Proper Pipetting Techniques

Pipetting liquids is a core part of day-to-day lab workflows. Technicians must use the right techniques and follow pipetting best practices to ensure accurate and precise results. Whether working with aqueous samples, bodily fluids, hazardous solutions or other liquids, use the following techniques and best practices for proper pipetting.

Pipette Types and Their Uses

Pipettes come in many styles but typically fall under one of two categories — air displacement or positive displacement pipettes. A pipette's technology affects the necessary techniques. Both options have a piston inside a cylinder or capillary to move the liquid, but they use different mechanics. Here's an overview of air displacement pipettes vs. positive displacement pipettes:

• Air displacement pipettes: These general-use pipettes maintain some air between the piston and the liquid. While they offer exceptional accuracy for standard aqueous applications, they can introduce variation with some samples. This variation occurs due to factors like temperature, viscosity and atmospheric pressure, which can alter the volume of the liquid.
• Positive displacement pipettes: The piston directly contacts the liquid and is integrated with the disposable pipette tip. Positive displacement pipettes are the most accurate options, and they work well for viscous and volatile liquids. They're also practical for specialized applications like PCR, hazardous samples and DNA/RNA solutions. The pipette design prevents aerosol contamination and sample-to-sample cross-contamination.

Both air displacement and positive displacement pipettes often use tips made of polypropylene (PP), a disposal material that helps prevent cross-contamination. You'll also find polyethylene (PE) or glass pipette tips. Standard pipettes vary in size and typically handle volumes from 1 mL to 50 mL. Micropipettes support smaller volumes between about 0.2 mL and 1 mL, while electronic pipettes automate most tasks in the process. We can also categorize pipette tips by whether they are sterile or filtered. A filter helps keep samples sterile by preventing aerosols from forming in them.

Understanding Liquid Properties in Pipetting

The proper pipette techniques and supplies vary based on several liquid properties. Viscosity, volatility and temperature are particularly influential in handling requirements. A positive displacement pipette can eliminate many of these issues, but you can also experience effective results with air displacement pipetting. You'll need to take these extra precautions:

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• Viscous liquids: These liquids enter the pipette tip more slowly and may stick to its wall during dispensing. You may need to use slower speeds or reverse pipette modes to ensure accuracy and precision.
• Volatile compounds: These liquids can evaporate quickly and reduce the delivery volume. With air displacement methods, you can minimize this problem by pre-wetting the pipette tip, using reverse mode and working quickly.
• Differing temperatures: Differences between the pipette and sample temperatures can cause variations in the dispensed liquid amounts. Minimizing handling and ensuring equal temperatures help ensure consistent delivery.

Pipetting Techniques and Best Practices

The pipetting process can heavily influence the volume of liquid in your reservoir. Follow these guidelines for standard pipetting techniques to eliminate variation in your research.

Basic Steps for Pipetting

Forward pipetting is the standard method used with both air displacement and positive displacement pipettes. For proper forward pipetting, follow these steps:

1. Establish temperature equilibrium: Allow the pipette, pipette tip and liquids to reach ambient temperature when working with air displacement pipettes. Equal temperatures remove variation due to the liquid's temperature-dependent properties.
2. Pre-wet the pipette tip: Aspirate and dispense the liquid 1-3 times. This step helps prevent evaporation in the air space of the pipette by increasing humidity.
3. Aspirate the liquid: Insert the pipette tip into the liquid and draw it up to the first stop. If using reverse pipetting or another method, you may need to use another stop on the pipette. Ensure an appropriate immersion depth. If the pipette is not immersed enough, it can aspirate air. If it is immersed too far, the volume of air in the pipette tip becomes compressed and can aspirate too much liquid. Aim to keep the pipette tip just below the meniscus of the liquid.
4. Pause after dispensing liquid: Each time you aspirate, liquid flows briefly into the pipette tip after you release the plunger. Pause for about a second while the pipette tip is still in the liquid.
5. Remove the pipette vertically: Hold the pipette as straight as possible while aspirating liquid, with no more than about a 20° angle. Pull it up from the center. Removing the pipette at an angle can cause too much liquid to enter the pipette tip.
6. Dispense the liquid: Gently press the plunger through the first stop and to the second to entirely dispense the liquid in forward pipetting. You may notice droplets on the end of the pipette tip. Slide the end of the pipette tip along the vessel's side to remove the droplet. You can also touch the droplet to the surface of the liquid. This option avoids residual liquid on the side of the vessel and may be necessary for smaller volumes.

Reverse pipetting is slightly more complex and can only occur in air displacement pipettes. This process starts by pressing the plunger to the second stop. Insert the pipette and release the plunger fully so the tip has twice as much volume as desired. To dispense the liquid, press the plunger gently to the first stop. With the plunger still held, dispense the remaining liquid into the original solution or dispose of it. Release the plunger fully.

General Pipetting Best Practices

Throughout this process, follow these best practices for pipetting to ensure accurate, precise results:

• Keep your speed consistent: Press and release the plunger as consistently as possible. Use gentle movements.
• Avoid over-handling the pipette and pipette tip: Body heat can affect the temperature equilibrium, so minimize handling where possible. Keep a light grip, use gloves and rest the pipette on a stand when not in use.
• Clean and inspect the pipette and pipette tip before use: Clean your pipette appropriately and get rid of any dirty pipette tips immediately after use.
• Prevent contamination between the pipette and sample: Sterilized tips and sterilized filtered tips can help prevent pipette-to-sample contamination, as can changing tips after each sample. Minimize sample-to-pipette contamination by always holding the pipette vertically, slowly releasing the plunger and using filter tips. Positive displacement pipettes also prevent contamination.
• Prevent contamination between samples: To avoid sample-to-sample contamination, swap pipette tips after every sample and clean or autoclave the pipette as needed.
• Choose appropriate pipette tips: Use the correct type of pipette tip to create an airtight seal. Tips should have high-quality materials, uniform size and shape, and proper packaging.
• Pay attention to ergonomics: Avoid fatigue by taking regular breaks, occasionally setting the pipette on a stand and working at a relaxed pace. Understanding the importance of ergonomics supports pipetting accuracy and precision. Consider using an electronic pipette for large-volume tasks.

Proper Handling and Care of Pipettes

Appropriate handling and cleaning practices support reliable results and help your pipette last longer. Inspect the pipette and pipette tips with each use to monitor for damage. Perform calibration and maintenance at least annually or more frequently for heavy use. Follow your pipette manufacturer's instructions for more precise intervals.

Between uses, follow best practices for pipette storage to prevent contamination and damage. Never store the pipette horizontally. Vertical storage keeps liquid from reaching the pipette's piston and inner components, where it can cause corrosion. It also prevents the piston lubricant from collecting on one side. Do not store the pipette with the pipette tip attached for the same reasons. Using a pipette stand between uses can help avoid horizontal storage, along with preventing drops and cross-contamination from work surfaces.

Achieve Accurate, Precise Pipetting With Supplies From Crystalgen

Accuracy and precision are essential for most pipetting tasks. Improper technique can quickly hurt the quality of your research, but it's only one part of the equation. You'll also need dependable supplies. Crystalgen carries a wide range of reliable pipetting materials, including pipette tips, stands and electronic pipette controllers. We simplify lab supply purchasing so you can focus on more important tasks. Shop pipette tips online, or reach out to us with any questions on pipetting supplies.