Tips to Improve Your Pipetting Technique and Accuracy

Pipetting is a fundamental laboratory skill in which good technique is critical but not always strictly followed for a variety of reasons, including lack of training, heavy workload, and distractions in the lab. By slowing down, staying focused, and following the tips in this article, you can improve your technique and significantly enhance both accuracy and precision.

This article features best practices from pipetting experts. Their collective expertise not only addresses best practices but also covers common pitfalls and invaluable workarounds and solutions. Read on for their advice on a wide range of pipetting-related topics, which will help you gain a deeper understanding of effective pipetting, ensuring more reliable and reproducible experimental results.

Our expert panel is composed of Joni Ake, Product Manager, Pipetting & Dispensing, Sartorius; Noelia Teliz, Product Specialist, INTEGRA Biosciences; and Rebecca Lampert, Product Manager BRANDTECH. Many of their tips overlap but frequently highlight different aspects so they are included in their entirety below.

Proper pipetting technique

When asked for recommended steps to ensure accuracy and precision in pipetting, Lampert from BRANDTECH summed up her recommendations succinctly: “Choose a high-quality pipette and select a pipette with your intended volume in the pipette’s middle to upper volume range; Ensure that the pipette tips are compatible for that pipette; Pre-wet the pipette tip; Follow proper aspiration and dispensing technique; Maintain consistent pipetting speed and technique for accurate and repeatable results; Maintain and calibrate the pipettes.”

Ake from Sartorius expanded on those tips with the following:

• “Operate plunger smoothly and consistently for both aspiration and dispensing, remember to utilize blow-out at the end of dispensing
• Pre-wet the system (pipette + tip) 3 to 5 times
• Hold the pipette in a vertical position during aspiration, tilt for dispensing
• For aspiration immerse tip 2–3 mm (≤ 1000 µL), 5–6 mm with large volumes
• Wait 1–2 seconds before lifting the tip from the liquid after aspiration to allow the liquid to stabilize. This is especially important for larger volumes, like 1,000 µL and above.
• Choose a pipette with a nominal volume as close to the target volume as possible. With adjustable pipettes it is easier for the user the larger volume you use, e.g., with 10–100 µL pipette it is much easier to accurately and repeatable pipette 100 µL than it is 10 µL.”

Teliz from INTEGRA added even more detail:

“Maintain consistent pipette angle: When pipetting, the angle of the pipette can play an important role in obtaining good results. If possible, hold the pipette at a constant angle throughout the entire process. Ideally, the angle at which the pipette is held should not exceed 20 degrees. For very small volumes of 30 µL or less, the straighter the pipette, the better. With changing the angle, the hydrostatic pressure inside the tip varies. As a result, the aspiration volume will be inconsistent.

Immerse the tip only 2–3 mm when aspirating: A common mistake made during aspiration is to immerse the pipette tip as deep as possible in the source vessel. As this increases the risk of liquid droplets clinging to the outside of the pipette tip, it is best to immerse the pipette tip just below the liquid’s surface. It is recommended to insert the tip only 2–3 mm into the source liquid to allow the desired volume to be aspirated. An exception to this is the aspiration of extremely low volumes such as 0.2 µL. Liquid retained on the outside of the tip can result in an inaccurate dispense.

Touch off after dispense: After a dispense, you will often see a droplet at the end of your tip. As this droplet belongs in the dispense, you should use one of the following three methods to remove your pipette from the target vessel.

• Side wall touch off: Remove the pipette tip by sliding the tip end along the sidewall of the vessel. This is the standard method and is recommended to achieve the most accurate dispense.
• Surface touch off: Remove the tip by touching off the liquid droplet on the surface of the liquid in the container. This technique is recommended when dispensing less than 1 µL as a neat transfer. Touching the droplet to the liquid draws the small droplet out of the pipette tip which ensures accurate delivery.
• Into liquid dispense: If the dispense was made directly into the liquid it is considered a wet-dispense and a touch off is not required. This method is ideal for small-volume dispensing to ensure the liquid does not remain on the side of the vessel.

Pre-wet your pipette tips: Temperature variations between pipettes, tips, and liquids can affect the volume of the air cushion inside the pipette, leading to volume variations and, ultimately, inconsistent measurements. After loading tips onto your pipette, aspirate and dispense the nominal volume three times. This will equilibrate temperature differences and humidify the dead air space inside the pipette and tip. When neglecting the pre-wetting procedure, the first few dispenses tend to deliver less volume due to evaporation. The evaporation can also cause droplet formation on the tip end, as vapor pressure increases and liquid is forced out of the tip.

Discard first and last dispense when repeat dispensing: Following aspiration, the mechanical parts inside an electronic pipette need to change direction before dispensing. When performing repeat dispenses, this can result in the first dispense being too low in volume, and the final dispense ultimately including the accumulated error of all previous dispenses. Discarding the first and last dispense helps to eliminate inconsistencies that can arise due to variations in initial dispensing pressure or residual liquid in the tip. This practice ensures that your data is based on the most reliable aliquots.”

Calibration

There was not as much agreement on some of the details of how often pipettes should be calibrated and maintained to ensure optimal performance, but all four experts agreed calibration was important.

According to Lampert from BRANDTECH, the frequency of pipette calibration depends on many factors. “We often see labs calibrating their pipettes every 6–12 months to ensure repeatable and reproducible results. Pipette maintenance includes tasks such as checking for wear and tear like leaks and cracks as well as cleaning and greasing pipettes per the manufacturer’s instructions. Regular maintenance and calibration can help to prevent errors in your data, which can be difficult to identify the root cause of. Maintenance and regular calibration, particularly to ISO 8655, allows for optimal results.”

Ake from Sartorius recommends a two-tier program to ensure continuous monitoring of pipetting deviations. This program includes:

• “In-lab cleaning and testing routines, with pipette performance checked in-lab regularly, for instance, daily, weekly, or monthly
• Regular calibration and maintenance of pipettes performed at an accredited service laboratory, annually, biannually, or even every 3–4 months.”

Teliz from INTEGRA has a more detailed approach: “Calibration frequency depends on pipette usage, type of samples or reagents being handled, and environmental conditions. We recommend an annual or semi-annual pipette calibration. However, before deciding on a calibration frequency, laboratory staff should always check that their SOPs or the regulations of a regulatory body do not already specify a calibration frequency that they must comply with. If no frequency is defined by a regulatory body or the SOPs, ‘as found’ readings can be useful to define a suitable calibration frequency. When we receive your pipette for calibration, we take readings from that pipette before we do any adjustments. This data is provided as "as found" readings. If you see that the majority of your pipettes fail in "as found" readings before calibration when implementing an annual calibration frequency, it might be better to have them calibrated every 6 months.”

Pipette tip selection

Pipette tip selection was a topic that the experts were mostly in agreement on.

Lampert from BRANDTECH explains that because there are many different types of pipette tips on the market, it is important to choose a pipette tip that has a proper fit with the pipette you’d like to use. “Improper tip fit can negatively impact the accuracy of your data. Of the many types of pipette tips available, it is important to consider the following factors: volume range, sterility, if a filter is needed, tip shape and design, etc.”

Ake from Sartorius adds that you should only use tips that are guaranteed by the tip manufacturer to work with your pipette. “Pipette and tip are a system that works together.” In addition, she provides the following tips:

• “If pipette and/or sample contamination is a risk, I would recommend using filtered tips. As the filter is built into the tip, it's exchanged every time a tip is exchanged, greatly reducing the cross-contamination risk.
• For work requiring sterile tips, pre-sterilized tips are a no-brainer to choose. Most of the time you can sterilize tips by yourself, but the hassle of e.g., autoclaving usually is not worth it considering the price difference between standard and pre-sterilized tips.
• Choose specialty tips, like extended length or wide orifice ones for the applications that benefit from them. Extended-length tips help avoid placing the end of the pipette inside when using long tubes, reducing contamination risk. Wide orifice tips help repeatable pipette liquid with larger particles in them.”

Teliz from INTEGRA agrees about using filtered tips to avoid contamination issues. “Every time you aspirate liquid, aerosols are generated inside the pipette tip. If you don't use filter tips, these aerosols may contaminate your pipette and, consequently, your next samples—even if you change tips in between. For example, when performing PCR applications, the cross-contamination of samples by aerosols in the pipette could lead to false positive results, as even the smallest quantities of DNA from a previous sample could be amplified. It's particularly important to use filter pipette tips when handling liquids that could damage your pipette, such as radio-labeled or corrosive samples, for both your own safety and the lifetime of your pipette.”

Pipetting errors to watch out for

There are a few common pipetting errors to look out for. 

Pipetting errors to avoid shared by Lampert from BRANDTECH include:

• “A properly fitting tip should easily fit onto the nose cone of the pipette without jamming or rocking the pipette back and forth. Consistently jamming or rocking the pipette can make it difficult to achieve a proper seal between the nose cone and tips. This can wear the nose cones over time and cause a loose-fitting tip and leaking or even tip ejection issues.
• Incomplete dispensing (not going to the second stop).
• Inconsistent technique (varying angles, immersion depths, pre-wetting, pipetting speed for more difficult liquids).”

Ake from Sartorius adds the following:

• “Using incompatible tips, which can easily cause many tens of percentages of error.
• Using (forward) pipetting technique for everything and all kinds of liquids, as in many cases reverse pipetting yields much better accuracy and repeatability.
• Being too quick with larger volumes. When pipetting, typically, over 1 mL volumes you need to give some time for the liquid to stabilize, in both aspiration and dispensing.
• Not checking and/or calibrating pipettes regularly. Routine checking in between the calibrations can help eliminate faulty pipettes from use faster than relying on annual calibration cycles (which is very common).
• Not formally training the staff for pipetting. Often, user-derived variance is the largest when it comes to pipetting. Standardizing the way you pipette helps eliminate a lot of the user-derived variance.”

And Teliz from INTEGRA adds two more:

• “Incorrect volume range: The volume range of your pipette can have an impact on both accuracy and precision. Air displacement pipettes show the best performance between 35% and 100% of their nominal volume. The closer the dispense volume is to the total volume of the pipette, the better your results. Below 35%, the volume of dead air in the pipette becomes quite large, and the risk of inaccurate and imprecise dispensing increases. On top of that, pipetting within the optimal volume range is less technique dependent and reduces user-related errors.
• Calibrate based on liquid density: Pipettes are normally tested and calibrated at the factory with distilled water at room temperature. Pipetting liquids with different densities results in inaccurate dispenses. Recalibrate your pipette if the liquid has a considerably different density than water.”

Viscous liquids, volatile solvents, and small volumes

Pipetting viscous liquids, volatile solvents, and small volumes presents a unique set of challenges. These factors often demand specialized techniques and equipment adjustments to ensure accurate pipetting. Our experts provide a handful of recommendations below.

Viscous liquids

According to Lampert at BRANDTECH, “The manufacturer’s pipetting accuracy and CV% are based on an aqueous liquid type. Any liquids that significantly deviate from aqueous solutions, such as more viscous or foaming liquids, may need different techniques. One option is reverse pipetting. If the liquid type does not aspirate and dispense accurately with reverse pipetting, it may be time to consider a positive displacement pipette or positive displacement repeater. Another option for viscous liquids is using wide bore tips or pipetting very slowly.”

Viscous samples usually enter the tip more slowly than other liquids and tend to stick to the tip wall when being dispensed because of their low elasticity. Therefore, Teliz from INTEGRA recommends the following techniques when pipetting viscous liquids:

• “Viscous samples should be aspirated and dispensed at slower speeds. In addition, pausing after every aspiration or dispense gives the liquid more time to smoothly move into or out of the tip.
• To avoid a situation where you dispense volumes that are too low because of liquid adhering to the tip's inside wall, you can use an electronic pipette with reverse mode. As it aspirates the selected volume plus an extra dispense that will be discarded, it is ideal to compensate for the retained liquid. An additional benefit of reverse pipetting is that there is no blow-out at the end. This prevents viscous liquids from starting to foam or forming air bubbles.
• Wide bore and low retention tips can also help with the smooth transfer of liquid samples ensuring maximum liquid recovery.”

Best practices for pipetting the most common viscous solutions using electronic pipettes

DMSO

• Use low-retention tips
• Use reverse pipetting
• Keep the tip in the solution for 2–3 seconds after aspiration and dispensing
• Use a pipetting speed of about 450 µL/sec or less for higher DMSO concentrations. Higher pipetting speeds may be used for lower DMSO concentrations

Glycerol

• Use wide bore tips
• Use reverse pipetting
• Keep the tip in the solution for 2–3 seconds after aspiration and dispensing
• Use a pipetting speed of about 300 µL/sec. Low glycerol concentrations are less viscous, and can be pipetted at higher speeds.

Tween 20

• Use wide bore tips
• Use reverse pipetting
• Use a pipetting speed of about 100 µL/sec

Viscous liquids pipetting best practices provided by INTEGRA

Volatile liquids

Volatile liquids can be difficult to pipette accurately.

Lampert from BRANDTECH notes that: “For volatile solvents, low temperature helps to avoid evaporation and using filtered tips prevents chemicals from going into the pipette, which can damage the internal mechanisms of the pipette.”

Ake from Sartorius notes that “with volatile solutions, the issue typically is dripping. This is due to evaporation causing an increase in vapor pressure. The key here is to stabilize the air between the liquid and piston seal. This can be achieved by pre-wetting the pipette-tip combination properly before pipetting. This can require up to 10 pre-wet cycles.”

Teliz from INTEGRA elaborates: “As they evaporate faster than aqueous solutions, tips often start to drip. To prevent dripping tips, you should pre-wet your pipette tip to humidify the dead air space. This can be achieved by aspirating and dispensing the full volume of your tip three times using the liquid you will be pipetting. Additionally, volatile solutions should be pipetted quickly and with an electronic pipette offering a reverse pipetting mode. The reverse pipetting mode incorporates a larger sample volume to minimize the effect of evaporation on the actual volume to be delivered.”

Small volumes

According to Ake from Sartorius, for small volumes, the biggest challenges come from liquid remaining in the tip after dispensing, or smaller than desired delivery because of evaporation. “Evaporation can be handled by pre-wetting the tip before pipetting, or by utilizing reverse pipetting or multi-dispensing modes (excess volume mitigates the error in actual delivery). You can minimize the liquid remaining in the tip (only applies to normal pipetting mode where you don't have excess volume) with a pipette with good blow-out function and high quality tips. Reverse pipetting can also help, as the excess volume is supposed to remain in the tip. If reverse pipetting is used to deliver small volumes, it might be a good idea to test or calibrate the pipette in reverse mode to ensure the best possible results.”