A simple method for validation and verification of pipettes mounted on automated liquid handlers

We have implemented a simple, inexpensive, and fast procedure for validation and verification of the performance of pipettes mounted on automated liquid handlers (ALHs) as necessary for laboratories accredited under ISO 17025. A six- or seven-step serial dilution of OrangeG was prepared in quadruplicates in a flat-bottom 96-well microtiter plate, manually using calibrated pipettes. Each pipette of the liquid handler (1-8) dispensed a selected volume (1-200 μL) of OrangeG eight times into the wells of the microtiter plate. All wells contained a total of 200 μL liquid. The absorbance was read, and the dispensed volume of each pipette was calculated based on a plot of volume and absorbance of a known set of OrangeG dilutions. Finally, the percent inaccuracy (%d) and the imprecision (%CV) of each pipette were calculated. Using predefined acceptance criteria, each pipette was then either approved or failed. Failed pipettes were either repaired or the volume deviation was compensated for by applying a calibration curve in the liquid-handler software. We have implemented the procedure on a Sias Xantus, an MWGt TheONYX, four Tecan Freedom EVO, a Biomek NX Span-8, and four Biomek 3000 robots, and the methods are freely available. In conclusion, we have set up a simple, inexpensive, and fast solution for the continuous validation of ALHs used for accredited work according to the ISO 17025 standard. The method is easy to use for aqueous solutions but requires a spectrophotometer that can read microtiter plates.