How to pipette - the essential guide to good technique

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How to pipette - the essential guide to good technique
There are basically two types of laboratory pipette:
  • Air displacement
  • Positive displacement
Air displacement pipettes are most commonly used in the laboratory for volumes greater than 100µl. The Principles of Air displacement pipetting - [caption id="attachment_8343" align="alignnone" width="300"]Air displacement principles Air displacement principles[/caption]
  • Air displacement is adequate for any water based and non viscous fluids
  • There is an air gap between the fluid and the plunger so there is no direct contact with the instrument
  • The pipette can dispense either by forward or reverse pipetting techniques depending on the required accuracy and precision
Forward Pipetting technique - Most commonly used pipetting technique that expels an exact volume of liquid from the pipette tip repeatedly. Careful technique is essential as the plunger is only depressed as far as the first stop when drawing up liquid and dispensing it. The technique must be reproducible and with smaller volumes this can be difficult to achieve. [caption id="attachment_8345" align="alignleft" width="193"]Forward pipetting technique Forward Pipetting Technique[/caption]     Procedure:
  • Depress plunger to first stop
  • Immerse tip vertically into liquid and smoothly release the plunger
  • Depress plunger button to first stop to dispense liquid into receiving vessel
  • Depress button fully to second stop to expel the remaining liquid from the tip
      Reverse Pipetting Techniques Reverse pipetting techniques are only possible with air displacement pipettes. The liquid is aspirated in excess of the selected volume but only a set volume is dispensed. This improves precision with liquids such as viscous, volatile or foaming samples, and with smaller volumes below 100µl. [caption id="attachment_8347" align="alignleft" width="193"]Reverse pipetting technique Reverse pipetting technique[/caption]     Procedure:
  • Depress plunger button to second stop
  • Immerse tip vertically into the liquid and smoothly release plunger button
  • Depress plunger to first stop only to release liquid into vessel
  • Discard remaining liquid by depressing to second stop
      Recommended Pipetting Procedures - [caption id="attachment_8349" align="alignleft" width="300"]Good techniques Good techniques[/caption]  
  •       Pre- wet tip x 2 before dispensing
  •       Dip the tip 2-4mm into the liquid
  •       Smooth aspiration both touching and not touching the side wall, (sometimes used with micrototre plate assays)
    Positive Displacement Pipettes [caption id="attachment_8353" align="alignleft" width="300"]Positive displacement technique Positive displacement technique[/caption]
  • preferred for viscous, volatile or corrosive samples
  • Direct contact between plunger and sample
  • Works by replacing both plunger and capillary for each sample
  • Liquid is forced out by the plunger through the capillary when dispensing
  • best for small volume precious samples
  Camlab have a comprehensive range of both air displacement and positive displacement pipettes online from both Socorex and Eppendorf.    
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piero zucchelli
Hi, i need to point out i work for - despite the vested interest i think it's important to highlight that thanks to our robots we can literally study and precisely evaluate the impact of the various approaches. First of all, reverse pipetting is far superior for foamy liquids than forward pipetting. Then, the most important trick: the difference between calibration labs and standard labs is the evinronmental humidity (70% for calibration labs). If you aspirate 1 uL and "just" 1 nL of water evaporates inside the tip, your liquid will go down microliter. Why? 1 nL of liquid water is about 1 uL of water vapour in volume. So the mandatory trick to deal with small volumes in a standard lab is: aspirate, mix up and down if you like, then dispense in the source, EXTRACT the tip from the liquid, let the air enter and restore the atmospheric pressure (together with the vapour) and THEN enter again into the source, to aspirate your volume...