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Welcome to the laboratory.

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This video is a demonstration

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of the standard[br]plate count technique,

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also known as serial dilutions,

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and a calculation[br]of the colony forming units per mil.

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The aim[br]of the standard plate count is

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to determine the number[br]of living,

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that is viable,[br]cells in a culture.

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So, in theory,[br]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[br]to one colony forming

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on your plate.

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So when performing[br]serial dilutions,

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you need to use[br]aseptic technique.

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So that means you need[br]to make sure

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that your bench has been wiped down[br]with disinfectant

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and a Bunsen turned on.

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You've been provided[br]with five McCartney bottles,

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each containing nine mils[br]of diluent.

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Diluent is just another word[br]for dilution solution.

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So that is the solution

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

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On your bench,[br]you should also have a culture

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of Micrococcus luteus.

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So you can see[br]this McCartney bottle

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contains a bacterial culture,

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it's cloudy,[br]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[br]we're going to calculate

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the number of viable cells for

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by completing[br]the serial dilution.

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So we're going to perform ten[br]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[br]is label each

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of the McCartney bottles[br]with the correct dilution factor.

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So to do that,[br]just grab your texter

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and label each of the bottles[br]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[br]in ten dilutions,

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that means we need[br]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[br]in ten dilution.

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And then serially dilute that

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into the other bottles[br]using one mil.

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So you need your pipette,

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which measures[br]1000 microliters,

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

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Make sure[br]that your Bunsen flame is turned

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to the blue flame[br]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[br]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,[br]we need to transfer one mil

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of the stock culture[br]to our first bottle

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to create the one[br]in ten dilution.

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So using the crook[br]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[br]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[br]of stock culture.

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Flame the neck of the bottle[br]and replace the lid.

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Once you've done that,[br]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[br]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[br]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[br]thoroughly mixed.

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So to perform the ten[br]to the minus two dilution,

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we need to grab a new tip[br]on our pipette,

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and aseptically transfer one mil

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from the ten[br]to the minus one bottle.

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So using the crook[br]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[br]to the minus two bottle,

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flame the neck of the bottle,[br]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[br]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[br]by removing the lid

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of our ten[br]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,[br]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[br]right up to ten to the minus five.

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So it's really important[br]when you are performing

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a serial dilution

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that it is[br]a one-person operation.

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So one person in your group

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should be[br]serially diluting the solution,

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just to ensure that it stays nice[br]and sterile and clean,

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and that nothing[br]becomes contaminated,

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and you don't get mixed up

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with what solution[br]has been transferred

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to which tube.

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[no monologue]

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When you've completed[br]the dilutions,

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make sure[br]that you do give your last bottle

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

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If you have a look[br]at your dilution series,

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you can note two things.

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You will see that the ten[br]to the minus one dilution

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is still quite cloudy.

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And then from ten[br]to the minus two onwards,

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

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

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ten to the one[br]to ten to the minus four,

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contain nine mils of solution

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because we were[br]transferring one mil.

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And then our final ten[br]to the minus five bottle

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contains ten mils of solution.

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So it is okay to leave[br]that extra one mil remaining

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in the ten[br]to the minus five dilution.

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It shouldn't affect[br]your actual spread plate

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or the number[br]of colonies that will grow

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for the ten[br]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[br]to the minus five dilution.

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So using the crook[br]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,[br]replace the lid,

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and then inject that tip[br]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[br]are complete.

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You will be able to move on[br]to spread plating them.