Welcome to the laboratory.
This video is a demonstration
of the standard plate count technique,
also known as serial dilutions,
and a calculation of the colony forming units per mil.
The aim of the standard plate count is
to determine the number of living,
that is viable, cells in a culture.
So, in theory, one bacterial or yeast cell
should give rise to one colony,
and it's the same for fungi.
One spore or one mycelium unit
should be equivalent to one colony forming
on your plate.
So when performing serial dilutions,
you need to use aseptic technique.
So that means you need to make sure
that your bench has been wiped down with disinfectant
and a Bunsen turned on.
You've been provided with five McCartney bottles,
each containing nine mils of diluent.
Diluent is just another word for dilution solution.
So that is the solution
that you're going to perform your serial dilutions in.
On your bench, you should also have a culture
of Micrococcus luteus.
So you can see this McCartney bottle
contains a bacterial culture,
it's cloudy, and Micrococcus is also yellow in color.
So this is our stock solution.
And this is the solution we're going to calculate
the number of viable cells for
by completing the serial dilution.
So we're going to perform ten to the minus one dilution.
So one in ten dilutions,
from ten to the minus one
to ten to the minus five.
So the first thing you should do is label each
of the McCartney bottles with the correct dilution factor.
So to do that, just grab your texter
and label each of the bottles with the dilution factor.
So, ten to the minus one,
ten to the minus two,
ten to the minus three,
ten to the minus four,
and finally,
ten to the minus five.
So if we're performing one in ten dilutions,
that means we need to add 1000 microliters,
or one mil, of the stock culture
to our first tube to collect--
or to create a one in ten dilution.
And then serially dilute that
into the other bottles using one mil.
So you need your pipette,
which measures 1000 microliters,
so make sure it's set to the correct volume.
Make sure that your Bunsen flame is turned
to the blue flame because we do need to work aseptically.
And you also need your blue tips.
So the blue tips go with the pipette
that measures 1000 microliters.
So pop a tip on to your pipette,
and then working aseptically, we need to transfer one mil
of the stock culture to our first bottle
to create the one in ten dilution.
So using the crook of your finger,
loosen the lid of the bottle.
Flame the neck of the bottle,
collect one mil.
Flame the neck of the bottle.
Replace the lid.
Grab your ten to the minus one bottle.
Remove the lid.
Flame the neck of the bottle.
Transfer the one mil of stock culture.
Flame the neck of the bottle and replace the lid.
Once you've done that, you can eject your pipette tip
into the pipette bucket.
Before we do the next dilution,
we need to make sure that our ten
to the minus one dilution is mixed properly.
So to do that, we use the vortex.
So turn the vortex on,
and then hold the bottle in the vortex
until the solution swirls
around for a couple of seconds.
And then it should be thoroughly mixed.
So to perform the ten to the minus two dilution,
we need to grab a new tip on our pipette,
and aseptically transfer one mil
from the ten to the minus one bottle.
So using the crook of your finger,
remove the lid of the bottle,
flame the neck of the bottle.
Collect one mil.
Flame the neck of the bottle.
Replace the lid.
Loosen the lid of the ten to the minus two bottle,
flame the neck of the bottle, transfer the one mil,
flame the neck of the bottle,
replace the lid,
and then eject your pipette tube.
Again, we need to make sure
this solution is mixed properly.
So we use the vortex,
give it a quick mix
for a few seconds,
and then it's ready to perform
the ten to the minus three dilution.
So we collect a fresh tip.
Using the crook of our finger,
remove the lid of the bottle,
flame the neck of the bottle.
Collect one mil.
Flame the neck of the bottle,
replace the lid,
and transfer it by removing the lid
of our ten to the minus three bottle,
flaming the neck of the bottle,
transferring one mil,
flaming the neck of the bottle, and replacing the lid.
Eject your pipette tip.
Give it a mix on the vortex.
And then keep performing
the dilutions
from ten to the three right up to ten to the minus five.
So it's really important when you are performing
a serial dilution
that it is a one-person operation.
So one person in your group
should be serially diluting the solution,
just to ensure that it stays nice and sterile and clean,
and that nothing becomes contaminated,
and you don't get mixed up
with what solution has been transferred
to which tube.
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When you've completed the dilutions,
make sure that you do give your last bottle
the ten to the minus five dilution, a mix as well.
If you have a look at your dilution series,
you can note two things.
You will see that the ten to the minus one dilution
is still quite cloudy.
And then from ten to the minus two onwards,
it's very difficult to see cells suspended in there.
The other thing you might note is that each of the tubes,
ten to the one to ten to the minus four,
contain nine mils of solution
because we were transferring one mil.
And then our final ten to the minus five bottle
contains ten mils of solution.
So it is okay to leave that extra one mil remaining
in the ten to the minus five dilution.
It shouldn't affect your actual spread plate
or the number of colonies that will grow
for the ten to the minus five dilution.
But if you are a perfectionist,
you can remove that one mil.
So what you need to do is,
using a nice clean fresh tip,
aseptically remove one mil
from the ten to the minus five dilution.
So using the crook of your finger,
remove the lid of the bottle,
flame the neck of the bottle,
collect one mil.
Flame the neck of the bottle, replace the lid,
and then inject that tip with the one mil of solution
into your pipette container.
So now...
your serial dilutions are complete.
You will be able to move on to spread plating them.