Precise and repeated dispensing of small and large volumes of liquids is central to ‘wet lab research’ in biological laboratories. Over the past few decades wet lab research has become increasingly fast-paced and hi-tech, covering the spectrum between low and high throughput assays that bank on the quick and accurate transfer of milliliter to sub-microliter volumes of diverse liquid samples and reagents. The manual pipette is therefore a universal fixture in all wet labs.
A number of errors can creep into your experiment during liquid handing that can be classified into systematic errors (accuracy) that measure how close the volume measured is to the desired reading, and random errors (precision) that measure how close the dispensed volume is to other dispensations of the same reading, assessing the reproducibility of the device1. A specific measure of transferred liquid can contribute to both systematic and random errors.
Factors Impacting Pipetting
Just like a magnificent rendition of Mozart by a world famous symphony can be ruined by an erring cell phone, the masterful accuracy and precision of the best pipettes can be compromised by a rise in the temperature, the angle at which you tilt the tip in the reagent, the force and speed with which you press and release the plunger, failing to consider the physical properties of the liquid, or a wobbly tip at the pipette’s nose. Given the high frequency of liquid handling in any wet lab, and the virtual impossibility of detecting miniscule discrepancies between individual pipetting dispensations by the naked eye that can have a significant impact on your results, it is crucial to take precautions against potential sources of variability rather than rectify a problem post hoc. An overlooked variable can result in expensive re-do’s, loss of precious samples and reagents, and irrecoverable delays.
Environmental factors like temperature, humidity, barometric pressure, partial pressure, altitude, vibrations, and air currents can affect not only the rate of evaporation of liquids and thereby the volume of liquid aspirated and dispensed while pipetting, but can also impact calibrations done under non-standard conditions. Generally, increased temperature and decreased relative humidity reduce the delivered volume2. To ensure maximum accuracy, it is best to test and optimize pipettes for the local environment.
When it comes to tip selection, it pays to match the type of tip to the type of application you intend to use it for. Extended length tips, such as Thermo Scientific ClipTip 300 Ext, are particularly useful when you need to reach the bottom of a long vial, or load a gel with deep wells. They also reduce the risk of contamination by lowering the likelihood of contact between the vial wall and the pipette. Thermo Scientific ART Solvent-safe carbon filter tips protect the pipette, and the sample from corrosive fumes and vapors. Wide orifice tips are best suited for transferring cells because they prevent shearing by reducing resistance to flow. Individually wrapped tips are apt for strict aseptic liquid transfers. Accuracy in transferring small volumes can be increased by using tips designed for the purpose, preferably in conjunction with the pipette, such as the Thermo Scientific Finntip Flex pipette tips designed specifically for Finnpipette pipettes1. Using speciality tips designed for particular applications can increase productivity, accuracy, and precision, while reducing cost, experimental time, and the risk of contamination.
After aspirating the sample, it’s best to pull the pipette straight out of the sample without touching the tip to the sides of the container. This is particularly important in the case of small volumes as surface tension causes the sample volume to vary if the exit angles are not comparable. Moreover, touching the tip to the wall of the sample can cause liquid to seep out of the tip, reducing the volume aspirated.
The seal between the tip base and the pipette shaft is crucial to aspirating and dispensing intended volumes. Conventional pipettes rely on the friction between tip base and pipette shaft to form a secure attachment, a system heavily reliant on user-exerted force. The new Thermo Scientific ClipTip pipetting system, available in both manual and electronic pipettes, circumvents this problem by introducing a flange on the surface of the pipette shaft that precisely fits the protruding clip on specially designed ClipTip tips3,4. This system allows for a more ergonomically comfortable experience and improves data reproducibility.
Plunger Speed and Force
To prevent repetitive strain injury (RSI) and as part of the Good Laboratory Pipetting (GLP) practice, it is important to use a minimum of force when pipetting. Releasing the plunger slowly in a smooth motion during aspiration not only reduces thumb strain, it also prevents the formation of bubbles in the aspirated liquid. The formation of tiny bubbles, particularly in viscous liquids or detergents, can significantly reduce the volume of liquid aspirated and transferred. It's also important to pipette parallel samples at the same speed for optimal accuracy and precision. Using electronic pipettes with adjustable speed will eliminate any variation in pipetting speed.
Liquid Retention in the Tip
When dispensing viscous liquids (e.g., oils), low surface tension-liquids (e.g., detergents), or very small volumes, a substantial portion of the liquid adheres to the inner and outer walls of the pipette tip even after you’ve pressed the plunger down completely to expel the liquid. This naturally affects accuracy, with the interior-retained liquid reducing the volume dispensed and exterior-retained liquid increasing it. Tips labelled as "low-retention tips" (e.g., Thermo Scientific ART barrier tips) can be used in such cases to deliver more accurate volumes and conserve reagents5.
All else remaining constant, the smaller the volume dispensed the greater the risk of error2. To prevent the repeated dispensing of very small volumes, use larger volumes wherever possible. This can be accomplished by using master-mixes for assays such as PCRs, where all reagents except for the DNA sample, are combined and dispensed, avoiding the need to transfer very small volumes. Pipettes that are specially designed for pipetting small volumes (0.1-2 µl) are also available.
The physical properties of the transferred liquid (e.g., viscosity, volatility, solubility, and temperature) may cause significant liquid handling discrepancies if not accounted for6. Commonly used laboratory pipettes work on an air displacement principle where an air space is maintained between the liquid in the tip and the piston. Dense liquids exert a greater force on the air space, resulting in a smaller volume being aspirated into the tip. Volatile liquids when aspirated into the tip evaporate into the tip’s air space, causing the air space to expand and the liquid to leak from the tip. Regulating pipetting speed and technique can reduce the effects of a liquid’s physical properties or the volume dispensed.
The standard pipettes commonly used in the wet lab may have overlapping ranges of operation. For example, some 200 µl pipettes can handle volumes as small as 1 µl. Generally, whenever possible, it is best to choose a pipette where your desired volume is closer to the upper range of the pipette to increase accuracy.
The Importance of Good Technique
Pipetting is physically and mentally challenging and laborious, particularly for large sample sizes in complex, multi-component assays. good technique is paramount whether using single or multichannel instruments. Surprisingly, given the complexity of precise and safe pipetting, very little training is offered and students are often expected to learn on the fly. Here are some practical steps you can take to preserve your health and improve the quality of your data.
Forward and Reverse Pipetting Techniques
When transferring common liquids, a forward pipetting technique is recommended. First press the plunger to the first stop, submerge the tip into the liquid then slowly aspirate the set volume to prevent bubbles and inadvertent suction into the pipette's shaft. The first stop is changeable and is determined by the amount you set the pipette to. If you go beyond the first stop while aspirating you're pulling up more that the volume you've set your pipette to. When dispensing touch the tip to the wall of the tube, press the plunger to the first stop, and then slowly to the final stop, expelling all the liquid from the tip. The second stop provides an extra push that is sometimes needed to expel the entire volume from the tip. For highly viscous liquids or solutions likely to form bubbles you might use a reverse-technique where you press the plunger all the way down before you submerge the tip into the viscous liquid, aspirate very slowly allowing extra time for the viscous liquid to rise into the tip, and when dispensing, press the plunger only to the first stop, allowing a portion of the liquid to remain in the tip which was not part of the set measure, and can be discarded or returned to the reagent tube.
Positive Displacement Repeat Pipette Considerations
Repeat pipettes, such as the Thermo Scientific Finnpipette Stepper Pipette, utilize positive displacement pipetting. These pipettes are ideal to dispense a single specified volume into multiple receptacles. Instead of a plunger, these pipettes possess a filling and dispensing lever and are used in conjunction with specialized large volume syringe-type tips, precluding the need for repeated aspirations and thereby saving the user time and effort. In positive displacement pipettes, the piston is part of the tip and in direct contact with the liquid. This makes positive displacement pipettes ideal for pipetting viscous and volatile liquids.
Since pipetting at the lab bench is inherently a repetitive task, it is important to be mindful of your posture. Before beginning your experiment, set up your workstation to minimize movement. Sit or stand keeping the spine erect and the neck stress-free while pipetting. Take regular breaks to relieve any physical or mental stress. Don’t attach the tips too tightly as repeated tip-ejection will exhaust your thumb. For repetitive pipetting tasks, electronic pipettes with motorized pipetting action are recommended. Some electronic pipettes, such as the Thermo Scientific E1-ClipTip, also use motorized tip ejection. When transferring samples between different labware formats such as 96-well plates and microcentrifuge tubes, a Thermo Scientific E1-ClipTip Equalizer multichannel pipette with adjustable tip spacing can reduce both time required and tiresome repetition by up to 90% compared to single channel pipettes7. Pipetting systems with interlocking tip attachment mechanisms such as Thermo Scientific E1-ClipTip and F1-ClipTip pipettes allow significantly lower tip attachment and ejection forces compared to traditional friction based systems reducing the impact on your wrist while attaching tips, and the strain on your thumb while ejecting tips3,4.
Beware of Contamination
Contaminants are everywhere, whether visible or not. Sometimes the best pipetting techniques, and the most skilfully executed experiments are brought to naught by contaminants in the sample or reagents. Although you’re not expected to be donned in a containment suit, being mindful of some simple rules while transferring liquids can radically reduce the risk of contamination in your experiments.
- Before starting your experiment, wipe down all instruments, gloves, and your entire workstation with 70% alcohol to remove dust and debris, or wipe down with bleach if working with microorganisms.
- When working with RNA do not touch anything with your bare hands that are teaming with RNA cleaving enzymes, and wipe down everything with an RNase-inhibitor.
- Place pipettes upright in stands to prevent dropping them, knocking them around, or touching them with bare hands.
- When aspirating liquids from large reagent bottles never immerse the shaft of the pipette into the liquid to prevent contamination, and to preserve the health and safety of your pipette.
- When possible use filter tips to prevent cross-contamination. Thermo Scientific’s ART universal pipette tips fitted with a self-sealing barrier block vapors or aerosols from crossing the filter by eliminating contamination of pipettes and sample cross-contamination during pipetting.
- Do not touch the pipette tip even with gloved hands to remove it from the nose of the pipette. Always use the ejector to remove tips.
- Always have the receptacle for the liquid ready before you aspirate the liquid from its container. This precludes the possibility of having to place the pipette on its side while getting the receptacle ready, as this may cause the liquid to trickle into the interior of the pipette.
- Do not reuse tips. Tips are designed for single-use only. Any manipulation to reuse tips destroys the metrological characteristics of the polypropylene or polycarbonate material, making the volume of transferred liquid unreliable.
Maintenance and Calibration
Pipettes, like all equipment are subject to wear and tear. Based on the duration and frequency of use, the best of pipettes undergo some shift in optimal performance. Routine maintenance and calibration is the best way to keep your pipettes in tip-top shape. Calibration of a pipette is the process of determining the difference between the average volume of a series of measurements and the set volume on the pipette’s display. Adjustment of a pipette involves changing the internal settings of the pipette so that the volume set on the pipette’s display matches the actual volume measured. Pamper your pipettes with periodic care and maintenance. Set up a schedule for getting your lab’s pipettes professionally cleaned and ensure their optimal performance by routine maintenance and service. Periodic, preventative maintenance every 6 to 12 months, based on the frequency of use, can pinpoint problems in a pipette before experiments are affected. This preventative action is vital because minute discrepancies in liquid volumes cannot be detected by the naked eye, resulting in data inaccuracies before the user realizes that there could be an issue.
The Art of Precision Pipetting
Accurate and precise manual pipetting approximates the diligence and dedication of an art form, whether using a single-channel, multichannel, or repeat-dispenser pipette. Being mindful of the multifarious causes that allow discrepancies to creep into your pipetting, and taking preventive measures by committing to established good practice guidelines and periodic calibrations, will ensure the quality and reproducibility of your data.
- Thermo Fisher Scientific. Finland. “Improve pipetting performance with pipette systems: Thermo Scientific Finnpipette F2 pipettes and Thermo Scientific Finntip Flex pipette tips,” Application Note, APLTF2Finntip0615, 2015.
- G. Rodrigues, D. Rumery, “Extreme Pipetting II,” PFQ, Wiley Periodicals, Inc. September 2007.
- Thermo Fisher Scientific, USA. “Breakthrough ClipTip technology,” 2014.
- S. Koivisto, S. Berghäll. Thermo Scientific. “ClipTip Technology—Part 1. Transform Your Daily Pipetting” Application Note, ANHPF1ClipTip0912, 2012.
- P. Heimler. Thermo Fisher Scientific, USA “Choosing the Best Pipette Tip for your Application” ANHPTTipSelect0612, 2012.
- S. Koivisto, Thermo Fisher Scientific. “Effect of Liquid Properties in Pipetting Liquid Handling Note – No. 1” AN-HP-PIPETTING1-1109. Lit. no. 151 7160. 2009.
- Thermo Fisher Scientific. “Is there an easier and more efficient way to transfer liquids between various labware formats, than using traditional handheld pipettes?” SMHPE1CT0214, 2014.
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