Accurate micropipetting technique

Accurate micropipetting technique is the habit that makes a stated microlitre volume the volume the tube actually received. The decision this page supports is how to handle an air-displacement pipette so that forward pipetting of aqueous reagents is repeatable, and when that technique is the wrong tool. Checking the same pipette on a balance between formal calibrations is the companion note, checking a pipette between calibrations.

Pipettes as instruments are in the scientific instruments catalogue. Tips and tubes are in the laboratory consumables catalogue. A specification, including any calibration document you need named, goes to the quote request. A family name is not a statement that a particular pipette has been adjusted.

The volume the experiment thinks it received

Most molecular measurements assume the pipette did what the dial said. Enzyme units, standard curves and PCR master mixes are all ratios. A pipette that delivers short on a small setting will not announce itself with a drip if the error is a thin air cushion rather than a leak. The experiment simply moves. Quantitative PCR is one place the community has written this down: the MIQE guidelines treat pipette performance as part of what makes a quantification publishable. The principle is older than that paper and wider than PCR. If the volume is the data, the pipette is a measuring instrument. If the volume is "about a millilitre of wash", a serological pipette or a cylinder may be the honest tool, and a micropipette is theatre.

Air displacement

In an air-displacement pipette a piston moves, a cushion of air transmits that movement, and the liquid stays in the tip. The pipette never intends to pull liquid into the barrel. That cushion is why the instrument is sensitive to temperature, to atmospheric pressure, to a wet barrel, and to a tip that does not seal. It is also why volatile solvents vaporise into the cushion and drip, and why viscous liquids lag behind the piston.

A positive-displacement pipette puts a piston in contact with the liquid, usually via a capillary tip. It is the better class for viscous, dense or volatile liquids, and it is a different calibration. Do not "correct" an air-displacement pipette for glycerol by a private factor scribbled on the handle and then use that factor for water.

The tip is part of the metrology. It must match the cone. Filter tips protect the barrel from aerosols and change the air path slightly; if you calibrated with one tip family, routine work that must match the calibration uses that family. Pre-wetting the tip, for aqueous forward pipetting, fills the air with vapour so the first dispense is not the odd one that evaporates into a dry cushion. Skip pre-wetting only when the method says the first stroke is discarded anyway.

Forward pipetting

Set the volume. Seat the tip with a firm press, not a pounding that bends the shaft. Hold the pipette vertical to aspirate. Press the plunger to the first stop, dip the tip a few millimetres under the surface, and release the plunger smoothly. A deep plunge carries liquid on the outside of the tip, which then wipes off into the destination and was never in the calibrated stroke. A shallow dip that breaks the surface sucks air. Pause briefly at the end of aspiration so viscous drag can catch up. If you see a bubble, expel and try again. A bubble is an air volume wearing a liquid costume.

Withdraw the tip along the wall or straight up, according to a single habit. Move to the receiving vessel. Touch the tip to the inner wall above the liquid, or just at the surface, as your method standardises. Press to the first stop. Pause. Press to the second stop to blow out. Still holding the plunger down, remove the tip, then let the plunger rise. Releasing the plunger inside the liquid pulls the delivery back up.

That sequence is forward pipetting. Reverse pipetting aspirates by pressing past the first stop, toward the second, and then delivers only to the first stop, leaving the blow-out volume in the tip. It improves repeatability for foaming, volatile and some viscous liquids, and it is a poor way to dispense the last of a precious standard you thought you had measured by the dial. Do not mix the two techniques in one calibration check. The Addgene protocol collection shows how often molecular methods simply say "pipette" and leave this choice unwritten. Your method should not.

Multichannel pipettes add a new failure: one loose tip, one bent piston, a plate that rocks, and twelve volumes that are not the volume you think you replicated. Seat all tips. Watch the tips enter the wells together. If one channel lags, stop using that channel for a quantitative plate.

When the balance disagrees

A gravimetric check weighs water delivered into a vessel and converts mass to volume with the density of water at that temperature. That is the skeleton of a calibration. The full procedure, the balances, and the documentary standards live in metrology practice. NIST's calibration-procedure collection is a public index into that world. Your quality system names the limits and the interval. This article will not invent an acceptable percentage.

Between those intervals, a quick check can tell you that a pipette has been dropped, that a piston is corroded, or that a new tip brand does not seal. How to do that check without pretending it is a full calibration is the companion article. If the check fails, the branch is simple. Stop using the pipette for quantitative work. Do not hand it to a student with a warning to "be careful". Careful technique cannot repair a leaking seal.

Record the pipette identity in the method when the volume is critical. "P200" is a range, not a serial number.

Failure modes that feel like biology

A master mix that is short because the water pipette drifts will look like an enzyme failure. A standard curve that flattens because small volumes were aspirated at an angle will look like saturation. Droplets on the outside of the tip, wiped on the rim, add an uncalibrated extra that matters most at the smallest settings. Laying a pipette down with liquid in the tip runs fluid into the barrel. That fluid corrodes the piston and then every later volume is wrong. Keep pipettes upright, tips off when you are not using them, and out of solvent pools.

Never rotate the volume dial past the range. The stop you feel is a mechanical limit. Forcing it is a repair bill and a silent bias until then. Do not set a 10-microlitre pipette to 1 microlitre and expect the percentage error of the middle of the range. Use a pipette whose middle includes the volume you care about.

Safety

The liquid is the hazard, not the piston. Acids, solvents, cultures and radioactive tracers do not become mild because the volume is small. Aerosols from blowing out a culture belong in the containment your assessment named, with a filter tip if that assessment asks for one. Do not mouth-pipette. Do not point a tip at a person while you seat it. A pipette is not a syringe for clinical dosing, and this page is not medical advice. Tips that touched hazardous material are waste, not a rinsed item returned to the box.

Temperature, humidity and the room you have

Calibration temperatures are often near 20 Celsius. Many working benches are warmer, and a room that is 30 Celsius with a fan on the balance will not reproduce a temperate calibration mass. Equilibrate water, tips and the pipette. Keep the balance out of the draught and out of direct sun. Humidity changes evaporation of small droplets during a slow check; work steadily and cap the vessel. In a humid week, condensation on a cold tip rack dilutes the first samples unless you dry the rack. A power cut does not decalibrate a piston, but it stops the balance and the air-conditioning that made yesterday's check comparable. Repeat the check when the room returns, if the volume is the result. Altitude changes air density and matters for laboratories far above the place where the pipette was adjusted. If you moved a pipette between cities of very different elevation, treat it as needing a check before you trust the old certificate.

What to send with an enquiry

State the volume range, the liquid class, single or multichannel, filter tips, and whether the specification must include a calibration document or only the instrument. Use the scientific instruments catalogue, the laboratory consumables catalogue for tips, and the quote request. Ask for the written range and tip cone. Do not ask a page to promise that a named unit has already been adjusted for your bench.