Pipetting

Awareness level only. This lesson explains the ideas so you recognise them. It does not qualify you to do this work. Before you use lab equipment or handle chemicals, biological materials or patient samples for real, you need the training your employer or institution requires, and you must work under supervision until they sign you off.

Why it matters

Biology and clinical labs move small volumes all day: 2 µL of DNA into a reaction, 50 µL of serum into a cuvette, 200 µL of standard into each well of a plate. A micropipette makes this possible, but it is easy to use badly. Holding it at an angle, plunging too fast or using the wrong stop can each cause errors of several percent, and those errors are invisible until a control fails.

How an air-displacement micropipette works

Most lab micropipettes are air-displacement pipettes. The plunger moves a piston inside the barrel. When you release the plunger, the piston rises and the air cushion beneath it pulls liquid into a disposable tip. The liquid never touches the pipette itself, only the tip.

Pipettes come in sizes. Common names follow the maximum volume:

Choose the smallest pipette that covers your volume. Accuracy and precision are best in the upper part of a pipette's range. Pipetting 15 µL with a P20 is better than with a P200. Never turn the volume dial past the pipette's limits: it can damage the mechanism.

The two stops

Press the plunger slowly and you will feel two points of resistance:

• First stop: the calibrated volume. You press to here before aspirating.
• Second stop (blow-out): pushes out the last drop when dispensing.

Forward pipetting (the standard method)

1. Set the volume and attach a fresh tip firmly.
2. Hold the pipette vertically. Press the plunger to the first stop.
3. Immerse the tip just below the surface, a few millimetres (about 1 mm for very small volumes, a few mm for large ones). Too deep coats the outside of the tip with extra liquid.
4. Release the plunger slowly and smoothly. Wait a second for the liquid to finish entering the tip.
5. Withdraw the tip, touching it against the wall of the vessel if your SOP allows, to remove drops on the outside.
6. Dispense by pressing to the first stop with the tip against the wall of the receiving vessel, then to the second stop to blow out the rest.
7. Keep the plunger pressed while you remove the tip from the liquid, then release it. Eject the tip.

Reverse pipetting

For viscous liquids (glycerol, serum) or liquids that foam (detergent solutions), labs often use reverse pipetting: press to the second stop before aspirating, then dispense only to the first stop, leaving the excess in the tip. It reduces bubbles and film errors. Your SOP will say which method to use.

Habits that improve accuracy

• Pre-wet the tip by aspirating and dispensing the liquid two or three times before the measured transfer.
• Let the pipette, tips and liquid reach the same temperature.
• Keep a consistent rhythm and plunger speed.
• Change tips between samples to avoid carry-over.
• Never lay a pipette on its side or turn it upside down with liquid in the tip: liquid can run into the barrel and contaminate it.

Checking a pipette: the gravimetric method

Labs check pipettes by weighing water, because 1 µL of water at about 20 °C weighs very close to 1 mg. The full procedure (ISO 8655) corrects for temperature and air pressure, but the idea is simple.

Worked example

Ten 100 µL dispenses of water weigh (in mg): 99.6, 100.1, 99.8, 99.9, 100.2, 99.7, 99.9, 100.0, 99.8, 99.6.

• Mean: 99.86 mg. Converting with a factor of about 1.003 µL per mg gives about 100.16 µL.
• Systematic error (accuracy): 100.16 minus 100 = +0.16 µL, or +0.16 percent.
• Random error (precision): the standard deviation is about 0.20 mg, so the coefficient of variation (CV) is 0.20 / 99.86 = about 0.20 percent.

Your lab compares these numbers with acceptance limits for that pipette size. If a pipette fails, it is labelled out of service and sent for adjustment.

Common mistakes

• Aspirating at an angle instead of vertically.
• Pressing past the first stop before aspirating (this draws up too much in forward pipetting).
• Letting the plunger snap back, which splashes liquid into the barrel and forms bubbles.
• Using a P1000 at 50 µL.
• Reusing a tip between different samples.
• Skipping the scheduled pipette check because the pipette "seems fine".

Quick recap

• Pick the smallest pipette that covers the volume, and stay within its range.
• First stop to aspirate, second stop to blow out (forward pipetting).
• Vertical, shallow, slow and smooth; pre-wet the tip; change tips.
• Pipettes are checked by weighing water: mean gives accuracy, CV gives precision.

Try it

Open the Do tab to analyse a pipette check on paper, then practise the plunger motions with plain water if you have access to a teaching lab.