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Good pipetting technique is essential for many biologists, but it can be hard to get right. When I take 1 µl of liquid using a micropipette, I seem to always take less than 1 µl, and that amount is variable/inconsistent. I would like to know what could be wrong.

What is the proper pipetting technique? Could it be that the pipette is inaccurate?

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    $\begingroup$ Welcome to Biology.SE. You should try to give more info about the moment at which you realize you did not piped exactly one microlitre. Do you see directly in the pipette that you did not pipette 1 microlitre. Or do you sometimes have some leftover in the pipette and sometimes not. Do you think the pipette you are using might be in cause? $\endgroup$
    – Remi.b
    Feb 9, 2016 at 4:57
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    $\begingroup$ I wouldn't be surprised if your pipette needs some service. Some of the parts (rubber rings) that are important for proper function wear out and need to be replaced every now and then. $\endgroup$
    – Chris ♦
    Feb 9, 2016 at 7:51
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    $\begingroup$ I've retracted my close vote, good pipetting technique is essential in a lot of biology. General pipetting technique information would be of use. $\endgroup$
    – rg255
    Feb 9, 2016 at 8:14
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    $\begingroup$ This post can be made a community wiki but I'll leave that decision with the OP and @rg255 $\endgroup$
    – WYSIWYG
    Feb 9, 2016 at 8:45
  • $\begingroup$ What pipette did you use? Using a 10-ml one won't help you. Use the smallest one you can find. Also, when you use one single pipette to combine all solutions and that one pipette has a systematic error (i.e., calibration needed, but not done) then there is no issue, as it is all about relative volumes. If you use multiple pipettes to combine fluids into a reaction mixture, then trouble arises. $\endgroup$
    – AliceD ♦
    Feb 9, 2016 at 10:21

2 Answers 2

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Here's some step-by-step advice on good pipetting technique:

  1. Make sure the pipette is set for the correct volume
  2. Ensure the tip is firmly attached
  3. Keep the pipette vertical when pipetting
  4. Slowly and smoothly depress the plunger to the correct point (the first stop position)
  5. Insert the tip in to the liquid to be pipetted. Ensure the tip is properly immersed in the liquid, but not touching/near any surfaces of the container
  6. Slowly and smoothly release the plunger back, keeping the pipette upright, and tip immersed, and making sure no bubbles form - you can try pre-wetting (see video linked below, around 3:00)
  7. Withdraw the pipette from the liquid
  8. Carefully dispense the liquid in to the desired container by steadily depressing the plunger to the second stop position (fully depressed), making sure all liquid is dispensed without spills or splashes.

"Put the tip in a 45-degree angle onto the wall of the container; then start pushing down up until the first stop; then push through to the second stop; keep the plunger depressed and remove pipette and check whether the drop is in the container and pipette tip is empty" - Christiaan

  1. Discard the tip using the release plunger

Also check you've got the right size/type of tip, and are using a suitable pipette (e.g. don't use a 10-100 µl pipette for 120 µl)

If this still causes incorrect measures then you could need to get your pipette calibrated.

Test a pipette by taking several samples. If the pipetted volume is consistent but incorrect (e.g. always 10% under the target) it suggests the pipette needs calibrating, it's probably not the technique that's wrong. If it is inconsistent (both under and over pipetting) then it's either technique or the pipette (bad/damaged pipettes can be inconsistent, poorly but good calibrated pipettes will more likely be consistent and only need calibrating).

Try using multiple pipettes, if they are each consistent but variance is among them (pipette 1 is always under by ~10%, pipette 2 is always over by 15%, pipette 3 is always over by ~2%...) then your technique is good, and you need better pipettes. Also get an experienced pipetter to check your technique and try the pipettes themselves.

Calibration can be done internally by lab techs or by external specialists, speak to your lab responsible person.

Also see this video which is well explained.

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    $\begingroup$ Additional point: care should be taken while pipetting surfactants and viscous liquids. Surfactant containing liquids should be properly mixed prior to dispensing and mixing by pipetting just before dispensing (with the same tip) should be avoided. Multiple rounds of pipetting creates bubbles inside the tip and leads to picking up of lower volumes. Viscous liquids should be gently pipetted and care should be taken to not let the liquid stick on the exterior of the tip. $\endgroup$
    – WYSIWYG
    Feb 9, 2016 at 8:13
  • $\begingroup$ It's also a good idea to practice your pipetting with serial dilutions of colored compounds, then measure the absorbance of each one and plot it. If your slope is off, you need to keep practicing, or fix your pipettes. $\endgroup$
    – user137
    Feb 9, 2016 at 10:11
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    $\begingroup$ It's been a long time since I held one, but ref. step 8) - shouldn't you always put the tip in a 45-degree angle onto the wall of the container; then start pushing down up until the first stop; then push through to the second stop; keep the plunger depressed and remove pipette and check whether the drop is in the container and pipette tip is empty? Then give a few small taps to the container to make sure the drop is at the bottom of the container? $\endgroup$
    – AliceD ♦
    Feb 9, 2016 at 10:19
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first of all i need to disclose i work for Andrew Alliance, but i answer for the sake of providing information and not for commercial purposes. What you experience depends - as we measured with our robot - from the humidity in the environment. if you have 1 nL of liquid water evaporating inside the tip, becasue the environmental humidity is low, it will become 1 ul of water vapour above the liquid level, so your sample will go down by the equivalent of 1 uL. you can verify this phenomenon by extracting the tip from the liquid, recovering air pressure and keeping the humidity inside the tip, and re-aspirating the liquid. This is actually why calibration laboratories work at 70% ambient humidity...

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  • $\begingroup$ Interesting detail, I have never thought about it. $\endgroup$
    – Chris ♦
    Aug 28, 2017 at 12:11

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