Reverse pipetting is a modified pipetting technique where you aspirate more liquid than you need, dispense only the target volume, and discard the excess left in the tip. It’s the go-to method for handling liquids that don’t behave well with standard (forward) pipetting, including viscous, volatile, or foaming solutions. The difference comes down to when you push past the blowout stop on the plunger.
How It Differs From Forward Pipetting
In forward (standard) pipetting, you press the plunger to the first stop before aspirating, draw up exactly the volume you need, then push through both the first and second stops to dispense every drop. The second stop, called the blowout, forces out any remaining liquid.
Reverse pipetting flips this sequence. You press the plunger all the way to the second (blowout) stop before aspirating, which means you draw up more liquid than the set volume. When you dispense, you only push to the first stop, delivering the exact target volume while a small surplus stays behind in the tip. That leftover liquid gets discarded into a waste container, and the tip is disposed of.
This matters because the blowout step in forward pipetting is where most accuracy problems happen with tricky liquids. By removing the blowout from the dispensing step entirely, reverse pipetting sidesteps the issues that cause volume errors.
Step-by-Step Technique
The process uses the same air displacement pipette you’d use for any other work. No special equipment is needed.
- Set the volume on your pipette as you normally would.
- Depress the plunger fully past the first stop to the second (blowout) stop before touching the liquid.
- Immerse the tip just below the surface of the liquid at a near-vertical angle, roughly 90 degrees. Avoid plunging the tip deep into the solution.
- Release the plunger slowly and smoothly back to its resting position. The tip now holds more liquid than your set volume.
- Dispense into your target vessel by pressing the plunger down gently to the first stop only. Do not push further.
- Discard the residual liquid remaining in the tip into a waste container, then eject the tip.
Speed matters at every step. Releasing the plunger too quickly during aspiration can introduce air bubbles, especially with thick solutions. Pressing too fast during dispensing can splash or create aerosols.
When to Use Reverse Pipetting
Reverse pipetting is recommended for any liquid that clings to the tip wall, evaporates quickly, or tends to foam. Each of these problems causes volume errors in a different way, and the technique addresses all three.
Viscous liquids like glycerol and DMSO coat the inside of the tip and resist being fully expelled during a normal blowout. With reverse pipetting, that clinging residue is simply the surplus you planned to throw away. Your dispensed volume remains accurate.
Volatile liquids like ethanol evaporate slightly inside the tip between aspiration and dispensing, which means you deliver less than you intended. Reverse pipetting compensates because any evaporative loss comes out of the surplus left in the tip, not from the volume you dispense.
Foaming liquids like solutions containing Tween 20 create bubbles when air is forced through them during the blowout step. Since reverse pipetting stops at the first stop and never blows air into the sample, foaming is minimized.
Other common applications include pipetting protein solutions, detergent-containing buffers, and serum samples.
When Forward Pipetting Is Better
For standard aqueous solutions like water, buffers, diluted acids, and diluted bases, forward pipetting is the recommended technique. Pipettes are calibrated using pure water and the forward method, so aqueous solutions naturally behave as expected with that approach. Reverse pipetting wastes a small amount of reagent with every transfer (the discarded surplus), which adds up when working with expensive solutions that don’t actually need the technique.
Tips for Better Accuracy
Temperature affects both your pipette and your samples. The standard working temperature for micropipetting is 21.5°C (plus or minus 1°C). If your samples have been refrigerated or frozen, let them equilibrate at room temperature for about 20 minutes before pipetting. Cold liquids change the air volume inside the pipette barrel, throwing off accuracy regardless of which technique you use.
Tip immersion depth is a common source of error. Position the tip just below the liquid surface. Going too deep coats the outside of the tip with extra liquid that gets carried over to your receiving vessel, inflating the volume you transfer. Going too shallow risks aspirating air instead of liquid.
Pre-wetting the tip by aspirating and dispensing the liquid once or twice before your actual transfer helps condition the tip’s interior surface, especially with viscous solutions. This reduces the amount of liquid that adheres to a dry tip on the first draw.
Finally, keep the pipette vertical during aspiration. Tilting it allows liquid to creep up the inside of the tip or reach the pipette’s internal components, which can contaminate both the instrument and your sample.