Accurate liquid handling is the most frequent manual operation in any laboratory. Errors at this stage propagate through every subsequent step, making pipetting technique and solution preparation critical for reproducibility.
Pipetting Principles
Air-displacement pipettes work by creating a cushion of air between the piston and the liquid. The volume is set mechanically or digitally, and the piston displaces a corresponding volume of air to aspirate or dispense liquid.
For accurate pipetting, hold the pipette vertically during aspiration. Immerse the tip 2–4 mm below the meniscus for microliter volumes, deeper for larger volumes. Depress the plunger to the first stop, draw liquid slowly and smoothly, then withdraw the tip along the wall of the container. Dispense by depressing to the first stop, pause, then depress to the second stop to blow out any remaining liquid.
Common Pipetting Errors
- Pre-wetting: aspirating and dispensing the liquid 2–3 times before the actual transfer humidifies the air cushion and reduces evaporation loss.
- Temperature effects: the air cushion expands or contracts with temperature. Both pipette and liquid should be at room temperature.
- Viscous liquids: reverse pipetting (depress to second stop for aspiration, dispense to first stop) improves accuracy for viscous or foaming solutions.
- Tip selection: use the correct tip for the pipette brand. Low-retention tips reduce sample loss for proteins and detergents.
Preparing Solutions
Mass/volume percent solutions are prepared by dissolving a weighed mass of solute in solvent and bringing to the final volume. Molar solutions are prepared by calculating moles from the molecular weight, dissolving, and adjusting to volume in a volumetric flask.
Always add solute to a partial volume of solvent, swirl to dissolve, then bring to the final mark. Never add solvent to dry solute in a volumetric flask — the volume may overshoot.
Serial and Stock Dilutions
A stock solution is a concentrated solution that is diluted to working concentration. The dilution factor is the ratio of final volume to aliquot volume:
C₁V₁ = C₂V₂
Serial dilutions are a stepwise dilution series where each step dilutes by a constant factor (typically 10-fold or 2-fold). This is the most efficient way to produce a logarithmic concentration range for standard curves, MIC assays, or cytotoxicity testing.
When preparing a dilution series, mix thoroughly at each step and change the pipette tip between transfers to avoid carryover.
