How to achieve error-free pipetting for accurate results by Noelia Teliz.
Pipetting is one of the most common laboratory tasks, forming the cornerstone of scientific research and clinical diagnostics. The impact of pipetting errors is often underestimated, but even slight discrepancies in precision – arising from both the instrument and the user – can accumulate over the course of a workflow, resulting in substantial inaccuracies in the final data. Pipetting by hand takes years of practice to perfect, but there are ways to quickly improve the accuracy of manual liquid handling for more reliable results.
Manual Pipetting best practices
Firstly, changes in pipetting angle will affect the hydrostatic pressure within the pipette tip, resulting in an inconsistent aspiration volume. Holding a pipette at an angle of 20 degrees or less from the vertical is therefore paramount for consistent liquid aspiration and dispensing, and for very small volumes of ≤30, the straighter the pipette, the better. After dispensing, there will often be a droplet left behind at the end of the pipette tip. A simple way to make sure the whole sample is dispensed into the target vessel is to perform a tip touch when removing the pipette from the vessel. There are three ways of doing this: side wall touch off, surface touch off, and direct dispensing into a liquid.
Another common source of error is caused by temperature differences between pipettes, tips and liquids. These can affect the air cushion inside the pipette, leading to volume variations. Incorporating a prewetting step into a pipetting routine helps to ensure that all labware and liquids are in equilibrium, largely overcoming this issue and providing more confidence in the accuracy of measurements. Some electronic handheld pipettes can even be programmed to include a prewetting step, making it easier to achieve precise and consistent results. During repeat dispenses, the first dispense may be too low in volume, and the final dispense could include all the accumulated errors of the previous dispenses. Discarding the first and last dispenses helps to eliminate these inconsistencies, resulting in more reliable aliquots.
Ensuring accurate volume control
Each pipette has an optimal volume range; for an air displacement pipette, this is typically 35 to 100 per cent of the device’s nominal volume. Staying within this range is therefore crucial for reducing the amount of air in the cushion, minimising errors and maintaining the best possible accuracy. In addition, poorly fitting tips can lead to leaking, quickly causing inaccurate aliquot volumes. Tips may also become misaligned or fall off, interrupting workflows and slowing down liquid handling steps. It’s essential to use high quality, compatible pipette tips – ideally from the same manufacturer as the pipette itself, rather than from a universal supplier – to avoid these common issues and promote consistent volume control.
Handling non-aqueous liquids
The calibration of air displacement in micropipettes is performed in a controlled environment with water, so pipetting viscous and volatile non-aqueous liquids – such as glycerol, DMSO, ethanol or Tween 20 – can affect the accuracy of the end result. Viscous liquids attach to the wall of the tip, making it difficult to expel the full volume, while the rapid evaporation of volatile liquids can significantly alter the pipetting volume. Specific techniques are required to ensure the best results when working with these more difficult liquid types.
1 Viscous liquids
a) Hold the pipette upright
b) Use a slower pipetting speed
c) Do not immerse the tip too far into the sample reservoir to reduce the risk of carryover
d) Use low retention, wide bore tips to enable liquids to enter more easily
e) Perform reverse pipetting to prevent incomplete filling or emptying of a pipette tip
f) Keep the pipette tip in the solution for longer, 2-3 seconds after aspiration and dispensing, to allow complete liquid transfer
2 Volatile liquids
a) Hold pipette as upright as possible
b) Use a faster pipetting speed and work quickly to prevent evaporation
c) Prewet to equilibrate air pressure and humidity to reduce evaporation
Preventative device maintenance
Pipettes can become less accurate and may cause sample contamination without the proper care, maintenance and storage. The best way to prevent this source of error and frustration is to clean and maintain pipettes regularly. The outside of a pipette should be cleaned daily with a lint-free cloth and 70 percent ethanol, and it’s important to perform a leak test and validate the pipetting volumes on a monthly basis to make sure that the pipette is working as intended. If its accuracy and precision are not within specifications, the device needs to be calibrated before further use. Calibration carried out at least every 12 months will help to keep pipettes working at their best. In addition, if the pipette is dropped, then it should immediately be checked for damage and recalibrated before use to ensure it still meets the specifications. Cleaning, maintaining and regularly calibrating pipettes will give them a long lifetime of reliable activity, and also helps to keep users safe from potential malfunction and contamination.
In summary, good pipetting practices, combined with regular maintenance, help to maintain consistent pipetting, leading to fewer manual errors and contributing to more reliable and accurate results.
Noelia Teliz is with INTEGRA Biosciences
