Best Practices for the Use of Micropipettes

Oct 09, 2014

Pipetting techniqueWhen it comes to pipetting technique, minor variations can have major consequences.

These differences may seem normal and insignificant; perhaps some pipetting technique variation is even expected.  After all, when technicians enter the lab field they bring all sorts of training methods, education, preferences and experience with them, right?  Differences in technique can be attributed to a variety of factors, but it’s important to recognize what’s at stake.  Incorrect, incomplete or carelessly performed pipetting steps can – and do – directly impact test results, and might even affect patient outcomes.

What are some potential sources of over- or under-delivery when using manual pipettes?   Well, just like other forms of laboratory instrumentation, even a properly calibrated pipette performs only as well as its operator.

The American Laboratory article “Best Practices for the Use of Micropipets” discusses a number of considerations.  Each of them can affect pipetting accuracy and precision.  And, fortunately, all of them can be managed.

Evaporation, heat transfer and changes in barometric pressure

These factors can alter pipetted volumes by causing the air cushion above the sample liquids to expand or contract.   Errors are easily minimized by paying attention to environmental influences, and taking the necessary steps to monitor and control them.

  • Maintain temperature equilibrium while you work.    Allow time for liquids, instruments and ambient air temperatures to stabilize.
  • Release your grip.  To minimize transfer of body heat, avoid over-handling the pipette.  Either place it back on the rack in between dispenses, or hold it loosely.
  • Don’t lose sample to evaporation.  Humidify the captive air pocket in the tip by pre-wetting it several times.

First stop to aspirate, second stop to dispense

Forward, or standard, pipetting mode is generally the best method to use with aqueous solutions.  Reverse-mode pipetting can be useful for viscous or volatile liquids, but it generally over-delivers when using solutions similar to water.

Go with the flow

How is hydrodynamic flow affected by an operator’s technique?  Well, liquid sitting close to the walls of a container may behave differently from liquid at the center of the sample.  A few key steps will help technicians manage this potential source of error.

  • Keep the tip in a vertical position during aspiration, and as it exits the source container.
  • Avoid contact between the tip and the container wall during or after aspiration.
  • When aspirating, immerse tips to the right depth – too shallow, and you’ll risk introducing air into the sample.  But go too deep and you’ll increase the chance of extra liquid adhering to the outside of the tip.
  • As the sample is dispensed into the receiving container, the tip should not be immersed at all.  This will help avoid both over-delivery and cross-contamination.

Precision and accuracy are directly related to technique

Like most skills combining science with art, manual pipetting really does benefit from regular practice and attention to the finer points.

  • Keep the plunger speed and pressure consistent.
  • Pause briefly – and consistently – after aspirating, while tip remains immersed.
  • If at all possible, avoid the practice of tip wiping.  Technicians may do this out of habit, but there are very few valid reasons to wipe the tip.

Equipment choice makes a measurable difference

Even the best operator technique can be undermined by subpar hardware.  To avoid compromising important test results, make sure you’re using the right tools for the job.

  • Use only high-quality pipette tips.
  • Examine the tip before dispensing.
  • Use the right pipette size.

Any of these factors could potentially introduce error into test results, and multiple variations only compound the risk.  That’s why it makes sense to devote time and effort to develop standardized pipetting technique through training and coaching.

Read the full article for more detail, and to learn additional ways to improve technique.


Additional Resources

Learn more about pipetting technique

Download American Laboratory article “Best Practices for the Use of Micropipets”