Micro-course in Micropipettes
I’m sure you’ve seen this - a photo of someone in a lab coat holding a pipette; it’s basically become a universal symbol for “scientist.” Of course, not all scientists in all fields use pipettes, but they are some of the most common pieces of equipment in a wet lab. In general, pipettes are used to transfer liquids, here we will outline the basics of using a micropipette. Micropipettes are appropriately named as they are generally used for volumes in the microliter range - think ~1000 µL (aka 1 mL) to ~1.0 µL. Depending on the lab that you are in or that you are looking to join, you might use them often or just occasionally. Either way, it is a great idea to familiarize yourself with how they work and practice your skills. You never know when the ability to use a micropipette will come in handy!
First off, most people don’t say micropipette every time they refer to one, you can just say pipette, which is what we’ve done for most of this post. Second, the figure below shows a standard pipette with the key parts labeled for your reference. With this in mind, here are a few basic steps for getting to know your pipettes!
Find the right pipette for the volume you need. Pipettes are calibrated to a specific volume range. Commonly there are p2, p10, p200 and p1000 pipettes. The number associated with them is the maximum volume it will pipette. A common rule for pipette volume is that a pipette can go down to 10% of its maximum volume. For example, a p2 pipette can be used for 0.2 µL to 2 µL and a p1000 pipette can be used for 100 µL to 1000 µL. However, this is not always the case and if this rule does not apply then the volume range that the pipette is calibrated for should be written on the pipette.
Reading the pipette volume. Once you have determined the exact volume you need to pipette, you need to set the pipette to that volume appropriately. This can be a little tricky to get used to, so be patient with yourself. The first rule to remember is not to set the pipette below or above its volume range. This can cause damage to the pipette and make it less accurate. You set the volume by twisting the volume adjustment knob. Typically, pipettes have vertical numbers of one side that indicate the volume to which the pipette has been set. The top number is the decimal place of the maximum volume. For example, on a p1000 the top number is the thousands place, the number below that is the hundreds place and the third number is the ones place, with the tick marks on the very bottom being the first decimal place. On a p200 the top number is the hundreds place, and so on. See the figure below for example volume setting for four common sizes of pipettes.
Fit the right pipette tip on the pipette. Unlike some other types of pipettes, liquid should never enter your micropipette, instead you need to attach a pipette tip that you can then use to take up liquids. Different pipette sizes need different sized pipette tips. You want to make sure that the pipette tip is the right size and fits snugly on the end of your pipette. This greatly affects the accuracy of the pipette. There are a lot of different brands of pipettes and not all of them fit on every type of pipette tips, so make sure you check before starting your experiment.
First stop and second stop. One tricky thing about using a pipette is that there are two “stops”. This means that when you depress the plunger on the pipette you will feel resistance when you hit the first stop; this indicates the volume you set your pipette to measure for you. You can push down further and reach the second stop; this is extra air and will not give you an accurate measurement if you use it while taking up liquid. The second stop is there to help expel the liquid from the pipette tip. Therefore the order to pipette is as follows: push down to the first stop; put your pipette tip into the liquid; slowly release your thumb to suck up the liquid; move the pipette to the vessel you are transferring the liquid into; push down to the second stop to expel all the liquid out.
Figure 1: A) Schematic of a general micropipette with key parts labeled. B) View of the Volume Readout panels of four common sizes of pipettes. The volumes each is set to are written below the images. Created with BioRender.com
Helpful Tips and Tricks
Pipette slowly to ensure accuracy! In order to be super accurate, when you release your thumb to suck up the liquid make sure that you do so slowly and leave the pipette tip in the liquid for a second or two after you have fully released your thumb.
Note that not all liquids are the same viscosity. Thicker liquids require slower pipetting to accurately get all the volume, while thinner liquids (like ethanol) require faster pipetting because they will drip out of the pipette tip easily.
Keep the pipette vertical! When you have liquid in your pipette tip, make sure to keep the pipette vertical. If you lay it down or tip it upside down, the liquid can run into the pipette and ruin it.
No air bubbles! Make sure when you are pipetting that you are careful to not get air bubbles. If you do get an air bubble, try again or get a new pipette tip. Sometimes a little bit of liquid gets stuck in the tip and makes it harder to get the right volume.
Change your pipette tip between solutions and do not touch pipette tips on anything else! It is very easy to get contamination when pipetting. A good rule is to always change your pipette tip between solutions and sample tubes. This is really important. Never use a pipette tip to measure multiple different solutions. You will contaminate your stock solutions and your samples.
Accuracy VERSUS Precision
An important distinction in pipetting - and research in general - is the difference between accuracy and precision. Accuracy means that when you measure something you get the expected value or are within an acceptable range. Precision means that every time you measure something, you get the same or very similar value. For example, when using a pipette you want it to be accurate, i.e. you pipette 10 µL and you get 10µL, AND you want it to be precise, i.e. you pipette 10 µL three times and every time you get 10 µL. Accuracy and precision are especially important to keep in mind when doing sensitive experiments and they can help ensure the reproducibility of your results.
Test your pipetting accuracy and precision by weighing water!
You can test your skills and your pipette’s calibration by pipetting water onto a scale. 1 mL of water should weigh 1 gram. So if you pipette 100 µL of water that should weigh 0.1 grams. To test your accuracy and precision, pipette a volume 10 times and record the final volume. Then calculate your percent error
(Actual value - expected value / expected value) X 100%
Micropipettes are small but mighty pieces of equipment that greatly help scientists across a huge variety of fields. Hopefully, this post helps you familiarize yourself with this tool and allows you to go forth and pipette with confidence.