The accuracy and precision of pipetting can have a significant impact on downstream applications, and depending on the degree of error, poor pipetting practices can lead to inaccurate results and failed experiments. While necessary for many labs, manual pipetting carries a higher risk of error and contamination than automated liquid handling, and also raises concern about repetitive strain injury (RSI) for regular pipette users. However, routine manual pipetting can still be accurate, precise and safe by avoiding common mistakes and following best practices. Here are some of the “do’s” and “don’ts” of pipette operation to increase your confidence in your technique and results, and prevent RSI from repetitive pipetting tasks.
DON’T: Immerse the pipette tip too deep into sample solution
Excessive immersion of the pipette tip is a very common source of error, and can increase inaccuracy up to 2-fold or more (0.6-0.8% inaccuracy compared to 0.2-0.4% inaccuracy when immersion depth is ideal).1 This negative impact on accuracy is even more pronounced with very small sample volumes (ex. 1-10 µL) and in extreme cases, when the end of the pipette tip touches the bottom of the container, aspiration of the liquid is impeded and the tip can become damaged.
DO: Immerse the tip only as deep as necessary for effective aspiration of the desired volume
Ensure the tip is submerged sufficiently to prevent the opening from ending up above the liquid before the full volume is aspirated: according to standards set by ASTM International (formerly the American Society for Testing and Materials), an immersion depth of 2-3 mm is recommended for volumes of 1-100 µL, 2-4 mm for volumes of 101-1000 µL and 3 to 6 mm for for volumes of 1.1 to 10 mL.2 For extremely small volumes (less than 1µL) an immersion depth as small as 1 mm may be necessary.3 Consult your pipette’s manual or relevant standards if you are unsure of the ideal immersion depth for your application.
DON’T: Hold the pipette at an angle during aspiration
When the pipette tip is immersed at an angle too far from 90 degrees, too much liquid can be aspirated due to deviations in liquid level from when the pipette was calibrated.4 For example, at an immersion angle of 30 degrees from the vertical, overaspiration of up to 0.7% can occur. When combined with excessive immersion depth, aspirating at a wide angle can increase inaccuracy 3- to 5-fold (1-1.2% inaccuracy) compared with ideal conditions.1
DO: Hold the pipette as vertical and straight as possible during aspiration
Ideally, the tip would be immersed at exactly a 90-degree angle, but generally it is most important to ensure the immersion angle does not exceed 20 degrees from the vertical.4
DON’T: Pipette using a completely dry air displacement tip
Sample evaporation into the air cushion of an air displacement pipette tip can lead to a lower volume being dispensed from the tip than expected. Evaporation of the aspirated sample will be greater when it is drawn into a fresh, dry tip that has not been pre-wetted.5
DO: Pre-wet each pipette tip before pipetting
To pre-wet an air displacement pipette tip, the set volume of sample should be aspirated and dispensed multiple times; at least five times, according to International Organization for Standardization (ISO) recommendations. This can be a time-consuming process, and fewer pre-wetting rounds may be sufficient depending on the accuracy and precision requirements of the application; for example, many manufacturers recommend three pre-wetting cycles. Regardless, pre-wetting tips is essential as it increases humidity in the air cushion and thus prevents sample evaporation.
DON’T: Use an improper pipette tip for challenging samples
Air displacement pipetting is suitable for handling aqueous solutions under ambient conditions, but for samples with higher- or lower-than-ambient temperatures (such restriction enzymes, which must be kept cold), high viscosity (ex. glycerol) or high volatility (ex. methanol), air cushion pipette tips can run into problems.6 Low temperatures can shrink the air cushion, leading to over-delivery, while high temperatures can cause it to expand, leading to under-delivery. Viscous samples can be under-delivered due to air bubbles forming when they are drawn too quickly into an air displacement pipette tip, and volatile liquids are more susceptible to evaporation into the air cushion than other liquid.
DO: Use a positive displacement or other appropriate pipette tip for challenging samples
One of the best solutions for difficult liquid handling is the use of positive displacement pipette tips, which contain a piston that comes in direct contact with the sample. While more expensive and difficult to change than air cushion tips, positive displacement tips avoid the problems of air cushion expansion and contraction, air bubble formation in viscous liquids and evaporation of highly volatile liquids. However, some air displacement tips (such as those designed to have an especially small air cushion) can also mitigate problems associated with challenging liquid samples.
DON’T: Handle the pipette for long, continuous periods of time
Pipetting tasks can sometimes take hours to complete, but there are several negative effects associated with continuously pipetting without breaks for extended periods of time. RSI, which results from the repeated forces and strain of pipetting on the body — especially the thumb, hand and wrist – can have long-term health consequences, and the risks increase the longer these repetitive motions are performed without interruption.7 Additionally, holding the pipette for too long can impact accuracy and precision due to hand-warming, in which heat from the body is transferred to the pipette through the hand, causing a change in temperature that can impact results.
DO: Take regular breaks while pipetting
To prevent RSI, the U.S. Occupational Safety and Health Administration (OSHA) recommends taking a 1- to 2-minute break for every 20 minutes of pipetting. To avoid hand-warming effects, place the pipette in its stand between pipetting cycles instead of continuing to hold it.4
DON’T: Use excessive force to mount the pipette tip
Slamming, twisting or rocking motions used to attempt to form a seal between the tip and pipette can both increase RSI risk and cause damage to the tip. Users may struggle to easily mount a tip if the tips used are not a good fit with the pipetting device (for example, if the tips and pipettes are made by different manufacturers).
DO: Use well-fitting tips and use a minimal amount of force to mount them
Always ensure that the pipette tips you’re using are suitable for use with the pipetting device; well-fitting tips should easily form a seal without the need for slamming, twisting or rocking. When mounting tips, only use the minimum amount of force needed to form a seal in order to reduce strain on the hand, wrist and arm.
DON’T: Place a pipette on its side
Placing a pipette down on its side on the bench, especially with a tip attached, runs the risk of liquids or evaporated moisture leaking into the barrel, causing contamination, corrosion or other damage.
DO: Store the pipette upright in its stand
Any time you are not handling the pipette, even for a brief moment, secure it on its stand or rack, upright so that the tip end is always below the plunger end.
DON’T: Rush
It may be tempting to speed through a pipetting session, but rushing through steps can lead to inaccurate and imprecise results. Withdrawing the tip too quickly during aspiration can prevent all of the desired liquid volume from entering the tip.8 Additionally, aspirating samples too quickly, especially viscous samples, can cause air bubbles to form, and dispensing too quickly can result in under-delivery or loss of some sample due to splashing.
DO: Take adequate pauses and operate the plunger with a smooth, steady motion
To ensure complete aspiration of the desired volume, keep the tip immersed in the liquid for at least one second before removal. The plunger should be pressed and released slowly and without excessive force, especially when handling viscous liquids. Slowing down can make it easier to achieve a steady and consistent pipetting rhythm, which is essential to ensure that results are precise and repeatable.
References
- "Good Laboratory Pipetting Practices," Thermo Scientific (2016). https://assets.thermofisher.com/TFS-Assets/LPD/manuals/Good-Lab-Pipetting-Guide-1517630-01-Brochure-EN.pdf
- "Good pipetting technique—don't go in too deep (or too shallow)," Article by Candie Gilman, Artel. https://www.artel.co/good-pipetting-technique-dont-go-in-too-deep-or-too-shallow/
- "Guide to Pipetting Third Edition," Gilson (2018). https://www.gilson.com/pub/media/docs/GuideToPipettingE.pdf
- "Pipetting Tips and Tricks," Mettler Toledo (2013). https://www.mt.com/dam/RAININ/PDFs/Pipetting_Tips_Tricks.pdf
- "Is your pipetting technique causing problems?," Article by Candie Gilman, Artel. https://www.artel.co/is-your-pipetting-technique-causing-problems/
- "Tech Compare: Air vs. Positive Displacement Pipetting," Article by Laura French, Labcompare (2022). https://www.labcompare.com/10-Featured-Articles/591341-Tech-Compare-Air-vs-Positive-Displacement-Pipetting/
- "5 Ways to Reduce Pipetting Strain," Article by Laura French, Labcompare (2022). https://www.labcompare.com/10-Featured-Articles/592969-Tips-for-Reducing-Pipetting-Strain/
- "Pause & Effect: How timing is critical for good pipetting technique," Article by Candie Gilman, Artel. https://www.artel.co/pause-effect-how-timing-is-critical-for-good-pipetting-technique/