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In Order to Use a Pipette: Placement, Technique, and Troubleshooting

Pipettes are fundamental tools in various scientific disciplines, from biology and chemistry to medicine and environmental science. Mastering pipette usage is crucial for accurate and reliable experimental results. This comprehensive guide delves into the proper placement and technique for using a pipette, along with troubleshooting common issues. We’ll cover various pipette types and offer tips for maintaining accuracy and precision.

Understanding Pipette Types and Their Applications

Before we discuss pipette placement, it's vital to understand the different types of pipettes and their specific applications. The most common types include:

1. Micropipettes (or micropipettors):

These are used for transferring volumes ranging from microliters (µL) to milliliters (mL). Micropipettes are further classified based on their volume range:

• P20: Typically dispenses volumes between 2 and 20 µL.
• P200: Typically dispenses volumes between 20 and 200 µL.
• P1000: Typically dispenses volumes between 100 and 1000 µL (1 mL).

Choosing the correct micropipette for the desired volume is critical for accuracy. Using a P200 to dispense 5 µL will lead to significant error.

2. Macropipettes (or serological pipettes):

These are larger pipettes used for transferring milliliter volumes. They are often graduated, allowing for the dispensing of various volumes within their range. Macropipettes are generally used with a pipette bulb or a pipette filler for aspiration and dispensing.

3. Pasteur Pipettes:

These are simple glass or plastic pipettes used for transferring small volumes of liquids without precision. They are often disposable and are commonly used in less demanding applications.

4. Electronic Pipettes:

These pipettes offer advanced features like motorized aspiration and dispensing, adjustable dispensing speeds, and programmable dispensing protocols. They provide enhanced precision and reduce the risk of repetitive strain injuries.

Proper Pipette Placement and Technique: A Step-by-Step Guide

The accurate placement of a pipette is paramount for achieving reliable results. Here’s a detailed step-by-step guide, focusing on micropipettes as they require the most precise handling:

1. Choosing the Right Pipette:

Select the micropipette with a volume range encompassing your desired volume. Avoid using a pipette at the extremes of its range for optimal accuracy.

2. Setting the Desired Volume:

Turn the volume adjustment knob to the desired volume. Ensure the volume is set correctly within the pipette's operational range. Different pipettes have slightly different mechanisms for setting the volume—consult your pipette's manual for specific instructions.

3. Attaching a Pipette Tip:

Firmly press the pipette onto a clean, appropriately sized pipette tip. Ensure a secure and airtight seal between the pipette and the tip to prevent leakage.

4. Aspirating the Liquid (Drawing up the Sample):

• Pre-wetting the Tip (Important): Before drawing up the sample, aspirate and dispense the desired volume into a waste container a few times to ensure the tip is properly wetted and to eliminate any air bubbles. This also helps to calibrate the pipette for better accuracy.
• Aspirating the Sample: Slowly and gently depress the plunger to the first stop. Immerse the tip into the liquid at a slight angle (approximately 45 degrees). Slowly release the plunger to draw the liquid into the tip. Avoid drawing liquid too high into the tip to prevent contamination.
• Checking for Bubbles and Leaks: Check the tip for any air bubbles or signs of leakage. If present, discard the tip and repeat steps 3 and 4.

5. Dispensing the Liquid:

• Dispensing: Touch the tip to the side wall of the receiving vessel at a slight angle. Slowly depress the plunger to the first stop to dispense the majority of the liquid.
• Expelling Residual Liquid: Depress the plunger to the second stop to expel any remaining liquid within the tip.
• Ejecting the Tip: After dispensing, press the tip ejector button to safely dispose of the used tip into an appropriate waste container.

Common Mistakes to Avoid:

• Incorrect Volume Setting: Double-check the volume before and after use.
• Using the Wrong Pipette: Always use a pipette that is appropriately sized for the volume you are working with.
• Insufficient Tip Immersion: Do not immerse the tip too shallowly, as this can lead to inaccurate dispensing.
• Aspirating Too Quickly: This can lead to air bubbles in the sample and inaccurate measurements.
• Touching the Tip to the Bottom of the Vessel: This can contaminate the sample and alter the accuracy.
• Improper Tip Ejection: Always use the ejector button to avoid accidental needle sticks or contamination.

Advanced Pipetting Techniques:

• Reverse Pipetting: Used when dispensing viscous liquids or those that tend to form bubbles. This technique involves depressing the plunger to the second stop before immersing the tip in the sample.
• Serial Dilution: This method involves diluting a solution several times. Pipetting plays a vital role here, ensuring the accuracy of each step.

Troubleshooting Common Pipetting Issues:

• Inconsistent Dispensing: Check for air bubbles in the tip, verify the correct volume setting, ensure a proper tip seal, and ensure the pipette is calibrated and functioning properly.
• Inaccurate Volume Dispensing: Ensure the pipette is correctly calibrated, inspect for tip leaks, confirm the correct volume setting, and use the correct pipette for the targeted volume.
• Pipette Jams: Avoid forcing the plunger— this might damage the pipette. Carefully check for any debris obstructing the mechanism.
• Tip Leaks: Check for any damage or cracks in the pipette tip or a poor seal between the tip and pipette.

Maintaining Pipette Accuracy:

Regular maintenance is vital for ensuring the longevity and accuracy of your pipettes.

• Calibration: Pipettes should be calibrated regularly (at least once a year or more frequently depending on usage) by a qualified technician to ensure accurate dispensing.
• Cleaning: Clean your pipettes regularly with appropriate cleaning solutions, depending on the type of liquid used. Always follow the manufacturer's instructions.
• Storage: Store pipettes in a clean, dry, and dust-free area to prevent damage or contamination.

Conclusion:

Proper pipette placement and technique are essential for accurate and reliable results in any laboratory setting. By understanding the different pipette types, following the step-by-step procedures outlined above, and addressing common troubleshooting issues, you can enhance your pipetting skills significantly. Remember that consistent practice and careful attention to detail are crucial for mastering this vital laboratory skill. Regular calibration and maintenance of your pipettes will ensure they remain accurate and reliable instruments throughout their lifespan. Always consult the manufacturer's instructions for specific recommendations and safety precautions.