The Art of Pipetting - Top Tips

In wet laboratory research, using pipettes is a daily staple for scientists and researchers. Pipette technique, like most things when you first walk into the laboratory, is passed on from the initial person who shows you the process. The art of pipetting, unlike other technical instruments used in laboratories, does not have quality controls as part of its routine use and usually the only aspect of pipetting which is calibrated is its servicing. Depending on laboratory funding and usage of the pipettes, the frequency and quality of this will vary between laboratories. The variation in technique and servicing can contribute to a lack of reproducibility observed in scientific findings and highlights the importance of teaching correct pipette technique in a standardised, practical way, early on in a scientist’s career. It is easy to see how experienced scientists can become offended when pipette training is suggested, but it is the core technique of being in the laboratory and science is about finding best practice, optimising techniques and handling equipment in the manner it has been designed to be used. So, these are our hands-on pipetting tips, which are something we discuss (and practise) in our Pipetting Skills course as part of the Laboratory Skills training series, in the Advance Training Centre at MRC Harwell Institute in Oxfordshire.

Ergonomics

Although the health and safety involved with pipetting is often rushed or improperly discussed, it is a vitally important consideration. Dismissal can contribute to chronic pain and poor pipetting performance. By its very nature, pipetting is a repetitive technique which can be performed daily and for long hours, even in this era of automation. Pipetting is a skill that most researchers do without active consideration until they start developing some discomfort. Frequently, the first sign that something is wrong is feeling pain in your thumb, elbow, hand, wrist, neck or shoulders. If ignored it can continue until it becomes more frequent and a repetitive strain injury (RSI) develops. Therefore, when pain develops, do not carry on. Stop and investigate, as your body is trying to tell you something.
Commonly, RSI of these regions of the body is caused by poor posture, repetition and thumb force. Being informed of injuries and risk factors before you begin working in the laboratory and selecting ergonomic pipettes will help to avoid injury. As with all techniques, it is best to instil good working practices early on so that it becomes second nature and minimises the risk of RSI. We undergo H&S assessment for setting up our workstations in the office and working with display screen equipment, so the same care should apply for laboratory training and pipetting. Before beginning to pipette you need to consider the following:
•    Aim to ensure that tasks, equipment and environment suit individual needs.
•    Ensure recovery time between sessions to prevent arm or hand RSI by taking regular breaks or changing tasks after 30 minutes.
•    Hand, wrist or arm injuries can lead to pipetting errors, so use ergonomically designed pipettes to reduce the force applied. Plunger and tip ejection can lead to thumb, finger and wrist strains. The force required to perform these actions will depend on the age and make of the pipette so this needs taking into consideration. Do not forget to adjust the volume using two hands, not a thumb twist!
•    Consider using multi-channel pipettes to reduce the number of steps required. Additionally, electronic pipettes are a sensible choice for people with existing conditions affecting hand movement or grip as these reduce the steps further.
•    Ensure your work area is suitable to maintain good posture whilst working to avoid excessive wrist flexion or extension, awkward neck positions or slouching forward. Having adjustable worktops and chairs to cater to the individual is the ideal scenario.

Choosing the right pipette for the job

There are many types of pipettes, including manual, electronic, single-channel, multi-channel, repeat dispensers. Choosing the right pipette depends on several factors.
The liquid to be pipetted can have a large bearing on pipette choice and whether to use ‘forward’ or ‘reverse’ pipetting techniques. Larger volumes or aqueous solutions frequently use air displacement pipettes and ‘forward’ pipetting, which involves a blow out in the final step to ensure all liquid has been dispensed. With volatile or highly viscous samples, positive displacement pipettes are considered more suitable. ‘Reverse’ pipetting, where an excess volume is aspirated, the desired volume is dispensed and surplus disposed of is often used for biological, foaming (protein/detergent rich), oily/viscous fluids and small volumes. Electronic and multi-dispensing pipettes use reverse pipetting routinely. When performed with a manual pipette, it is important to ensure they are calibrated as such. Pipettes are only accurate for forward or reverse pipetting and should be labelled appropriately.

The volume and number of samples will have a large impact on the work. To set up a single 96 well PCR plate can take 576 individual pipetting steps with a manual single channel pipette (6 reagents x 96 wells). Whilst with a manual 8 channel pipette this is reduced to 72 steps or 48 steps with a 12 channel pipette. The reduction in steps dramatically reduces the impact with regards to RSI risks and time, whilst additionally improving the accuracy of pipetting and productivity. Using electronic pipettes can further increase efficiency.
As is always the case in science, cost will be a factor. Manual pipettes are cheaper and simpler to use than an electronic pipette. The latter can give more reliable results as user inconsistencies are minimised by their motorised piston, they are less reliant on technique and often have multiple functions. These benefits can be further incorporated to include electronic multi-channel pipettes. To increase efficiency and reduce pipetting errors when working between different well formats the use of Adjustable Spacer pipettes are a suitable option to consider.

Factors affecting accuracy and precision

Whilst the pipette is a tool, the way we use it can affect its exactness in measuring liquid samples. ‘Accuracy’ and ‘precision’ are important terms in pipetting. Accuracy describes how close the real volume of the liquid delivered is to the set volume on the pipette. Precision defines how similar repeated measurements of the same volume are to each other. Therefore, it is possible to be precise but not accurate, or accurate and not precise, as shown in Figure 1.
Figure 1. Accuracy and precision in pipetting. It is possible to be precise but not accurate, or accurate and not precise.
Some of the factors for poor accuracy and precision are demonstrated in Figure 2, where tip use, uncalibrated pipette use or poor pipetting technique can significantly change the delivered sample volume:

Figure 2. Factors affecting accuracy and precision.
It is a common enough event, albeit one no one wants to admit to, that scientists use the tips they have available. Incorrect tip use reduces the quality of the liquid uptake by the pipette as it compromises the air seal of the pipette-tip system. Good techniques to practice include:
•    Use racked tips to minimise the risk of damaging the tip-cone. Usually, the best performing tip is the manufacturer’s tip.
•    Pre-rinse tips to improve precision and accuracy when using forward pipetting.
•    Avoid tilting the pipette whilst aspirating as this can increase the volume in the tip to greater than set. Immerse at a vertical angle for aspiration at a depth of 2 mm.
•    Eject tips using the tip-ejector button only, not with fingers.
•    Ensure pipettes are in good condition. Wear and tear can affect accuracy, particularly on metal pistons exposed to corrosives and piston ‘O’ rings perish over time.
Laboratory conditions can additionally affect accuracy and precision. Changes in environmental temperature, humidity and air pressure can affect the real volume of sample uptake and density of the liquid. Dry air can result in sample evaporation inside the pipette leading to inaccuracy in the volumes measured and pipette contamination. We recommend pipettes are calibrated in the laboratory conditions they are used under.
Accurate and precise pipettes are critical for reproducible results; we recommend the following habits to routinely follow:
•    Regularly clean, maintain and calibrate pipettes for best results but do not disassemble the tip-cone or piston for cleaning as it will need recalibrating.
•    Make use of annual pipette clinics to service pipettes and send damaged or poorly performing pipettes for ad hoc calibration.
•    Store pipettes in an upright position to prevent liquid in the tips from damaging the seal.
•    Never lend out your pipettes.
•    The pipette itself should never ever touch any liquid.
•    Never overwind or underwind a pipette outside of its prescribed volume range.
•    Use the smallest pipette for any given volume (e.g. a 20 µl pipette for 15 µl, and not the 200 µl)
•    Consider training new pipette users and anyone wanting a refresher to enhance the integrity of the laboratory data.

About the Author

Lee Moir provides the Pipetting Skills course as part of the Laboratory Skills training series, in the Advance Training Centre at MRC Harwell Institute in Oxfordshire, UK.
The course can be booked online:
https://www.har.mrc.ac.uk/training/courses/pipetting/