your last bottle

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the ten to the minus five dilution,
a mix as well.

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If you have a look
at your dilution series,

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you can note two things.

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You will see that the ten
to the minus one dilution

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is still quite cloudy.

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And then from ten
to the minus two onwards,

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it's very difficult
to see cells suspended in there.

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The other thing you might note is
that each of the tubes,

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ten to the one
to ten to the minus four,

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contain nine mils of solution

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because we were
transferring one mil.

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And then our final ten
to the minus five bottle

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contains ten mils of solution.

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So it is okay to leave
that extra one mil remaining

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in the ten
to the minus five dilution.

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It shouldn't affect
your actual spread plate

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or the number
of colonies that will grow

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for the ten
to the minus five dilution.

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But if you are a perfectionist,

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you can remove that one mil.

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So what you need to do is,

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using a nice clean fresh tip,

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aseptically remove one mil

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from the ten
to the minus five dilution.

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So using the crook
of your finger,

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remove the lid of the bottle,

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flame the neck of the bottle,

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collect one mil.

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Flame the neck of the bottle,
replace the lid,

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and then inject that tip
with the one mil of solution

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into your pipette container.

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So now...

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your serial dilutions
are complete.

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You will be able to move on
to spread plating them.