Good Pipetting Practice: Better Results through Training and Support

Most life science applications include critical liquid handling steps where pipetted volume directly influences the result. If pipetting isn't consistent and accurate, then time, money, and material can go to waste. Accurate pipetting forms the foundation of reproducible work, which is essential to generating meaningful data.

The first step toward improving pipetting accuracy is to choose the right type of pipette and the proper volume range. For size, pick the lowest-volume pipette that will accommodate the volume you're pipetting. For example, if you're pipetting 2 µL and have a 5 µL pipette and a 20 µL pipette, you'll get better accuracy using the 5 µL. 

Check out this white paper on pipette selection to understand which pipette works best for the liquid and other factors unique to your situation: Selecting The Right Pipette

Once you have the right instrument, put your good pipetting technique into practice. Set up a routine testing schedule to maximize trust in your results over time, and, as with any precision instrument, make sure to institute a regular, expert service and calibration regimen.

The easiest way to quickly test a pipette's accuracy may be the Rainin SmartCheck. Find out more at mt.com/SmartCheck 

Choosing the right pipette involves evaluating your requirements. First, what kind of samples are you pipetting? Are they aqueous? Or are they viscous, volatile, foaming, corrosive, or hazardous? For aqueous and near-aqueous liquids, the most common air-displacement pipettes (also called "air cushion" pipettes) are a good choice. For viscous, volatile, and other unusual liquid types, a positive-displacement pipette would be a better choice for both accuracy and safety. 

What volume will you be pipetting? How many samples do you need to transfer from one vessel to another? Check out this white paper on pipette selection to understand which pipette works best depending on the liquid and other factors unique to your situation.

Pipetting the smallest possible volumes involves specialized instruments and techniques. A repeater pipette may be your best choice to repeat-dispense volumes down to 100 nL. Another pipette can also work for just a few dispenses. Make sure to use the smallest-size pipette that will accommodate the volume you wish to pipette. 

At very low volumes, pipetting technique has a proportionally greater impact on accuracy and reproducibility. Immersion depth, pipetting angle, and rate of aspiration are important to control. The Pipetting Techniques poster from Rainin can help.

Rainin NanoRep repeat-dispenses aliquots down to 100 nL. Find out more here: mt.com/NanoRep

The most important factor in accurate, reproducible multichannel pipetting is the integrity of the seal for every channel of the pipette. That means every tip fits perfectly on each shaft with zero air leakage. Different manufacturers achieve this in different ways, with varying ergonomic ease and sealing consistency levels. LTS LiteTouch System pipette tips from Rainin require the least force to load and eject from multichannel, and they seal consistently every time.

Click here to view Rainin's wide range of multichannel pipettes.

When aspirating, hold a pipette as close to vertical as possible, avoiding any angle greater than 20° off the Y axis. When dispensing, if you're touching off on the wall of the target vessel in order to fully pull all liquid from the tip, it's OK to exceed the 20° angle.

Forward pipetting is the standard pipetting technique and the best choice for aqueous samples. For viscous and volatile samples, reverse pipetting delivers better accuracy.

To forward pipette, push the plunger down to the first stop while holding the pipette outside the liquid. Then, immerse the tip 2-10mm deep into the liquid you are working with – no deeper. Release the plunger slowly to full extension to aspirate the complete liquid volume. To dispense into your target vessel, move the pipette into the vessel, then press the plunger at an even rate through the first stop all the way to the second stop – as far as the plunger goes. With the plunger fully pressed, lightly drag the tip of the pipette up the wall of the vessel to "touch off." This helps completely dispense the full liquid volume. Release the plunger, eject the pipette tip, load a new pipette tip, and repeat.

To reverse pipette, push the plunger all the way down through the first stop to the second stop – as far as the plunger goes. Then, immerse the tip in liquid and slowly and evenly release the plunger to aspirate. Move to the receiving vessel and press the plunger ONLY TO THE FIRST STOP to dispense the correct volume. A small residual volume will remain in the pipette tip. Move the pipette over a discard-dispense vessel and press the plunger the rest of the way down to the second stop, also called the "blowout" stop, to release any remaining liquid. 

Release the plunger, eject the pipette tip, load a new pipette tip, and repeat.

An air-displacement pipette can be used to pipette slightly viscous liquids, including blood. To achieve maximum accuracy, use the reverse pipetting technique described above. For more viscous liquids, such as 85% glycerol or Triton X-100, a positive displacement pipette offers the best accuracy and consistency.

Slightly volatile liquids such as ethanol can be pipetted with an air-displacement pipette using the above-mentioned reverse pipetting technique. However, positive-displacement pipettes are a better choice for volatile liquids. 

If using an air-displacement pipette, pre-rinse the pipette tip (aspirate and dispense) at least five times before pipetting your volume. This equilibrates the air inside the pipette, slowing the liquid's evaporation rate. Rapid evaporation of volatile liquids expands the air inside a pipette and forces liquid from the tip without any pressure being placed on the plunger, and this diminishes accurate volume delivery.

Volatile liquids such as acetonitrile are generally better handled with a positive displacement pipette.

Yes, pipetting with air-displacement pipettes can create aerosols. Aerosols can rise into the interior of the pipette and contaminate the interior walls and piston. Aerosolization occurs more readily when the plunger is released too rapidly during aspiration and can also occur in the opposite direction outside the pipette with overly swift dispensing. 

To minimize aerosols, aspirate and dispense slowly and evenly. Use filtered tips. 

Positive-displacement pipettes use syringe-style tips that prevent aerosolization. A positive-displacement pipette is the best choice for pipetting hazardous liquids and any liquid of elevated viscosity or volatility.

Very cold or hot liquids change the air pressure within air-displacement pipettes, leading to inaccurate pipetting. 

Positive-displacement pipettes, with syringe-style tips that have no air pocket between liquid and piston, are not affected by different temperatures of liquids. Positive-displacement pipettes will deliver accurate results with non-room temperature liquids.

If you must pipette a very hot or cold liquid with an air-displacement pipette, do not pre-rinse the pipette tip—this is typically the best practice for the highest accuracy. Instead, aspirate and dispense expeditiously to minimize the effect of the liquid's temperature on the pipette's interior air gap.

To avoid bubbles and foaming when pipetting, do not immerse the tip more than 10mm in the liquid reservoir, and aspirate slowly.

Reverse pipetting offers additional protection because, before aspirating, you fully press the plunger to the 2nd stop, so there is no chance of injecting air into the liquid sample. For more on reverse pipetting, visit this page. 

A positive-displacement pipette does not create bubbles or foam when interacting with liquid because its syringe-style tip does not present air for the liquid to interact with.

Decontaminate a pipette by wiping it with a wet cloth and a detergent like isopropanol or a 10% bleach solution. Avoid the micrometer's clear plastic window.

Autoclaving may be another option, but check your pipette's instrument specifications. If it is autoclavable, is the entire instrument autoclaved or only the liquid end?  Often, only the liquid end can be autoclaved.

For more on cleaning a pipette, get the Cleaning A Pipette poster.

Calibration is the controlled and documented measurement of a pipette's systematic error rate (accuracy) and random error rate (precision). In many laboratory environments, pipette performance must be regularly tested and documented by law under specific conditions certified by an accredited body. 

ISO-certified providers calibrate pipettes in a controlled setting. Results are securely documented on a calibration certificate, adding to an audit-ready performance history for each instrument. When adjustments are necessary, ISO-certified calibration labs provide expert service with manufacturer-recommended parts and lubricants. 

Rainin and METTLER TOLEDO are the world's largest pipette service organization with quick turnaround from mail-in and onsite service options. Service labs are ISO 17025 certified, and procedures follow the 2022 update to ISO 8655. 

Pipette accuracy—or trueness, as it´s called now—is the difference between the mean volume of all measurements (4-10 measurements) and the set volume. 

Precision is the standard deviation of all measurements performed. How far apart from each reading are the others?

A pipette's uncertainty is the quantitative term for its overall accuracy. It is a combination of trueness (a measure of systematic error) and precision (a measure of random error). 

The equation for uncertainty is: 

Uncertainty = Systematic Error + Random Error * k. k (coverage factor or Z-score) depends on the number of measurements. k=3.31 for four measurements and 2 for ten measurements.

Store pipettes vertically, hanging from a shelf or pipette stand. Keep pipettes clean and free of any contaminants. Keep pipette tip racks closed.