In the medical laboratory, the measurement of volumes is often done in microliters (µL) and nanoliters (nL).
Pipettes are essential tools in controlling small volumes of liquid, making accurate pipetting a crucial skill in biotechnology.
Definition of Pipette
A pipette is a laboratory device used to measure out or dispense small amounts of liquid, typically in volumes of milliliters (mL) or microliters (μL).
The term "pipette" originates from a French word that translates to "small pipe."
Principle of Micropipettes
Air Displacement Pipette: The volume of air that is displaced within the pipette correlates to the selected aspiration volume.
Positive Displacement Pipette: This type utilizes a plastic tip that integrates an internal piston, ensuring precise dispensing of samples.
Types of Pipettes
Single-channel Pipettes: Designed to dispense one sample volume at a time.
Multichannel Pipettes: Capable of dispensing multiple samples simultaneously, which streamlines parallel processing tasks.
Pasteur Pipette: Used primarily for transfer of liquids without precise volume measurement.
Serological Pipettes: Used for transferring larger volumes of liquid, providing increased precision and measurement.
Classifications of Pipettes
Single-channel vs. Multichannel:
Single-channel allows for only one liquid transfer at a time.
Multichannel enables simultaneous transfer, usually in blocks of eight, twelve, or more.
Manual vs. Electronic Pipettes:
Manual pipettes require hand operation, while electronic pipettes automate the dispensing process for higher accuracy and convenience.
Fixed vs. Adjustable Volume Pipettes:
Fixed pipettes are set to a specific volume, while adjustable pipettes can be set to various desired volumes within their range.
Parts of a Micropipette
Plunger: Controls the movement of the piston within the pipette.
Volume Adjusting Knob: Allows for setting the desired volume to dispense.
Tip Ejector Button and Ejector Arm: Facilitates the removal of the pipette tip post-dispensing.
Digital Volume Display Window: Displays the set volume for accurate dispensing.
Plastic Shaft and Tip Cone: Serves as the entry point for the liquid to be aspirated.
Pipetting Techniques
Forward Filling: A technique where the sample is drawn into the pipette from the stock solution and dispensed into the receiving vessel.
Reverse Filling: Used to re-aspirate or refill the pipette with new liquid after partial dispensing.
First Stop and Second Stop: Refers to the two levels of plunger movement: the first is for aspirating, and the second for expelling contents.
Accuracy and Precision
Definitions:
Accurate: Measurement that is close to the true value.
Precise: Measurements that yield consistent results when repeated.
Scenarios depicted demonstrating various degrees of accuracy and precision, indicating:
Accurate and precise.
Accurate but not precise.
Precise but not accurate.
Neither accurate nor precise.
Calibration of Pipette
Gravimetric Method: The weight of a known volume can be measured to determine accuracy.
For instance, 100μL=1mg, 1000μL=1g.
Colorimetric Method: Utilizes color change or intensity to quantify a sample's volume based on a calibration curve.
Common Sources of Pipetting Error and Mitigation Strategies
Poor Technique: Leads to inconsistent volumes and aerosol generation.
Mitigation: Use smooth, consistent motions; pre-wet tips; immerse tips to the correct depth; select the appropriate pipette for the volume.
Thermal Effects: Air displacement pipettes are sensitive to temperature changes, leading to over-delivery.
Mitigation: Equilibrate all reagents and the pipette to room temperature before use.
Wrong Tip Usage: Using incorrect tips can create a poor seal, leading to volume inaccuracies.
Mitigation: Always use high-quality, manufacturer-recommended tips.
Not Calibrating Regularly: Pipettes can drift out of specification.
Mitigation: Implement regular professional calibration and frequent self-checks using gravimetric analysis.
Human Fatigue: Over time, fatigue can diminish precision and accuracy of pipetting.
Mitigation: Utilize electronic pipettes for high-throughput tasks and ensure regular breaks are taken to maintain performance.
Immersion Depth and Angle
Describes how the immersion depth affects accuracy:
1 cm immersion: 0.2-0.4% inaccuracy.
3 cm immersion: 0.6-0.8% inaccuracy.
4 cm immersion: 1-1.2% inaccuracy.
Immersion angle should ideally remain vertical; deviations of over 20 degrees can lead to inaccuracies due to excess liquid entering the tip.
Pipetting Improvement Techniques
Dispensing Techniques: Contacting the wall of the tube while dispensing ensures no sample remains clinging to the orifice; this technique can enhance accuracy by up to 1%.
Aspiration Techniques: Maintaining a consistent rhythm during liquid aspiration prevents splashing and loss of sample volume, improving accuracy by up to 5%.
Pre-rinsing/Pre-wetting: Releasing the plunger preemptively a few times helps to neutralize capillary effects and equalizes temperature, improving accuracy by up to 0.2%.
Angle of Immersion: Should remain as vertical as possible, ideally avoiding deviations greater than 20 degrees, to decrease inaccuracies caused by excess liquid in the tip.
Conclusion
As laboratory demands for accuracy and precision increase, mastering effective pipetting techniques becomes essential for reliable results in experimental and clinical settings.