Pipetting Procedures and Tips to Run an Accurate Assay

Even small variations in pipetting procedures may seriously impact assay results. This is of particular significance if the findings may be relevant for clinical or manufacturing decisions, such as in the case of the Limulus amebocyte lysate (LAL) assay used to assess samples for endotoxin contamination. However, imprecisions have been detected in a substantial proportion of individuals performing manual pipetting, and especially when pipetting small volumes. In one study, the mean intra-individual pipetting imprecision was found to be 5.7%, 0.8%, and 0.2% for pipetting 10 μL, 100 μL, and 1 mL, respectively. Moreover, the mean inter-individual imprecision was 8.1%, 1.1%, and 0.4% for pipetting 10 μL, 100 μL, and 1 mL, respectively.1 

Characteristics of a proper pipetting technique

All steps of the pipetting procedure, including preparation, liquid uptake (aspiration), and liquid discharge should be considered to ensure the use of a proper technique.2 Excessive handling of the pipette and tip should be minimized to prevent contamination. It is recommended to pre-wet the pipette tip by pipetting the selected volume up and down. For liquid uptake, the pipette tip should be immersed a few millimeters below the liquid’s surface, and the liquid should be aspirated slowly and evenly. After aspiration, the experimenter should pause for 1–3 s to let the liquid rise up in the tip. Consistent speed and plunger pressure should be applied throughout the liquid discharge. Finally, the tip should be examined before and after each liquid discharge to ensure that the full volume has been dispensed.

In addition to the described characteristics of a proper pipetting technique, adhering to the following tips would aid the delivery of consistent and reliable results:

  1. Select a pipetting technique suitable for each solution – Standard (forward) mode pipetting is used for aqueous solutions, which are the majority of solutions used in laboratory experiments. Reverse mode pipetting is recommended for liquids with high viscosity or volatility.
  2. Select a pipette with an optimal volume range – Using a pipette with an optimal volume range for the planned experiments reduces the risk of user-related errors. For the commonly employed air displacement pipettes, this range is reportedly 35%–100% of the nominal value.
  3. Handle and maintain pipettes with care – Pipettes should be checked for contamination daily. When not in use, they should be stored on a designated pipette stand to prevent damage and should be kept away from potentially harmful reagents.
  4. Perform regular calibration – Pipettes should be calibrated at regular intervals to maintain their precision and accuracy within the recommended range (commonly ± 5% of the specifications).3 Calibration should be performed under conditions that resemble the actual experimental conditions as much as possible. The recommended calibration interval depends both on the application and frequency of pipette use.
  5. Ensure pipetting technique training of the laboratory personnel – Teaching experimenters a proper pipetting technique is a prerequisite to obtain reliable experimental results. This notion is supported by the findings of a study conducted with the personnel of a forensic laboratory, which showed that pipetting technique training improved pipetting accuracy.4
  6. If possible, work at a temperature equilibrium – If the experimental conditions and requirements permit it, maintaining the pipette, tips, and solution at ambient temperatures may positively influence the accuracy of the results.
  7. Discard the first and last dispense – When multiple aliquots are dispensed, it is recommended to discard the first and final aliquot.
  8. Design a thorough and efficient experimental plan – An efficient experimental plan would help prevent user-related errors, especially when pipetting long series of aliquots into microplates. Web applications that facilitate experimental planning, such as Pipette Show, are being developed.5


Literature sources

  1. Lippi G, Lima-Oliveira G, Brocco G, Bassi A, Salvagno GL. Estimating the intra- and inter-individual imprecision of manual pipetting. Clin Chem Lab Med. 2017;55(7):962-966. doi: 10.1515/cclm-2016-0810.
  2. Ewald K. Impact of pipetting techniques on precision and accuracy. Eppendorf Userguide. No 20, May 2015. Available online at: https://www.eppendorf.com/uploads/media/USERGUIDE_20_GB_Final_25.pdf.
  3. Sanders ER. Aseptic laboratory techniques: volume transfers with serological pipettes and micropipettors. J Vis Exp. 2012;(63):2754. doi: 10.3791/2754.
  4. Epstein DM, Tebbett IR, Boyd SE. Eliminating sources of pipetting error in the forensic laboratory. Forensic Science Communications. 2003;5(4).
  5. Falk J, Mendler M, Kabisch J. Pipette Show: An open source web application to support pipetting into microplates. ACS Synth Biol. 2022;11(2):996-999. doi: 10.1021/acssynbio.1c00494.