How to use serological pipets
How to use serological pipets and pipet controllers
Volumetric measurements using serological pipets are a part of daily life in the lab. Despite their simplicity, there are best practices to follow to ensure accurate and precise liquid measurement, ideally with a pipet controller such as the PIPETBOY GENIUS with repeat dispense mode and overfill protection.
Start with the basics
Accuracy and precision
What is meant by accuracy and precision? Accuracy refers to how close a dispensed volume is to its specified value and is determined by the design and quality of the instrument and consumables. Precision is a measure of repeatability – the ability to pipette multiple aliquots of a specified volume with the same result each time – and is largely dependent on following best pipetting practices.
Serological pipets are volumetric tools designed for the accurate transfer of liquids in the milliliter range. They are usually calibrated with deionized water 'to deliver' (TD, Ex) with an accuracy of ±2 % at 20 °C; this cannot be improved on, even with perfect liquid handling practices. These essential laboratory tools are calibrated to take into account the wetting residue remaining in the pipet after liquid transfer, as the volume aspirated is slightly more than that dispensed. A blow-out is mandatory to ensure all the liquid is delivered. Some liquid will subsequently accumulate at the tip after the blow out, but this is not part of the dispense volume. It is important to realize that, since the calibration/reference temperature is 20 °C, any deviation from this temperature will require correction to maintain pipetting accuracy and precision; users must aspirate liquid to slightly above or below the pipet graduation mark to compensate for under or over delivery when pipetting at temperatures other than 20 °C.
Following best practices
Temperature
It is important to allow time for the liquid to equilibrate to the environmental temperature before pipetting, and to consider the influence of temperature on the expansion of both air and liquid. Air expansion is influenced by the temperature of the liquid being pipetted; the more air there is in the tip, the greater the influence.
Pipetting warm or cold liquids at room temperature without equilibration leads to different degrees of expansion of the air within the serological pipet, which may not match with the graduations. Ideally, the pipet, pipet controller and liquid should be equilibrated to the same temperature. This avoids significant temperature differences between the pipet and the liquid. Pre-wetting of the pipet by aspirating and dispensing the liquid two or three times helps to equilibrate the environment inside the tip, to reduce the extent of any error.
Liquid density
Aspiration of a liquid is influenced by its volumetric mass density – the mass-to-volume ratio – commonly referred to as density. Serological pipets are calibrated using deionized water, but may be used to pipette liquids of differing densities. For example, they may be used to aspirate and dispense non-aqueous fluids with a lower or higher density than water. It may therefore be necessary to aspirate more or less liquid to compensate for differences in density. The volume dispensed can be check-weighed using a laboratory balance to ensure accuracy.
Non-aqueous liquids
The pipetting technique needs to be adjusted according to liquid properties such as viscosity and volatility to ensure proper handling. For example, volatile liquids evaporate rapidly, so aspirating and dispensing as swiftly as possible, as well as prewetting, is beneficial. The opposite is true for viscous liquids, where it is important to allow sufficient time to ensure complete aspiration and dispensing of the required volume and slow aspiration and dispense speeds are relevant.
Altitude
Different geographic altitudes have different air pressures. This can impact the results if the pipet is used at a location with a significantly different altitude from the calibration site. Aspirate a small, defined volume of water and weigh it on a balance to check for any deviation, then adjust the volume dispensed as necessary.
The correct pipet volume
Dead air volume has a major effect on pipetting accuracy, as the air may expand with rising temperature. The smaller the dispense volume, the more important this becomes. Choose the smallest pipet possible to minimize dead air volume. For example, use a 10 ml (or even 5 ml with extended volume) pipet to dispense 8 ml of liquid. Serological pipets are only as precise as their smallest increment, which is indicated on the upper end of the pipet. The smallest volume you can dispense is indicated at the upper end of the serological pipet; 1 ml in 1/100 means that the smallest increment is 0.01 ml.
Reading the liquid meniscus
The meniscus describes a curvature of the surface of a liquid, which may be upwards or downwards, and is caused by the interplay between the adhesive and cohesive forces of the liquid and the diameter of the serological pipet. The correct volume is usually established by reading the concave meniscus, where slightly more liquid is aspirated than required before dispensing to the graduation line. The pipet must be held in an upright position at eye level, so that the graduation ring appears as a line, reading the volume with the lowest point of the meniscus touching the upper edge of the graduation mark. The meniscus will appear darker and be easier to read in front of a light background.
Pipet controller hints and tips
The pipet controller must be set up prior to pipetting, as below.
For 1 and 2 ml pipets:
- Preset the pipetting speed to minimum, to avoid overfilling
- Depending on the pipet controller, turn around the silicon holder in the sterile module (nose piece) to ensure that the pipet is held securely.
For 5-100 ml pipets:
- Preset the pipetting speed to maximum, or choose the speed that works best for your pipetting volume and sample.
- When working with 25-100 ml pipets, choose ones with an anti-drip design, if possible, to reduce the risk of spills.
Note: With INTEGRA’s PIPETBOY pipet controllers, the speed of aspiration and dispense can be controlled by applying pressure to the pipet controller buttons until the defined speed is reached. In addition, the maximum speed can be preset using the thumb wheel.
For all serological pipets
Unpack the pipet from the upper end to keep it sterile as long as possible, then attach it to the pipet controller. Immerse the tip into the liquid and pre-wet by aspirating and dispensing the full volume of the pipet two or three times. Aspirate slightly more than the desired liquid volume, then wipe the outside of the pipet to remove any remaining liquid. Holding the pipet at eye level, dispense to waste until the bottom of the meniscus reaches the correct graduation. Dispense all remaining liquid into the target vessel while touching the tip on the vessel wall, then blow-out a small volume of air to ensure complete liquid delivery.
Note: INTEGRA PIPETBOYs allow drop-by-drop dispensing, for a highly controlled liquid dispense and low shear forces when working with sensitive cells or viscous liquids.
Performing repeat dispenses with a pipet controller
Depending on the application, it is often useful to aspirate one large volume, then dispense multiple smaller aliquots. Traditional pipet controllers require visually controlled dispensing of the required aliquots according to the graduation marks on the pipet body. Alternatively, you can use a pipet controller with a repeat dispense mode. INTEGRA’s PIPETBOY GENIUS allows users to dispense multiple aliquots of the same user-defined volume at the press of a button, without the need for visual inspection.*
*Only when used in combination with INTEGRA serological pipets.
Pipet controller maintenance to avoid contamination
Regularly clean pipet controllers with a cloth moistened with soapy water or 70 % ethanol, and replace the pipette mount and filter rubber in the sterile module if they are damaged. The sterile module can usually be autoclaved. The hydrophobic filter prevents excess liquid entering the device, and should be replaced every three months or if it has been contaminated by overfilling the pipet. INTEGRA’s PIPETBOY GENIUS offers overfill protection to reduce the risk of filters needing to be replaced due to unintentional contact with the liquid being pipetted.
Note: 0.2 and 0.45 µm, sterile and non-sterile filters are available for the INTEGRA PIPETBOY. Sterile 0.2 µm filters are recommended for delicate applications, such as cell culture.
Summary
It is important to follow the best practices outlined above to ensure accurate and precise serological pipetting; using a pipet controller will help provide greater control of the liquid level. However, even with best practices, it is not possible to improve on the calibrated accuracy of the serological pipet. Serological pipets should ideally be used with an electronic pipetting aid, as this is the most comfortable way to aspirate and dispense these volumes of liquid. INTEGRA serological pipets range between 1 and 100 ml in volume, with each pipet featuring a calibrated graduation that allows the liquid volume to be read. Color coding enables rapid identification of the pipet volume at a glance, and larger volume pipets** also incorporate an anti-drip design to improve accuracy and reproducibility by reducing the risk of liquid dripping from the pipet during transfers. These are perfectly complemented by INTEGRA PIPETBOY pipet controllers, to provide everything you need for meticulous serological pipetting.
Check out our PIPETBOY pipet controllers and serological pipet promo.
Note: Micropipettes are recommended when smaller dispense volumes are required. Read How to use micropipettes for more information.
**25, 50 and 100 ml