Forward vs reverse pipetting

Pipetting volatile, viscous, foaming, and oily liquids accurately and consistently can be a challenge due to the inherent characteristics of these liquids, which can cause them to stick to the pipette or drip. To overcome these challenges, it is important to follow best pipetting practices and consider the specific properties of the liquid being pipetted. This includes selecting an appropriate pipette and compatible pipette tips and using the correct pipetting technique. Forward pipetting and reverse pipetting are two commonly used techniques in the laboratory, each with its own set of advantages and disadvantages. In this guide, we provide practical advice on how to successfully pipette difficult liquids by accounting for their specific properties.

Forward pipetting

Forward pipetting involves aspirating liquid into the pipette and then releasing it into a receiving container.  A separate blowout step is used to completely empty the tip by pressing the plunger to the second stop. This technique is suitable for transferring liquids with low viscosity, such as water or solvent solutions, and is often used when transferring small volumes of liquid due to its precise control of the volume being transferred. To perform forward pipetting, the tip of the pipette is placed into the liquid, the liquid is drawn into the pipette, and the pipette is then carefully removed from the liquid. The pipette is then held vertically and the liquid is slowly released into the receiving container.

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Reverse pipetting

Reverse pipetting, on the other hand, involves aspirating an excess amount of the liquid into the pipette. When the plunger is pressed to the first stop, the desired target volume is dispensed, and the excess amount is left in the pipette tip. This excess volume can then be returned to the source container or discarded by pressing the plunger to the second stop. This technique, known as "blow-out" pipetting, can be particularly for transferring liquids with high viscosity, such as thick gels or oils, as it helps to prevent air bubbles from being trapped in the pipette. Reverse pipetting is also commonly used when transferring large volumes of liquid due to its efficient transfer method.

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Accuracy of reverse and forward pipetting

In terms of accuracy, forward pipetting is generally more accurate than reverse pipetting, especially when transferring small volumes of liquid. This is because forward pipetting allows for precise control of the volume being transferred and the liquid is not drawn back into the pipette, which can introduce errors. Reverse pipetting, on the other hand, is more prone to errors due to the liquid being drawn back into the pipette. However, if proper technique is used, reverse pipetting can still be accurate, especially when transferring larger volumes of liquid.

Efficiency of reverse and forward pipetting

In terms of efficiency, reverse pipetting is generally more efficient than forward pipetting, especially when transferring large volumes of liquid. This is because the technique allows for the efficient transfer of the liquid, and it is not necessary to refill the pipette multiple times to transfer the desired volume. Forward pipetting, on the other hand, can be time-consuming when transferring large volumes of liquid, as the pipette must be refilled multiple times.

In addition to the differences in technique and accuracy, it is also important to consider the potential contamination risks associated with each technique. Forward pipetting poses a higher risk of contamination, as the pipette tip is inserted into the liquid and then removed, potentially introducing contaminants from the environment. Reverse pipetting, on the other hand, poses a lower risk of contamination as the pipette tip is not inserted into the liquid and is only in contact with the receiving container.

Conclusion

In conclusion, both forward and reverse pipetting techniques have their own advantages and disadvantages, and the appropriate technique should be chosen based on the specific needs of the experiment. Forward pipetting is generally more accurate, especially when transferring small volumes of liquid, while reverse pipetting is generally more efficient, especially when transferring large volumes of liquid. It is also important to consider the potential contamination risks associated with each technique to follow the proper technique and use the appropriate pipette for the specific task to ensure accurate and efficient pipetting in the laboratory.