Pipetting Techniques: Forward, Reverse and Challenging Liquids

Forward and reverse pipetting differ mainly in the relationship between the liquid aspirated, the dispensing stroke and the residual volume in the tip. Reverse pipetting may be worth evaluating for viscous or foaming matrices, but it does not automatically make a difficult transfer correct. The choice needs verification with the actual combination of pipette, tip, liquid and technique. A valid instrument calibration alone does not demonstrate that the operator is delivering the required volume under working conditions. [1–3]

The concern here is operational: making the technique reproducible, recognising signs of error and distinguishing possible causes. The table and case are original GuideGxP proposals. They are not a universal procedure, a complete calibration protocol or experimental evidence of your laboratory’s performance.

1. A controlled instrument does not eliminate variation in technique

Before watching the operator’s hand, identify the transfer: liquid, selected volume, containers, tip and analytical consequence of an error. Distinguish the selected volume from the volume actually delivered. The certificate describes results under calibration conditions; performance during a work sequence also depends on factors that this check may not represent.

The PTB/DKD study of air-cushion pipettes considers environmental and handling influences, including tip conditioning, rhythm, operating force and heat from the hand. The operator’s contribution therefore cannot be reduced to a general instruction to “take care”. It needs to be translated into observable behaviours and documented conditions. [3]

A 2026 study of clinical laboratory personnel found performance differences between operators with different experience. It is cited for the principle of checking competence, not to transfer thresholds from its experiment into a GMP laboratory. Even extensive experience does not replace a demonstration relevant to the task. [4]

2. Forward pipetting: consistent aspiration and delivery

In the conventional arrangement of a compatible manual air-displacement pipette with two stops, forward pipetting prepares aspiration from the first stop and draws liquid by releasing the control in a controlled manner. Delivery uses the intended stroke and, where the configuration requires it, the final blow-out. The IRD teaching material distinguishes these movements from reverse pipetting. [2]

Before starting, identify the dosing control and the tip-ejection control. Do not automatically apply this description to electronic or positive-displacement instruments: modes and sequences must match the approved instructions for the system. A familiar movement on one pipette can have a different meaning on another.

Critical steps include entry of the tip into the liquid, continuity of aspiration, withdrawal from the surface and release of liquid into the receiving vessel. Look for bubbles, contact with the bottom, external droplets and any release of the control while the tip is still in the receiving liquid. A sequence may look fast and regular yet include unwanted re-aspiration.

Do not correct a questionable transfer by adding a drop “by eye”. Stop and manage the event according to the method. The criterion is not whether the tip looks empty, but whether the intended transfer has been performed and verified with adequate evidence.

3. Reverse pipetting: residual liquid is part of the technique

In the corresponding reverse arrangement of a compatible manual pipette, aspiration draws an additional quantity and dispensing stops at the position intended for the dose. Some liquid remains in the tip. This residual liquid must not automatically be expelled into the receiving vessel: delivering it with the dose would change the transfer. Subsequent handling follows the approved sequence, including disposal where required. [2]

This approach may be a candidate when wetting, viscosity or foam formation make forward pipetting problematic. It does not justify treating the selected and delivered volumes as equal for every matrix. The comparison must account for additional sample consumption and handling of residual liquid, especially where material is limited or cannot be returned to the source vessel.

Avoid two opposite errors: treating the intended residual liquid as a defect to be eliminated into the dose, or calling any incomplete dispensing operation “reverse pipetting”. Define the mode before the trial. If an electronic pipette offers programmes with similar names, record the programme actually used and its settings.

A study of ligand-binding assays observed that differences between manual techniques contributed to discrepancies in dilutions. The finding concerns that bioanalytical workflow and does not establish the universal superiority of one technique or of automation. Instead, it supports comparing the method under its intended conditions of use. [5]

4. Make operating variables observable

Immersion and angle. Maintain geometry consistent with the system and liquid level. A falling level during a series can change the operator’s movement without being noticed. Insufficient immersion can allow air to enter; inappropriate contact with the wall or bottom can obstruct aspiration. Do not adopt one numerical immersion depth for every tip and volume. [2]

Speed, pauses and rhythm. Guide the control without allowing it to spring back. Check whether aspiration has finished and whether the delivery sequence is repeatable. A useful pause for one matrix is not automatically suitable for a volatile matrix. During comparisons, also record interruptions and variable intervals between transfers; rhythm is among the handling influences considered by PTB/DKD. [3]

Pre-wetting and consumables. Tip conditioning must be consistent with the method, matrix and contamination risk. Do not improvise cycles or return liquid to the original bottle if the procedure does not permit it. A tip change can alter the surface condition: record when it occurs during a comparison and do not unknowingly alternate between different conditions. [2,3]

Thermal conditions. Consider liquid, instrument and ambient temperatures, as well as handling duration. The metrological study distinguishes these influences and the role of evaporation in the air cushion. This does not mean bringing every sample to room temperature: if stability or the method requires different conditions, verify the technique under those conditions. [3]

5. Challenging liquids: recognise the limits of the technique

For a viscous matrix, observe completion of aspiration, wetting of the inner walls and behaviour during delivery. Evaluate a compatible sequence and, where relevant, reverse pipetting; then verify the transfer. Slower operation may be a variable to investigate, not a guarantee of the correct volume.

For a volatile liquid, a droplet appearing without operation of the control does not necessarily demonstrate a mechanical leak. Evaporation can interact with the air-displacement system. A controlled comparison must distinguish matrix, consumable, seal integrity and environmental conditions. Do not automatically blame the instrument or assume that reverse pipetting will resolve every situation. [3]

For foaming liquids, observe whether aspiration or final blow-out introduces bubbles and makes the transfer unstable. Omitting a particular stage may form part of an approved reverse technique, but does not replace dose verification. At small volumes, also check whether the verification method has sufficient capability: a reading that cannot distinguish the relevant difference does not prove the absence of error.

If behaviour cannot be controlled in the selected configuration, consider a different transfer principle and its compatibility. NIST’s discussion of pipette performance limitations helps maintain this distinction. The response is not to persist with the same movement or arbitrarily adjust the indicated volume to conceal a problem that has not been understood. [6]

6. Tips, aerosols and unintended transfers

The tip must belong to the evaluated configuration and be fitted correctly. Check integrity, sealing and the replacement sequence. A tip that appears to fit does not by itself demonstrate equivalent performance. After an abnormal event, retain the identity of the combination used instead of changing several components simultaneously and losing the opportunity to distinguish the cause.

Prevent liquid from reaching parts not intended for contact, the tip from touching unrelated surfaces, and material from the receiving vessel from returning to the sample pathway. The risk of contamination through micropipettes and aerosols is also addressed in IRD’s institutional training. Adapt measures to the actual work: not every transfer requires the same arrangement, and a filter tip does not make poor technique harmless. [2]

GuideGxP proposes distinguishing three observations in the verification record: a volumetric event, a contamination event and an identification event. They may coexist but require different actions. An accurate dose delivered into the wrong vessel does not fulfil the purpose of the task.

7. An original table for distinguishing errors

The effects below are plausible, not automatic diagnoses. A discriminating check must keep other variables controlled and remain compatible with safety and the method. Adopt a correction only after verifying its effect.

8. Simulated case: two operators, the same pipette

Two analysts prepare a dilution with the same controlled pipette and obtain different results. The first hypothesis must not become “the instrument is faulty” or “the second operator is wrong”. In this simulated case, the supervisor records the liquid identity, volume, tips, containers, sequence, conditions and method used to detect the difference.

Observation reveals that one operator uses forward pipetting, while the other aspirates according to a reverse approach but also delivers the residual liquid. The team treats the sequence as a plausible cause to verify, not an already proven explanation. It also checks that the liquid and configuration are actually equivalent and that the comparison does not introduce other changes.

A sequence consistent with approved use is defined, both operators are trained, and a planned comparison is repeated with individual data and criteria established before testing. No improvement percentages are invented and no universal number of repetitions is imposed. If the difference persists, investigation continues into seal integrity, thermal conditions, matrix and the capability of the verification method.

The useful outcome is a documented decision: which hypotheses were supported or ruled out, which technique is permitted and which conditions require reassessment. Merely recording “training completed” is insufficient without showing what changed in the technique and observed performance.

9. Training and verification proportionate to the task

ISO 8655-10:2024 covers user guidance, competence, training and suitability for use of piston-operated volumetric apparatus. This article uses its public official scope without attributing clauses or tolerances that were not consulted. The reference confirms that user competence warrants assessment separate from the mere availability of the instrument. [1]

Prepare a record identifying the activity represented, configuration, matrix or test liquid, conditions, sequence, observer and assessment criterion. Define what the comparison measures. A water test may help assess part of the technique; it does not automatically demonstrate performance with a very different liquid.

If using gravimetric verification, an appropriate measurement system and mass-to-volume conversion relevant to the liquid and conditions are necessary; reading a number on a balance is insufficient. If using an analytical comparison, consider variation from the other stages. Retain observations and individual results, distinguishing systematic error from scatter without claiming a complete calibration that was not performed.

Reassess competence when the task changes or signs of difficulty appear: a new matrix, new tip, different mode or persistent differences. The frequency and extent of checks follow from risk and the applicable quality system. This article imposes no universal interval.

Operational conclusion

Make the mode, geometry, rhythm and handling of residual liquid explicit; then verify the transfer in the relevant configuration. Use observed errors to formulate hypotheses that can be compared, not to assign blame. The desired result is a documented technique supported by evidence, with a clear boundary beyond which the method or technology needs to change.

Continue in the Liquid Handling and Sample Preparation HUB and the article on choosing between air and positive displacement when the problem concerns the transfer principle.

Sources and consultation limits

Sources checked on 30 September 2026. ISO was consulted for scope and status, not in full; IRD is 2006 teaching material used for movement principles, without adopting its numerical values or intervals. PTB/DKD is a study with its own conditions. The clinical and bioanalytical studies were read through abstracts and, for the 2026 paper, indexed methods extracts: no full-text reading is claimed. NIST was consulted as a page and summary, not by watching the complete video. The table and case are original GuideGxP material.

1. ISO 8655-10:2024 — User guidance, competence, training and POVA suitability. Edition 1, February 2024.
2. Gauthier, CBGP-IRD — Organisation et fonctionnement d’un laboratoire de biologie moléculaire. Dakar training, September 2006, micropipette sections.
3. PTB/DKD — Experimental study on the calibration of piston-operated pipettes with air cushion. DKD-E 8-1, 2013 edition, revision 1; DOI 2025, content unchanged.
4. Özcan et al. — Pipetting Performance across Laboratory Personnel. J Appl Lab Med. 2026;11(3):460–469. DOI: 10.1093/jalm/jfag006.
5. Pandya et al. — Strategies to minimize variability and bias associated with manual pipetting. J Pharm Biomed Anal. 2010;53(3):623–630. DOI: 10.1016/j.jpba.2010.04.025.
6. Sander, NIST — Volumetric Transfer of Liquids. JResNIST 122:2, 2017. DOI: 10.6028/jres.122.002.