How to create serial dilutions

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.

[no monologue]

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.