Pipetting and pipette calibration

Principle

• Air-displacement micropipettes move a set volume of air to aspirate and dispense liquid; accuracy depends on technique and regular calibration.
• Accuracy is closeness of the mean to the set volume; precision (repeatability) is closeness of replicate volumes to each other, expressed as CV.

Reagents and equipment

• Adjustable and fixed micropipettes with matching tips, volumetric glassware (Class A), serological pipettes with pipette aids (never mouth pipetting).
• For calibration: analytical balance, distilled water, thermometer, evaporation trap, or a photometric (dye) calibration system.

Procedure

• Forward pipetting (standard for aqueous solutions): press to the first stop, aspirate, then dispense by pressing to the second stop (blow-out).
• Reverse pipetting (viscous, foaming or volatile liquids, and whole blood): press to the second stop, aspirate, dispense to the first stop only, leaving a small residue in the tip.
• Pre-rinse the tip with the liquid, hold the pipette nearly vertical, immerse the tip just below the surface, and aspirate slowly.
• Dispense against the vessel wall; use steady plunger action and avoid bubbles.
• Use the pipette within its range; accuracy worsens near the lowest setting.
• Gravimetric calibration (ISO 8655): weigh repeated dispenses of water at set volumes, convert mass to volume using a temperature-based factor (about 1.003 µL/mg at 20–25 °C), and calculate systematic error and CV against limits.
• Glassware: read the bottom of the meniscus at eye level; Class A volumetric pipettes are "to deliver" (TD) and should not be blown out unless marked.

Quality control and pitfalls

• Calibrate at set intervals (often every 3–12 months, per policy) and after damage or repair; label with status and due date.
• Errors: wrong or loose tips, liquid in the shaft, releasing the plunger quickly, pipetting at an angle, temperature differences between pipette and liquid.
• Store vertically; never lay down a pipette with liquid in the tip.

Clinical use

• Accurate measurement of samples, reagents, calibrators and controls in all laboratory sections.