Pipetting Techniques and Best Practices

Pipetting Techniques and Best Practices

Introduction to Pipetting

  • Pipetting is essential for providing sustenance to cells by dispensing solutions, mixing, and rinsing away unwanted substances.
  • Micropipettes are commonly used for dispensing solutions.
  • Instead of weighing powders or using concentrated stock solutions directly, it's more efficient to prepare various solutions and store them at different temperatures (refrigerated, room temperature, -20°C, -70°C) for later use.

What is a Pipette?

  • A pipette is a small apparatus, typically a narrow tube, used to draw fluid by suction for dispensing or measurement, retaining it by closing the upper end.
  • Historically, various designs of pipettes have existed.

Micropipette Suppliers and Types

  • The market now offers micropipettes from many different manufacturers at reasonable costs due to efficient shipping, especially from China.
  • Pipettors come in different colors and have a spindle at the end, along with numerical indicators and controls.

Manual vs. Automated Pipetting

  • Manual micropipettes are commonly used for practices.
  • Biotech and pharmaceutical companies use liquid handling machines with one or more pipettors for high-throughput screening of compounds.
  • Liquid handling machines may have tip rinsing capabilities.
  • For practicing, you can reuse the same tip, but for live cultures, always use new, sterile, and clean tips to ensure sample integrity, especially important, for example, in COVID testing.

Importance of Proper Equipment Usage

  • Equipment, whether inexpensive or expensive, is engineered for precision but requires proper usage to achieve it.
  • User manuals, even if lengthy should be consulted.
  • The key components of a pipettor are the plunger and the tip.
  • The plunger displaces air inside the pipette tip. Immersing the tip in solution and releasing the plunger creates a vacuum, drawing fluid up equivalent to the displaced air volume.
  • Dispensing involves pressing the plunger to the first stop to expel liquid, with a small amount remaining in the tip.
  • Pressing past the first stop to the second stop can expel more liquid.
  • After dispensing, either take another portion from the same solution or use a new tip to prevent contamination.

Micropipette Volume Measurement

  • Micropipettes are used to measure small volumes, typically less than one milliliter.
  • Different micropipettes are designed for different volume ranges.
  • Each pipette is marked to indicate its measurement range.
    • P1000: 100 microliters to 1000 microliters.
    • P20: 2 microliters to 20 microliters.
  • A window on the pipette displays the set volume.
    • On a P1000, digits represent thousands, hundreds, and tens places.
    • On a P20, digits represent tens, ones, and tenths places.
  • Example:
    • P1000 set to 950 microliters.
    • P20 set to 12.5 microliters.

Adjusting Micropipette Volume

  • To adjust the volume on a P20, gently pull up and turn the black wheel and push the black wheel back into its original position after adjustment.
  • Never adjust the pipette volume outside of its designed range to avoid damaging the micropipette.

Using Disposable Pipette Tips

  • Place a disposable plastic pipette tip onto the end of the pipette before aspirating fluid.
  • Different micropipettes use different types of tips.
  • Ensure the tip is firmly seated on the end of the micropipette.
  • Fluid is aspirated and expelled using the micropipette plunger.

Aspirating Fluid

  • Push the plunger down to the first stop.
  • Hold down the button as you place the pipette tip into the fluid.
  • Only the very end of the pipette tip needs to be immersed in the fluid.
  • Slowly release the button to draw the fluid up into the pipette.
  • Keep the pipette tip in the fluid until the plunger has completely regained its original position.
  • Draw the tip out of the fluid.
  • The volume of fluid taken up into the pipette tip matches the micropipette setting like 20 microliters.

Expelling Fluid

  • Place the pipette tip into the receiving tube.
  • Slowly push down on the button at the top of the plunger to the first stop to expel the liquid.
  • Push past the first stop to the second stop to remove the last bit of fluid from the pipette tip.
  • Hold down the button until the tip is no longer touching the fluid to avoid aspirating the fluid back up into the tip.

Pipetting Tips for Accuracy and Precision

  • Pre wetting the pipette tip:
    • Aspirate and dispense the sample back into the sample container or waste at least three times to pre wet the tip.
    • This increases humidity inside the tip and reduces sample loss due to evaporation.
  • Working at temperature equilibrium:
    • Volume dispensed varies with environmental conditions, like room temperature.
    • Allow liquids and the pipette to equilibrate to ambient temperature before use to minimize variation in dispense volume.
  • Examining the tip before dispensing:
    • If droplets are on the outside, dab carefully with a lint free cloth, avoiding the tip opening.
    • Avoid a full tip wipe to prevent the sample from wicking out.
  • Using standard mode pipetting:
    • Depress the plunger to the first stop, immerse the tip, and slowly release to aspirate.
    • Move to the transfer container and press the plunger to the first stop, then the second stop, to dispense all solution.
  • Reverse mode pipetting:
    • Depress the plunger past the first stop to aspirate more than the target volume.
    • Move to the transfer container and depress the plunger back to the first stop, delivering the target volume.
    • This leaves residual sample in the tip and is suitable for viscous or volatile solutions.
  • Pausing consistently after aspiration:
    • Slow, even plunger release and a one to two second pause after aspiration will allow the liquid to stabilize.
    • This is especially important for viscous solutions.
  • Vertical withdrawal during aspiration:
    • Always hold the pipette vertically during sample aspiration and avoid touching the sides of the container.
    • After sample aspiration, pull the pipette straight out of the liquid.
    • Surface tension effects may cause sample volumes to vary if the pipette is not pulled out vertically.
  • Minimizing handling of the pipette and tip:
    • Avoid overhandling to prevent body heat transfer, which affects temperature equilibrium.
    • Hold the pipette loosely or return it to a stand between transfers.
    • Wearing gloves can help reduce heat transfer.
  • Immersing the tip to the proper depth:
    • When aspirating, immerse the tip two to six millimeters below the meniscus, avoiding the container walls and bottom.
    • Inserting too far may cause excess droplets on the outside of the tip.
  • Using the correct pipette tip:
    • Choose the correct tip for the pipette being used.
    • Proper tip fit ensures a tight seal and minimizes sample loss due to leakage.
    • Avoid excessive force when placing a tip on the pipette to prevent damage.
  • Using consistent plunger speed and pressure:
    • Maintain constant steady plunger movement during both aspiration and delivery to prevent air from entering the process.

Additional Considerations

  • Pre wetting Tips:
    • Account for slight fluid drop remaining in the tip.
    • First aliquot may be slightly less volume than subsequent ones.
  • Reverse Pipetting:
    • Purposefully draw more than the volume at the first stop.
    • Touch the tip to the side of the container during dispensing.
  • Tip Wiping:
    • Routinely wipe the outside of the pipette tip on the inside of the source container to remove excess fluid.
  • Tip Attachment and Disposal:
    • Use a tip rack holder for secure attachment.
    • Use the side piece to eject the tip.
    • Keep the tip vertical to prevent residual fluid from going up into the pipettor.
  • Pipettor Maintenance:
    • Pipettors have a filter inside to prevent fluid from coming up, and replace them regularly.
  • Reverse Pipetting for Viscous Fluids:
    • Works better for sticky or viscous fluids.
  • Positive Displacement Pipettes:
    • Use an inner plunger all the way to the bottom to eliminate the air gap.

Lab Practice

  • The lab session will focus on getting comfortable with pipetting.
  • Practice both forward and reverse pipetting.
  • Measure accuracy using a balance.