Pipetting Best Practices

Appropriate pipetting techniques are often one of the first topics taught in introductory science classes, but many researchers would be surprised to find some lesser-known tips and tricks that can make your workflow more streamlined. Even though these devices are one of the most routinely used in the lab, correct maintenance and care can keep them functioning far beyond their average 7-year lifespan. 

From proper cleaning and temperature considerations to effective calibration protocols for accurate aliquots, keep reading to learn how to make your pipettes work at their maximum efficiency. Even pipetting pros can benefit from a refresher course!

Squeaky clean

Proper cleaning will look slightly different for every scientist, depending on your pipette style. However, there are a few basic rules and recommendations that experts suggest to maintain the longevity of your device. “To clean your pipettes, use 70% ethanol, especially if the pipettes will be used in a sterile environment such as a biosafety cabinet,” says Melissa Mulla, Product Specialist at CELLTREAT Scientific Products. “If your pipette has metal parts, avoid corrosive cleaning agents such as 10% bleach. If bleach is used, follow up with 70% ethanol to prevent corrosion. Some pipettes may be autoclaved but not all, so check with the manufacturer’s recommendations before attempting.”

However, it’s recommended to adjust your cleaning frequency depending on the chemicals or substances used in your samples. “For example, aqueous solutions and buffers require a thorough rinse of the contaminated pipette parts with distilled water,” explains Steve Beckett, Senior Product Specialist, Liquid Handling Solutions, UK & Ireland, Thermo Fisher Scientific, “while organic solvents require the contaminated parts to be immersed in a detergent solution before being rinsed with distilled water. The chemical compatibility of the pipette should always be checked prior to cleaning.”

Calibration consistency

No matter how much you think you can trust your eyesight, proper calibration ensures that you’re pipetting your desired quantities correctly each and every time. Always make sure to recalibrate your pipette if your sample has a significantly different density than water. Your pipetting speed should correlate with the viscosity of your sample—slower speeds for viscous liquids and quickly for volatile liquids. 

Pipettes and tips should work together in synchrony, so retain the same tip type and brand that was used during calibration. The pipette should be recalibrated immediately if a different brand or tip type is used. But what about daily upkeep? “If the pipette is used daily, it should be cleaned and lubricated at least every three months. Once cleaned, the pipette should be calibrated and the calibration adjusted if found to be outside the required accuracy limits,” according to Beckett.

Accuracy and precision

Your pipette should be aligned similarly for each sample dispersion at no more than 20 degrees. When pulling the pipette, don't touch the tip on the side of the container, as that can be problematic for small samples. After aspirating, pull the pipette straight out. Slow, steady pulls are essential for reproducibility and minimizing volume errors. 

“To ensure accuracy and precision when pipetting, always operate the plunger slowly enough to ensure the full volume is pulled into the tip,” adds Mulla. “Taking your time will also help mitigate the risk of creating air bubbles. Never rush when pipetting viscous liquids such as detergents, and since these liquids tend to stick to the outside of the tip, try not to submerge the tip any further into the detergent than necessary. For these thick liquids, you may also consider using a wide bore tip to facilitate pipetting.”

Additionally, pre-wetting the pipette tip helps to ensure that no remnants of the sample remain in the tip post-dispersion. “Many of the solutions we transfer are proteins, and proteins tend to be sticky,” Mulla continues. “Also, many solutions are in water, and the adhesive properties of water will cause molecules to remain behind after the liquid is dispensed from the pipette tip. By pre-wetting the tip, we are accounting for these molecular actions to ensure that the volume and concentration of sample or reagent that we are actually dispensing is more accurate.”

Sample temperature

If possible, ensure that your pipette, accessories, and sample are all at room temperature. Noelia Teliz, Junior Product Manager at INTEGRA Biosciences, elaborates, “The pipette, tips, and liquids should be equilibrated to the working temperature, as temperature differences lead to contraction or expansion of the air cushion inside the pipette tip and pipette, which can negatively impact the accuracy and precision of the dispense. Ideally, everything should be equilibrated to room temperature if possible.”

Even when working with room temperature samples, note that hot hands can heat up the pipette’s barrel. To err on the side of caution, always wear gloves, hold the pipette loosely, and return to its stand between uses.

Pipetting ergonomics

To avoid strain after repetitive pipetting, make sure you have an appropriate posture and an organized workspace to rest your arms on the table, if necessary. Make sure you are seated upright and sitting in a comfortable seat. Users often jam single-channel pipette tips onto the device with too much force, leading to increased hand strain and difficult ejections. Try utilizing a light double tap to quickly and reliably seat the tip using the pipette’s momentum without excess exertion that can contribute to fatigue.

Below is a list of tips and tricks from Thermo Fisher Scientific for further assistance on pipetting ergonomics:

• Clean and grease pipettes on a regularly scheduled basis
• Make sure you have pipette tips that fit correctly and eject easily
• Use minimal force when applying pipette tips
• Utilize manual pipettes with light plunger forces
• Choose a pipette that fits comfortably in your hand
• Use electronic pipettes if you use more than five microplates a day
• Minimize thumb strain by using electronic pipettes with index finger action and electronic tip injection

Conclusion

No matter how often you utilize pipettes, understanding how to use them at their optimum capacity ensures reliable and accurate measurements. These tips and tricks from the experts are designed to help researchers pipette with ease, with reliable and easily dispersed aliquots. Automation is often unavailable to all labs, so learning how to reduce strain while maintaining speed and efficiency is critical for productive workflows.