Forward vs Reverse Pipetting: Which Technique to Use and When
Pick by the job, not by habit
Burette vs pipette, side by side
Why reverse pipetting exists — the physics of the air cushion
Air-displacement pipettes work by compressing an air cushion above the sample. For water-like liquids at room temperature, the cushion behaves predictably and the calibration holds. For viscous liquids (glycerol, DMSO, serum), the liquid clings to the tip walls and drains slowly — forward pipetting under-delivers because not all the liquid exits before you release. For volatile liquids (ethanol, acetone, chloroform), solvent vapour expands the air cushion during aspiration — forward pipetting over-delivers. Reverse pipetting compensates by loading more than you need and dispensing only to the first stop, leaving the excess and the error in the tip.
Pre-wetting matters for both techniques
Before the first real aspiration, draw up and expel the liquid once (pre-wet). This equilibrates the air cushion with solvent vapour and coats the tip walls with liquid film. Without pre-wetting, the first dispense of a forward pipette is consistently low — a systematic error that disappears on the second draw. With reverse pipetting, pre-wetting is even more important because the excess volume left in the tip changes between a dry and a wet tip.
Three technique errors that cause silent inaccuracy
- Using forward technique on viscous liquids. The systematic under-delivery looks like a bad pipette. Switch to reverse — the problem disappears.
- Pressing to the second stop on forward pipetting. Pressing too far on aspiration draws an extra bubble of air into the tip, which then causes over-delivery on dispense.
- Blowing out the residual in reverse pipetting. The whole point of reverse technique is that the excess stays in the tip. Blowing it out defeats the correction and over-delivers.