Micropipette
What are they and why do we use them? What types are there?
I got about half-way before I realised it requires a central metal tube and I am still yet to source one…
The micropipette is the tool of the trade, a device specialised in handling small volumes of liquid. Pipettes come in a range of brands and qualities, but the important distinction you care about is the tolerance. This number on the pipette tells you the minimum and maximum volumes that the pipette can handle. The number tells you the maximum volume in microlitres (µl), and the minimum tolerance is 10% of that value. I would recommend a lab has at least one of each of the following pipettes;
P2: 0.2-2 µl
P20: 2-20 µl
P100: 10-100 µl
P200: 20-200 µl
P1000: 100-1000 µl
Every single one of these pipettes also come with their appropriately sized pipette tips. Pipette tips sometimes work with a couple of sizes of pipette, but this may result in some fluctuation in accuracy.
Pipette tips should be sterilised in an autoclavable box by autoclaving. The micropipette is not sterile, only the tip.
A micropipette can be quite expensive, but keep in mind that there are cheap brands available for <US$40. There is also the option of printing your own micropipette according to the specifications of this paper; Open Design 3D-Printable Adjustable Micropipette that Meets the ISO Standard for Accuracy.
When do you use?
Any time you wish to transfer a volume of liquid less than 1 ml. You will use the micropipette every day in every way. The following protocols rely heavily on skilled usage of the micropipette;
How do you use?
A micropipette should be used with sterilised tips. You can purchase pre-sterilised tip boxes, but the most self-sufficient labs use reloadable tip boxes with an autoclave. Reloading a tip box is a meditative/mind-numbing process.
Assuming you have nice sterile tips, your next concern should be selecting the correct pipette and setting it to the correct value. Select a pipette that will work for your intended volume, the most accurate pipette will have your volume in the centre of it’s tolerance spectrum;
P2: 0.2-2 µl
P20: 2-20 µl
P100: 10-100 µl
P200: 20-200 µl
P1000: 100-1000 µl
E.g. If you need to pipette 100 µl, you can use a P100, P200 or P1000, but the P200 will be the most accurate.
Once you’ve selected the correct pipette, you need to set it to the correct value. This is done by turning the plunger, or rotating it by the central barrel. Depending on the size of the pipette, it can be confusing at first as to which number means what because some use a decimal place while others do not. The easiest way to check is to turn the pipette all the way to the largest number, which should match the P-number (e.g. On a P100, it should stop turning just after 100 µl). You now know whether the numbers on your pipette include a decimal place (often marked in red if it’s at the bottom of the number) and can work backwards to determine how to set it to any number within the range. After a while you’ll instinctively know what the numbers on a pipette mean, but it’s very common to be confused early on since there really are logical inconsistencies in the system.
Once the pipette is set to the correct value, you need to know how to use it! There are 1-2 depressible buttons on your average pipette;
Some pipettes have a ‘tip ejector’ button, which shunts off your pipette tip into the plastic waste between each movement. A single sharp press is all you need to use this button and it is not strictly necessary since you can just pull the tip off with your gloved hands. This is simply a quality of life button.
The important button that is always there is the central button, the plunger which pushes the air in and out of the pipette tip, creating a vacuum of a specific volume (equal to the number you just set!) when released.
This button can be pushed down to the soft-point (1st stop), which can be physically felt in your thumb as you depress it. At the soft-point you can push down further but it feels more difficult to do so. The volume of liquid picked up by the pipette at the soft-point will be equal to the number on the side.
This button can also be pushed further to the hard-point (2nd stop), at this point you will be physically unable to push down any further and you will pull in a larger volume than the number on the side.
The soft-point is used for picking up liquids, while the hard-point is used for ejecting them. This is necessary because surface tension will hold a small quantity of liquid in the barrel of the pipette unless you can give it a little extra shove.
The process for moving a liquid is:
Add a new sterile tip by pushing the pipette down onto the tip in the box.
Depress the plunger to the soft-point with your thumb.
Dip the end of the sterile tip into the liquid you wish to pick up.
Slowly release your thumb, lifting up the desired volume of liquid. If you notice it bounce/jump or any bubbles get in, depress your thumb back to the soft-point and try again more slowly.
Lift the tip out of the liquid and move the pipette to the vessel you wish to add it to.
If any drips out, your pipette isn’t holding a vacuum and needs repair or more appropriately sized tips.
Dip the tip into the new liquid and press all the way down to the hard-point. Keeping your thumb depressed, remove the tip from the liquid. Release your thumb once it is clear.
Eject the tip into plastic waste and prepare to repeat with a new tip.
You can reuse tips in certain situations, but only if you don’t mind contaminating both the source and destination of the liquid.
It is possible to service a leaky or broken pipette. The most common cause of problems is the O-ring. Replacing this single part can double or triple the lifetime of some pipettes. If you can’t afford to buy your own, consider repairing old pipettes thrown out by a local university or donated from overseas. There are loads of faulty pipettes floating around if you know where to look.
Image credit: https://en.wikipedia.org/wiki/Air_displacement_pipette