To improve reproducibility; look first to pipetting

February 1, 2021
Est. Reading: 4 minutes
Gustavo Chavarria

With years of experience and expertise with pipettes, Gustavo Chavarria outlines pipetting factors and practices that lead to success and reproducibility in the lab…

Lab managers and PIs looking to improve the quality and consistency of their data should start with pipetting. Pipetting is fundamental to most life science labs, yet it is surprising how few researchers understand the importance of good pipetting practices to the success and reproducibility of their experiments. No matter how precise a pipette may be, it is the skill and knowledge of the user that ultimately determine the accuracy and reliability of the results.

Q: How did you come to be the global product manager for instruments?

A: My love of chemistry and math drew me to a career in STEM. Working in biochemistry and cell biology used both. I soon discovered that statistics was fundamental to effective experiment design, clinical trial data collection, and validation of the research results. I was working as a scientist for a company, developing bioanalytical assays to detect metabolites in human samples, when this product manager opportunity came up. Five years later, I’m still a product manager. I love my job because I learn the newest trends in the life sciences without doing the experiments while using data science to make better decisions and bring new solutions that help the current and upcoming generation of scientists.

Q: Pipetting seems so straight forward. How do errors slip into the process?

A: Pipetting is a manual process. As a result, it’s not surprising that it’s prone to small errors during the process. In some ways, pipetting is like driving a car. People 'know' how to drive, but many forget to turn on the blinker when changing lanes!

 Q: What are some of the most common mistakes and some simple steps to avoid them?

A: The three most common errors involve matching the pipette to the sample, maintaining a slow yet steady aspiration rate, and attaining the necessary inversion angle.

Q: What’s an effective way to improve pipetting technique overall?

A: Proper training is the key to better pipetting technique. It’s as critical as having a calibrated pipette or high-quality tips. Often people receive training when a new instrument, like a plate reader or sequencer, is purchased. Yet there is very little done to train lab workers on pipetting techniques and how to maintain these instruments. It would be ideal if new hires and lab members received formal training that explained the basics of pipetting, followed by a proficiency test to verify their skills against a standard curve or dedicated pipetting verification system. Annual refresher courses should be mandatory for all pipette users. In the long run, these simple practices will result in better and more consistent pipetting performance while reducing the costs associated with inconsistent or irreproducible results.

Q: Is there anything else you'd like to add about pipetting?

A: The pipette, pipette tip, and operator are three components that comprise a system: the pipetting system. Each part must be appropriate for the application to deliver the desired outcome: accurate volumes. An uncalibrated pipette can skew results by delivering inconsistent volumes. An incorrect or low-quality tip can contaminate or damage the sample. An improper technique can also deliver incorrect volumes. For these reasons, pipetting should always be considered as a system rather than as individual components.

Avoiding common errors

Matching the pipette to the sample

Not all samples are created equal. As a result, a forward-pipetting technique cannot be used at all times. A good rule of thumb for air displacement pipettes is to use a reverse technique for viscous and volatile liquids and a forward technique for aqueous-based solutions. Compared to air displacement pipettes, positive displacement pipettes offer greater accuracy and consistency when handling highly viscous or volatile liquids. 

Maintaining a slow yet steady aspiration rate

Releasing the plunger too quickly while aspirating can produce a ‘fountain effect’ or introduce air pockets inside the tip. These errors will affect pipetting accuracy through the formation of bubbles and inconsistent volumes, respectively. Splash-back can also occur, contaminating the pipette and/or damaging its internal working. Any of these mistakes will ultimately affect the delivered volume’s accuracy and will often make it necessary to repeat some or all of the protocol. 

Attaining the necessary inversion angle

Failing to hold the pipette as close to a vertical position as possible is not always easy, especially when pipetting small volumes. In these cases, it’s very common to hold the pipette and vessel at an angle, even at angles parallel to the bench. The pipette and vessel are often held this way to reach the bottom of a small vessel while looking through the content. Or, they’re held this way because it’s simply more comfortable for the lab worker’s arm to hold the pipette and vessel at an angle. Research shows that the problem with extreme angles of greater than 20 degrees is that they can significantly affect accuracy. Re-learning to hold the pipette as close to vertical can solve this.

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