For many of us the process of pipetting transitioned into a subconscious routine to which we devote little thought after only a little practice, despite quality assurance placing great emphasis on liquid handling, pipette accuracy, repair and maintenance. Nevertheless, ensuring the competence of pipette operators (lab employees) is a way-too-often often neglected activity. In reality, even highly experienced laboratory technicians rarely received formal pipetting training, making them prone to pipetting errors that can be avoided with a few simple steps.
As your laboratory’s demand for accuracy and precision increases, so does the importance of understanding and developing an optimal pipetting technique. Walk through the following 12 steps that will bring your pipetting to the next level, and see how many you have already mastered and integrated as part of your everyday pipetting routine.
How good are you?
1. Pre-Wet the Pipette Tip
Aspirate and expel any sample liquid at least three times before aspirating a sample for delivery. Evaporation within the tip can cause significant sample loss before delivery. Pre-wetting increases humidity within the tip, thus reducing any variation in sample evaporation. Using the same tip to deliver multiple samples without pre-wetting can result in a lower volume in the first few samples. The need to pre-wet increases when working with volatile samples (i.e. organic solvents).
2. Immerse the Tip to the Proper Depth During Aspiration
Before aspirating, immerse the tip adequately below the meniscus. Large volume pipettes (1-5 mL) should be immersed to 5-6 mm, while smaller volume pipettes should be immersed to 2-3 mm. Too little immersion, particularly with large volume pipettes, can lead to air aspiration. Too much immersion can cause samples to cling to the outside of the tip. Touching the container bottom with the tip may restrict aspiration.
3. Pause Consistently After Aspiration
Leave the tip still in the liquid for about one second after aspirating the sample. It takes a moment for the liquid in the tip to finish moving after the plunger stops, so failure to do so will cause the volume to be too low. Make this pauses as consistent as possible.
4. Use Consistent Plunger Pressure and Speed
Press down and release the plunger smoothly and consistently. Try to apply the same pressure and speed when aspirating and dispensing each sample. Repeatable actions produce repeatable results.
5. Pull the Pipette Straight Out
During sample aspiration always hold the pipette vertically and avoid touching the sides of the container. After sample aspiration pull the pipette straight out of the liquid from the centre of the container. This technique is especially important when pipetting small volumes (<50 µL). Holding the pipette at an angle as it is removed from the sample alters the volume aspirated. Touching the sides of the container can cause wicking and a loss of volume due to the effects of surface tension.
6. Examine the Tip BEFORE Dispensing a Sample
Before dispensing, carefully remove droplets on the outside of the tip with a lint-free cloth, being sure to stay clear of the tip opening. Absorbent material rapidly sucks the sample from the tip if it comes into contact at the opening, and unnecessary wiping of the tip increases the possibility of sample loss so use caution.
7. Examine the Tip AFTER Dispensing a Sample
While dispensing a sample, position the tip so that it touches the side of the container to deliver any residual sample remaining in the tip. Keep your thumb pressed on the second stop of the plunger and remove the tip to avoid sample re-aspiration into the pipette tip. Make sure that you see the sample leaving the tip.
8. Use Standard Mode Pipetting
Choose “standard (or forward) mode” pipetting rather than “reverse mode” for all aqueous samples, but you might consider it for particularly viscous or volatile samples. If the reverse mode is used with normal aqueous samples, the pipette tends to deliver more than the calibrated volume. On the other hand, using the standard mode with viscous or volatile samples may result in under-delivery.
9. Use the Appropriate Pipette
It is important to use a pipette with a volume range closest to the volume you plan to aspirate and dispense. The accuracy of your test will improve if there is a small difference between a pipette’s minimum volume and the volume being tested. For example, if you need to dispense 15 µL, a 1 mL pipette would be the wrong choice, whereas a 20 µL pipette would be ideal.
10. Use the Correct Pipette Tip
Use high quality tips intended for use with specific pipettes. In most cases, manufacturer tips perform well. Alternative brands are also acceptable if their performance has been proven with a specific pipette model. Mismatched tips and pipettes can result in inaccuracy, imprecision, or both. Quality tips provide an airtight seal without the need for excessive force, are made of superior materials and are free of molding defects, thus ensuring reliable liquid delivery.
11. Work at Ambient Temperature Equilibrium
Allow liquids and equipment to reach an equilibrium at an ambient temperature before you begin pipetting. The volume of a sample delivered by air displacement pipettes varies with air pressure, relative humidity and the liquid’s vapor pressure, all of which are temperature dependent properties. Working at a single, constant temperature minimizes this variation and improves overall precision.
12. Minimize Pipette Handling
Hold the pipette loosely, return it to the pipette stand or set it down when you are not pipetting. Always wear gloves to reduce body heat transfer to the pipette and avoid handling pipette tips or containers of samples yet to be pipetted. Body heat transferred during handling disturbs the temperature equilibrium, which can lead to variations in delivered volumes.
- Five Good Reasons The Argument for Pipetting Technique Training
- 10 Tips To Improve Your Pipetting Technique
- An Introduction to Microvolumetrics and Pipetting
- Getting the Best Results
- How to Improve Pipetting Technique
- Guide to Pipetting
- 10 Ways to Prevent Pipetting Errors
Urška Čepin, PhD, is the Head of Application Specialists at BioSistemika LLC. Her specialities are PCR and liquid handling.