>> Hi, everyone, and welcome to the Penguin Prof Channel.
Today, we're going to be looking at part
of the lab survival series, and I'm going to be talking
about the subtle art of pipetting.
You understand the basics of pipetting already,
if you've ever used, you guessed it, a straw.
Basically, the idea is very simple.
You create a pressure gradient, and it pulls
or pushes volume through the straw.
Now, don't get me wrong, we are not going to be using straws
in any kind of science lab.
We will not be pipetting by mouth.
But actually, just so you know, that was done back in the day.
We're talking a long time ago.
And, as you might imagine, some very unpleasant things happened
as a result of mouth pipetting.
Today, we use a variety of tools to cause the suction
that forces the fluids to move.
We use roller pumps and little squeezy balls.
I'm going to show you the roller pumps here in a little second.
And then, attached to these pumps, we have glass
or plastic pipettes, and they're going to be marked incrementally
so that you can measure volumes with fairly good accuracy.
This is a pretty typical one.
This is a 10 mL pipette, and as you can see, this is going
to tell you what the maximum volume is that you can dispense,
and it tells you what the increments are.
And, this means total delivered volume, and it is calibrated
at 20 degrees Celsius.
So, each numbered increment, so between, for example,
the 4 and the 3, this is 1 mL.
And then, each line increment, these little lines,
that's a tenth of a milliliter.
So, you really have to read these,
so that you understand exactly what you're pipetting,
and you know what lines you're shooting for.
One other example, this is a 1 mL pipette.
Each numbered increment is a tenth of a milliliter,
so between the 4 and the 3, here,
this is one-tenth of a milliliter.
And, each little line now represents one-hundredth
of a milliliter.
So, as I said, you need to pay attention
and read what's printed on that pipette,
so that you know exactly what you're getting.
When you actually use it, you want to make sure
that you measure from the bottom of the meniscus, right?
So, here's your little meniscus, and the volume is going
to be read from the base of this.
So, this would be 2.5 mL.
So, pipetting can take a little bit of practice, but you want
to make sure that you insert the pipette vertically
into your sample, and use a very smooth motion with your pump
to draw your fluid up the pipette.
And, wherever you have the bottom of the meniscus,
that's going to be the correct volume,
as you look into the pipette there.
Now, you can move it anywhere you like, and when you're ready
to dispense, you simply move the pump, either with your thumb
like this, or just go ahead and depress the plunger,
and eject your sample.
So, isn't that exciting?
What about really small volumes, though?
In physiology, as well as a lot of other fields,
we require very small volumes to be transferred
with fairly high accuracy.
The units that we use are often in microliters,
and microliters are one-millionth of a liter,
so 1000 microliters is equivalent to 1 mL,
and what we're going to use for that,
we have a variety of tools.
We have fixed-volume pipettors, and in most of your classes,
you will use these at some time or another.
But, the magic is really the adjustable-volume pipettors,
and specifically one that looks something like this.
I know, it is extremely magical.
These are your new best friends.
The Pipette Man, as it is sometimes called.
There is a variety of companies that make them,
and all the different tips, depending upon the size
of the micropipettor that you are using.
These are the parts of the beast.
This is actually a fairly expensive piece of equipment,
so please take some time and practice with it.
I always have my students practice with water
until they get the feel of it,
because it is sort of an acquired skill.
But, you can see the different parts,
and how you hold it in your hand.
And, at the very bottom, we have the disposable tip.
That's this part down here, and this is the only part
that will actually come in contact with your sample.
It really is a fairly sophisticated mechanism
for doing the same thing as a straw.
We've come a long way from the straw,
but basically what you're doing is displacing air,
which moves liquid, very much like a straw.
But, of course, with a very high degree of accuracy.
The hardest part of using a micropipettor is really knowing
which pipettor to use, and what the volume is
that you're going to dial in.
So, there are a variety of tools, a variety of models,
rather, that will help you
to dispense the volumes that you want.
But, you've got to pay attention to what you're using,
and how you dial them in.
So, this gives you the adjustable range of each
of the models, the recommended range.
In other words, this is the column that shows,
with high accuracy, what you can actually pipette.
And then, the smallest increment, here,
that you can actually adjust to.
Now, I know this is confusing, but you might want
to spend some time looking at this, and pressing pause,
so that you understand what this actually means.
You're going to see a little window with three numbers in it,
and the different models
of micropipettors will be dispensing different volumes,
depending upon what shows up in the window.
So, for example, if you're using a P10, and you dial in 075,
what you actually get is 7.5 microliters.
If you dial in the same numbers, 075, on a P100,
you get 75 microliters.
So, I really do encourage you to spend a lot of time,
I give my students a little handout
on what the different models are,
and what the dispensed volumes will be.
It does take a while.
Eventually, you're going to get to a point
where you don't even think about it, but in the beginning,
this can be really confusing, if you're doing an experiment
or a lab which requires you
to use multiple models all at one time.
To use the micropipettor, first of all,
you've got to pick the right one.
You've got to dial in the correct volume,
and then you've got to firmly place your tip
on the end of the pipettor.
And, you're going to press the plunger down,
that's a tongue-twister, there, to the first stop.
And then, you're going to hold it vertically,
and immerse it a couple millimeters into your fluid.
Don't go all the way to the bottom.
You're going to slowly release the plunger.
Do not allow it to snap.
So, you want to do it in a controlled fashion.
Snapping will change the volume
that you're actually going to get.
It will reduce your accuracy dramatically.
Then, you're going to withdraw the tip.
You're going to place the tip against the wall
of the receiving container, and you're going
to depress the plunger down.
You're going to feel a stop.
Keep going.
You're going to feel some resistance, and that's going
to actually push any little droplets that might be there
out of the tip, and with the plunger depressed, go ahead
and remove it from the tube, and then dispose of it
by pushing the ejector button.
And, here's a little video of that.
So, here I've got my P1000 micropipettor, and now I'm going
to make sure that the volume is set to what I want.
So, I'm going to dial in my desired volume.
And, you can see here that this is going to dispense a volume
of 350 microliters, as I've set it right now.
Next, you're going to get yourself a tip.
Make sure you're using the right tip for your micropipettor.
I'm going to depress the plunger, and then I'm going
to take my sample, and then slowly release the plunger,
and that's going to pull up my 350 microliters.
Next, I'm going to take the tube that I'm going
to be dispensing into.
I'm going to press, I'm going to insert the tip,
I'm going to press down all the way to the first stop,
and then to the second stop.
That's going to give a little bit more of a push,
you can see the bubbles there, to release any extra droplet.
And, there you have it.
As always, I hope this was helpful.
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Good luck.