Reverse Pipetting: How It Works, When to Use It, and Common Mistakes

Short answer: reverse pipetting loads more liquid than you need (press to second stop on aspirate), then dispenses only to the first stop — leaving the overage in the tip. The overage absorbs the viscosity, volatility, or foaming error that would cause forward pipetting to mis-deliver. Use it for glycerol, DMSO, ethanol, serum, detergents, and any liquid that clings, evaporates, or foams in a standard tip.

Pick by the job, not by habit

  • Glycerol, PEG, mineral oil (high viscosity)
    Reverse pipetting + low-retention tip
    Viscous liquid clings; reverse loads overage to absorb the drag error
  • DMSO, ethanol, acetone (volatile)
    Reverse pipetting + pre-wet
    Vapour expands air cushion; reverse overage compensates over-delivery
  • Serum, plasma, protein solutions
    Reverse pipetting + low-retention tip
    Sticky proteins coat tip walls; low-retention + reverse minimises loss
  • Detergents, surfactants (foaming)
    Reverse pipetting, aspirate slowly
    Fast aspiration creates foam; reverse with slow draw avoids bubbles
  • First dispense consistently lower than subsequent
    Pre-wet the tip before first draw
    Dry tip air-cushion mismatch; one pre-wet equalises all subsequent draws
  • Need to dispense same volume multiple times
    Electronic repeater pipette
    Built-in reverse mode; reduces fatigue and tip changes on repetitive work

Burette vs pipette, side by side

  • Aspirate: press to
    First stop
    Second stop (extra volume)
  • Volume in tip
    Target volume only
    Target + overage
  • Dispense: press to
    First stop (all liquid out)
    First stop only (overage stays)
  • Tip after dispense
    Empty; blow-out if TD marked
    Contains overage — discard, do not blow out
  • Best liquids
    Aqueous, water-like solutions
    Viscous, volatile, foaming liquids
  • Tip type
    Standard tips adequate
    Low-retention tips improve accuracy further

Step-by-step: reverse pipetting correctly

Step 1 — Pre-wet the tip. Aspirate and expel the liquid once before the real draw. This coats the tip walls and equilibrates the air cushion with solvent vapour. Skip this and the first dispense will be low.

Step 2 — Press to the second stop before aspirating. Unlike forward pipetting (first stop only), reverse pipetting requires pressing fully to the second stop before you put the tip in the liquid. This pre-loads extra air displacement so more liquid enters the tip.

Step 3 — Aspirate slowly. Draw the liquid up slowly and smoothly. Fast aspiration creates turbulence and bubbles, especially in foaming liquids.

Step 4 — Dispense to the first stop only. Press only to the first stop. The calibrated volume exits; the overage stays in the tip. Do not press further.

Step 5 — Discard the tip with the overage. The residual in the tip is not re-usable for accurate work — it contains the absorbed error. Discard and use a fresh tip for the next dispense.

Why the overage must not be blown out

The overage left in the tip after reverse pipetting is not clean excess — it has absorbed the viscosity drag, vapour equilibration, or foam from the aspiration. Blowing it out delivers an unpredictable volume and contaminates the next transfer. This is the single most common reverse-pipetting mistake in practice.

Low-retention tips improve reverse pipetting further

Standard tips leave a liquid film on their inner walls, which is especially problematic for viscous or protein-containing solutions. Low-retention (hydrophobic) tips reduce this film, so more of the aspirated volume actually exits on dispense. Combined with reverse technique, low-retention tips give the best accuracy on difficult liquids — at the cost of slightly higher consumable expense.

Standards

  • ISO 8655:2022
    Piston-operated volumetric apparatus; defines forward and reverse operating modes
  • ISO 8655-2
    Systematic and random error limits — reverse technique reduces systematic error on difficult liquids
  • ISO 8655-6
    Gravimetric test method — use to verify accuracy improvement from reverse technique

Frequently asked questions

  • What is reverse pipetting used for?
    For viscous, volatile, or foaming liquids where forward pipetting mis-delivers. The technique loads an overage and dispenses only to the first stop, leaving the error-absorbing excess in the tip.
  • Should I blow out the tip after reverse pipetting?
    No. The residual left in the tip after reverse pipetting is intentional — it absorbed the viscosity or vapour error. Blowing it out over-delivers and defeats the technique.
  • Does pre-wetting matter for reverse pipetting?
    Yes, especially for volatile liquids. Pre-wetting equilibrates the air cushion with solvent vapour before the real draw, so the overage volume is consistent from the first dispense.
  • When should I use low-retention tips with reverse pipetting?
    For protein solutions, serum, or any liquid that leaves a visible film on standard tip walls. Low-retention tips reduce the residual film and improve delivery accuracy on sticky liquids.
Need pipettes or low-retention tips for viscous or volatile work? Contact us for factory-direct supply.