The micropipette is a precision instrument used in laboratories worldwide to measure and transfer extremely small volumes of liquid, typically in the microliter range. This tool is fundamental to fields such as molecular biology, chemistry, and clinical diagnostics where accuracy in sample handling directly impacts experimental outcomes. Mastering the precise operation of this device is a foundational skill for achieving reliable and reproducible results.
Understanding the Micropipette and Volume Selection
A standard air-displacement micropipette consists of several basic components, including the plunger, the volume adjustment dial, the tip ejector, and the barrel, which holds the disposable tip. The plunger controls the internal piston that displaces air to draw up and expel liquid, and the dial sets the exact volume.
Selecting the correct pipette size is the first step toward accurate measurement. Micropipettes are designated by their maximum capacity, such as a P20 for up to 20 microliters (µL) or a P1000 for up to 1000 µL. Always choose the pipette whose range best accommodates your required volume, ideally operating within the upper 35% to 100% of the instrument’s designated volume for best accuracy. Turn the volume adjustment dial slowly to the desired setting, approaching the final value by dialing down to prevent mechanical stress on the internal components.
The Essential Steps for Accurate Pipetting
The process begins by firmly seating a new, clean tip onto the tip cone to ensure an airtight seal, which is necessary for the air-displacement mechanism to work correctly. Depress the plunger smoothly to the first stop, which is the calibrated point of resistance corresponding to the set volume. While holding the plunger at this first stop, immerse the tip a few millimeters into the source liquid—typically 2 to 3 mm—keeping the pipette as vertical as possible.
Aspirate the liquid by slowly and steadily releasing the plunger to its resting position. This slow, controlled movement prevents air bubbles, splashing, or drawing liquid into the barrel of the pipette. After aspiration, pause briefly to ensure the full volume has entered the tip. Carefully withdraw the tip from the source liquid, touching it against the side of the container to remove clinging droplets.
To dispense the liquid, place the tip against the inner wall of the receiving vessel at a slight angle. Depress the plunger smoothly to the first stop to release the measured volume. To ensure complete expulsion, continue pressing the plunger down to the second stop, which is the firm blow-out position. While keeping the plunger depressed to the second stop, slide the tip up the wall of the vessel to separate it, and then use the tip ejector to discard the used tip.
Avoiding Common Errors and Ensuring Precision
Several factors and common errors can compromise pipetting accuracy. One frequent issue is the temperature difference between the pipette, the tip, and the liquid itself. Pipetting a cold liquid with a room-temperature pipette can lead to under-delivery because the cooler air cushion inside the pipette contracts after aspiration. Allowing the liquid and equipment to equilibrate to ambient temperature helps minimize volume variations.
The speed of aspiration and dispensing also affects accuracy; plunging too quickly can cause liquid to splash, form bubbles, or lead to incomplete aspiration. Holding the pipette at a sharp angle during aspiration, especially greater than 20 degrees from vertical, can also result in inaccurate volumes.
Reverse Pipetting Technique
For liquids that are highly viscous, volatile, or prone to foaming, the standard forward technique may not be suitable. The reverse pipetting technique is used in these cases. This involves aspirating to the second stop and dispensing only to the first stop, leaving a small, excess volume in the tip to compensate for liquid clinging to the tip walls.
Proper Maintenance and Storage
Maintaining the micropipette ensures its long-term accuracy and reliability. If liquid accidentally enters the barrel, the instrument needs to be disassembled and thoroughly cleaned according to the manufacturer’s instructions, often involving rinsing components with distilled water or 70% isopropyl alcohol. Routine calibration checks, typically performed every six to twelve months, confirm that the pipette is dispensing the volume indicated on the dial within acceptable tolerances.
When the micropipette is not in use, it must be stored correctly to prevent damage to the internal mechanism. Micropipettes should always be stored vertically on a dedicated stand or rack. Storing the instrument horizontally can cause the internal piston lubricant to pool, impairing the piston’s smooth movement. If a tip is left attached after use, storing it vertically prevents residual liquid from wicking up into the internal components, which could cause corrosion or contamination.