Modern biology lab work is increasingly characterized by the manipulation of very small volumes and quantities of sample. Several factors explain this trend: high-value samples, the ability to detect changes accurately in low volumes, and the continuing replacement of radiolabeling with optical (particularly fluorescence) techniques that enable multiplexed assays. The venerable test tube gave way to vials, which have been replaced, for the most part, by microplates in which microliter reaction volumes and nanoliter reagent additions are quickly becoming the norm. Within these tiny volumes, factors like temperature, pipetting technique, and dispensing accuracy take on extreme significance.
Temperature uniformity becomes a significant factor in low-volume pipetting. Kyle Briggs, product manager at Biotix (San Diego, CA), relates his experiences during drip testing. “I could take a cold pipette, draw a roomtemperature sample and my hand would warm the air inside the pipette, causing expansion and making a droplet form at the tip.”
In a landmark 2007 article in Nature Methods, Gilson engineer Frédéric Millet stressed the importance of allowing samples, pipettes, and tips to equilibrate to ambient temperature. Millet had discovered that pipetting cold samples led to inconsistent volume delivery, which led him to the recommendation that operators pre-wet the tip before final aspiration/ dispensing, which “consists in aspirating and dispensing the sample liquid three or four times before aspirating for sample delivery. This technique, albeit inappropriate to enhance accuracy, permits [operators] to obtain a better reproducibility, a key factor in the most commonly used laboratory protocols involving repetitive pipetting tasks such as ELISA or PCR.”
Errors result from the fact that a liquid’s viscosity, and to a similar degree volume, changes with temperature. “So if the sample is very cold it will be very difficult to aspirate micro volumes,” Briggs adds. “The same goes if room temperature is too cold. For regular volumes, temperature does not really matter, but for 0.2 microliters, it certainly does.”
Gilson supplies additional tips for accurate low-volume pipetting:
- Accuracy depends on you: know your pipette, maintain it, and practice good technique.
- Consider a positive displacement pipette (vs. an air displacement model). Air displacement models are prone to temperature effects as air expands to a greater degree with temperatures than liquids. Downside: expense due to replacement of tip and displacement barrel.
- Service pipettes every six to 12 months; check and clean daily, and store them vertically.
- Best practices for pipettes include pipetting with a slow, smooth action, holding the pipette vertically during draw, shallow immersion of tips into samples, and pipetting onto sidewalls or into liquids instead of air.
- Check pipette accuracy on an analytical balance with liquid masses in the 0.1 g range. Variation greater than 0.5% deserves a check of equipment and/or technique. Keep in mind that analytical balances are far more accurate than any pipette.
- Dispense volumes appropriate for your instrument, but at the same time use the largest volume possible for your experiment. For example, consider diluting samples to deliver the same quantity of reagent in a larger volume. Good practice vs. best practice
Kent Koeman, director of pipette technique training at TTE Laboratories (Hopkinton, MA), agrees with Gilson on pre-wetting tips and offers further insight into pre-wetting and other finer points of technique.
“Pre-wetting serves to condition the inside of the tip to the properties of the liquid. A common mistake made during pre-wetting is not ensuring that the tip is visibly dry before aspiration. It’s important therefore to pre-wet by aspirating two or three times, then dispense completely into the source, so the tip is visibly dry.” New tips need not be used unless cross-contamination is an issue, but when they are, they should also be pre-wetted.
“Try to be consistent when installing the pipette tip. Jamming the tip can damage it,” says Koeman. Note that some pipette manufacturers have eliminated this variable by engineering spring-loaded tip cones. “We have observed that, especially with pipettes delivering higher volumes, installing the tip with greater force can lead to a higher measured volume.”
Koeman recommends holding pipettes at 90 degrees to the sample during aspiration, and 45 degrees for dispensing.
“Pipettes are made to work with the forces of gravity, so a 90-degree aspiration is important. We don’t see too much variation in the measured volume until the aspiration angle reaches approximately 60 degrees, but 90 degrees is best.” The goal during dispensing is complete voiding of the tip, which a 45-degree angle facilitates. “Precise delivery depends upon consistent technique, but accurate delivery depends more on correct technique. Combine the two and you have best practice.”
Koeman advises not handling pipettes when they are not in use, mostly for reasons of temperature control. “I don’t think this recommendation is overdoing it, not one bit.” He relates experiences at a QC training session at a major pharmaceutical company which was experiencing user-related pipette failures. “The data showed a clear change in volume when the pipettes were held more than two minutes.”
Accurate pipetting of very small volumes requires the same care and best practices users should follow for larger volumes, but to a somewhat greater degree. “Users must be aware of all the factors that influence measurements,” Koeman says. Users must remember the immutable physical truth that systematic error, which exists in any measurement, has the potential for greater impact at 5 microliters than at 500 microliters.
Similarly, calibration confers a level of trustworthiness that is only as good as the calibration method itself, so extra care must be taken at super-low volumes. For low-volume instruments, that means comparing results gravimetrically using a stationary six- or seven-place balance in a controlled environment.
The criticality of pipetting therefore comes into play. “All of our QC pharma customers require a proof-of-calibration testing, in the form of an ISO certificate, that their pipettes are performing within specifications. Some unregulated labs do not have these requirements, but should nevertheless always use pipettes that function correctly and deliver reliable results.”
For additional resources on pipettes including useful articles and a list of manufacturers, visit www.labmanager.com/pipettes