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Reliable, precise and trusted – however, a pipette performs only as well as the operator’s technique allows. As demand accuracy and precision increases, the importance of understanding and developing optimal pipetting technique is crucial. Pipette maestro Alison Rawlinson takes us through some simple does and don’ts to up your liquid handling game… 

Delivery volumes and data integrity can be affected and altered through differences in technique¹. The application fields for dispensing systems depend on the design of the system, as well as the physical limits inherent to the application. To meet the demanding requirements, in terms of precision and accuracy, the application primarily calls for a precise device with high quality tips and also, sufficient practical experience, correct handling and cleanliness of the dispensing systems.

The dispensing techniques used most frequently are forward pipetting, reverse pipetting and sequential dispensing

The dispensing techniques used most frequently are forward pipetting, reverse pipetting and sequential dispensing. While manual pipettes are only suitable for forward and reverse pipetting, electronic dispensing systems frequently cover all of the aforementioned functions. Selection of the dispensing technique suitable in each case may have a significant impact on the results. The technique of forward pipetting is suitable for aqueous solutions which may contain low concentrations of protein or detergents. Reverse pipetting may greatly improve the dispensing of viscous or foaming liquids, or when dispensing very small sample volumes. Here the liquid is aspirated with blow-out and dispensed without blow-out. A residual amount of liquid remains in the plastic tip, and it is subsequently discarded or returned to the aspirating vessel.

With sequential dispensing, different volumes of a solution are dispensed one after the other in a specified sequence. The aspirated liquid is distributed in defined steps and this technique is often used when processing long test series or filling microtest plates. When diluting liquids, an initial volume is first aspirated, followed by an air bubble and the second volume. Both volumes are then dispensed in one step. This dispensing technique increases throughput and reduces fatigue, compared with performing aspiration and dispensing twice with manual pipettes – particularly in the case of sizeable volumes and multichannel pipettes.

Pipettes are widely used tools across the laboratory and usually carefully chosen, however with pipette tips this is not always the case. Pipette tips are consumable items and not usually chosen with the same care or quality in mind. The standard ISO 8655:2002 recommends those working in the laboratory to use a pipette and pipette tips from the same manufacturer. Studies have suggested that a pipetting system working perfectly with a certain tip can exceed the permissible error tolerances when a tip from a different manufacturer is used.

Changing the tip for calibration measurements should not affect the calibration result if the tip-to-tip quality is consistently high

Furthermore, the calibration method can influence the performance of the pipetting system; it can be significant whether the calibration is done with or without tip change for each measured volume. Changing the tip for calibration measurements should not affect the calibration result if the tip-to-tip quality is consistently high. Autoclaving can impact the tip dimension as well as the calibration result, especially with small volumes. However, with high quality tips it is possible for tips to perform within permissible error tolerances, regardless of the calibration method or autoclaving, allowing the user to be free to choose the method most suitable for their application.

Calibration often has a large role in pipetting errors.

Technique is also vital. For air-cushion pipettes, the pipette tip should be selected to minimise the air cushion between the pipette piston and the surface of the liquid. The smaller the tip, the lower the air volume, and the greater the accuracy of the results. The following items should be taken into consideration during pipetting, irrespective of the dispensing technique used:

• When aspirating the liquid, the tip should only be immersed a few millimetres into the medium.
• The filled tip should be moved up against the wall of the vessel to avoid residues of liquid on the outside of the tip.
• Pre-wetting the tip two or three times will improve the accuracy and precision of the results.
• Liquid should be aspirated slowly and evenly.
• A waiting period of 1 to 3 seconds should be allowed for the liquid to rise in the tip.

The pipette should be held vertically during aspiration. On aspiration of the liquid, the hydrostatic pressure of the liquid column in the tip falls as the angle of inclination of the pipette increases. This will result in an increased aspiration volume². The deviation in volume can be estimated for any pipette volume and angles of inclination. With an angle of 45° diverging from the vertical the extra volume with a 1000?l pipette is 2.9?l or 0.29%. For a 100?l pipette at an angle of 60°, an extra volume of 0.53?l or 0.53% will result.

When pipetting small volumes, the influence of the pipetting technique can have considerable effects on the experimental result

The reason that different people do not pipette exactly the same volume with exactly the same pipette can be explained by factors such as different angles of inclination when aspirating liquid. Although the effect is small in each instance, the error may be much greater when all of the differing factors are added together. Choosing the correct pipetting technique helps to secure the accuracy and precision needed for reproducible and reliable results. When pipetting small volumes, the influence of the pipetting technique can have considerable effects on the experimental result.

Two main techniques exist and each should be applied depending on the sample liquid. Aqueous liquids can be dispensed using the well-known forward pipetting technique, but dispensing more challenging liquids like glycerol, ethanol or BSA solution requires a different approach called reverse pipetting. Additionally, general considerations like a low immersion depth and a vertical pipetting angle must be taken into account. The impact of immersion depth and angle on precision and accuracy can be significant, with a vertical angle and low immersion depth guaranteeing the most accurate pipetting result.

Author: Alison Rawlinson works at Eppendorf UK Ltd. She has more than ten years of research experience in Biochemistry and Molecular Microbiology. References 1. George Rodrigues Ph.D. 2006. How to Improve Pipetting Technique. https://www.qualitydigest.com/inside/metrology-article/how-improve-pipetting-technique 2. Lochner, K. H.; Ballweg, T.; Fahrenkrog, H.-H.: Untersuchungen zur Messgenauigkeit von Kolbenhubpipetten mit Luftpolster. In: Lab Med 20 (1996), Nr. 7/8, S. 430–440.