Micropipettes – Troubleshooting & DIY

Proper Care & Handling of Your Micropipettes

It’s hard to think of an instrument that’s more symbolic of modern science than micropipettes. They’re inexpensive, easy to use, and found in nearly every lab. But despite their apparent simplicity, most of their sources of error and malfunction aren’t very obvious. This page is intended as a quick reference of the most useful and esoteric information without making you waste your time skimming through non-essential information. Links to additional material are provided for anyone inclined to further reading.

Pipetting Dos and Don’ts

Do  Store the pipette vertically or at a downward angle in its intended holder.
Do  Always use a tip; pre-wet the tip; and change the tip before aspirating a different liquid.
Do  Clean the pipette exterior daily, and as required.
Do  Maintain stable temperature and humidity in your lab.
Do  Allow samples to equilibrate to room temperature before pipetting (when possible).

Don't  Lay the pipette down on the lab bench between uses.
Don't  Use the wrong size tip.
Don't  Turn the dial beyond the volume limits.
Don't  Drop the pipette.
Don't  Hold the pipette while it’s not in use.
Don't  Insert swabs into the pipette for cleaning.

Pipetting Technique

Pipetting is a skill that takes practice to master, and even pipette pros can benefit from an occasional review of the basics. Do you have flawless technique? Consider taking a moment to watch this excellent three minute video by Eppendorf to find out.


Troubleshooting your Micropipette

My pipette isn’t dispensing the right volumes consistently! (Poor accuracy/precision)

Errors, Contamination, & Overdue Maintenance
First, rule out user error by ensuring that everyone in the lab is pipetting with the same technique consistently. Next, consider the possibility of internal contamination. There will usually be visible clues – taking a dry, clean, lint-free white swab to the inside of the tip holder can be helpful. After this, consider the potential for internal wear. How old are the O-rings, and are they regularly exposed to organic solvents? How long has it been since your piston was lubricated with silicone grease? Your pipette may be overdue for basic maintenance.

Samples Requiring Special Techniques
Some samples may require you to use a special technique called reverse pipetting. These include:
Don't  Liquids with a tendency to foam.
Don't  Liquids with high viscosity.
Don't  Volumes of liquid near your pipette’s smallest deliverable amount.

Normal & Reverse Pipetting

How to Reverse Pipette
When pipetting normally, you press the plunger to the first stop, draw up your sample by releasing the plunger slowly, and then dispense the sample by pressing to the second stop to expel any lingering contents. In reverse pipetting, you begin by pressing the plunger all the way down to the second stop, and then draw up the sample by releasing the plunger slowly – completely. This places a reserve of liquid into the pipette tip that is greater than you intend to dispense. You then deliver the sample by only pressing the plunger to the first stop. The additional liquid will remain in the pipette tip. You can either return it to the original container or dispose of it into a waste receptacle as you see fit. Continue to hold the plunger at the first stop until you’re over the container you’ve chosen, and then press the plunger the rest of the way to the second stop before discarding the tip.

Off-Site Calibration
If you find that your pipette has good precision, but is delivering a consistently inaccurate volume, you will most likely need to start calibrating on-site. Most pipettes operate by air displacement, so a simple difference in location can bring your calibration out of spec. The density of the air in your lab is influenced by regional variations in air composition, combined with differences in elevation, humidity, and temperature. This can cause the air density at your lab to be very different from your calibration lab. Verdant only offers calibration on-site, because it’s the best practice.

When all else fails, blame the environment!
If you’ve ruled out contamination, your maintenance is up to date, your calibration is valid, everyone is consistently using the proper technique, then it may be your lab itself. If your lab’s environmental conditions are unstable from day to day, then your pipettes may be the first place you will see it. Micropipettes are high precision pneumatic instruments, which makes them especially vulnerable to lapses in environmental stability.

Contamination & Spills

Gradual contamination from proper use can be minimized by using filter tips. The negative pressures from pipetting will cause a small fraction of most samples to vaporize. Over time, this can lead to internal contamination of the pipette equivalent to a single spill. The filter tips form a line of defense by giving the sample somewhere else to deposit before reaching the internal components of your pipette. Evaluate the chemical properties of your samples to determine whether it’s prone to vaporize and deposit. If it is, you should pipette your samples with filter tips. If you’re not sure, you can always ask us.

Spills include any direct accidental contamination, such as pipetting with no tip, or laying the pipette on its side with a sample in the tip. If this happens, the pipette must be decontaminated, which will include checking and possibly repeating its calibration.

Important! The Forbidden Texts – Not for use in GLP/GMP labs Important!

Many pipettes are discarded after their calibration expires, because it’s often cheaper to replace than to re-calibrate. At Verdant, we strive to keep these out of landfills by bundling pipette coverage with other services at the same cost.

Outside of GLP/GMP settings, users can service their own pipettes as they see fit. But the absence of regulation doesn’t diminish the importance of a pipette’s accuracy and reproducibility. That’s why we’ve chosen to provide complete instructions on how to disassemble, clean, and calibrate a pipette.

Important! Disassembly procedures and calibration adjustment location vary from one manufacturer to another. It would be impossible to explain them all, so instruction manuals for most common pipette models are provided at the end of this section.

Decontaminating a pipette

Before servicing a pipette, it will need to be decontaminated, which will require that it be disassembled. And once a pipette has been taken apart, its calibration will need verified – and possibly repeated – before it’s put back into service. So there are three steps to a decontamination – cleaning, maintenance, and calibration. These steps should be performed every three months, or any time a pipette is either known or suspected to be contaminated. A full calibration (not just a check) should be performed annually.

Pipettes used for radioactive samples
Check the pipette for radioactivity using a calibrated Geiger counter. The standard maximum acceptable limits are 10 Bq for ɣ-radiation (gamma), 10 Bq for ß-radiation (beta), and 25 Bq for X-ray radiation. If the pipette exceeds these levels, it must be disposed of as radioactive waste in accordance with the above laws and policies. Otherwise, you may continue with the decontamination. There are no special procedures to reduce radioactivity. Cleaning a pipette contaminated by radioactive material only involves dilution and physical removal, by wiping the radioactive material away with a strong detergent and distilled water.

Important! If the institutional policies and laws governing your laboratory or physical location set a different maximum limit on radioactivity, always follow the lower (safer) limit.

Purchase or prepare a cleaning solution appropriate for your samples. This should be a mild industrial detergent which – depending on your sample – may include an oxidizing agent, a disinfectant, or an enzymatic cleaner. For pipettes which only deliver organic solvents, isopropyl alcohol followed by a 60 °C evaporation may be adequate.

1) First, soak lint-free tissues (such as Kimwipes) in the cleaning solution, and use them to wipe the body of the pipette.
2) Next, spray the disassembled components with the cleaning solution.
3) Dip a sponge-tip or lint-free swab in the cleaning solution, and then clean the inside of the tip-holder (the component to which the pipette tips attach).
4) Clean any O-rings and piston seals before discarding them into an appropriate receptacle for contaminated waste. These are to be replaced during reassembly.
5) Spray, wipe, or swab all remaining parts with the cleaning solution.
6) Repeat the above cleaning process for all components with distilled water (rinsing thoroughly where possible).
7) Follow this with isopropyl alcohol.
8) Leave the pipettes disassembled to dry in an oven set to 60 °C, or in a fume hood purged by high flow dry nitrogen for one hour. Otherwise, leave the pipettes covered by lint-free tissue, in a dust-free environment under positive pressure (such as a fume hood) if possible.

Important! When cleaning multiple pipettes at the same time, ensure that the parts are kept together with the original pipette. If you do swap parts or lose track, the pipette will need a complete re-calibration – not just a check.
Important! A sonicator bath can replace any physical cleaning steps above, saving yourself some labor and disposable cleaning items.
Important! Planning ahead can make your life easier: protein, DNA, or RNase samples are easiest to clean with an autoclave. If an autoclave safe pipette isn’t available, a mild detergent with an oxidizer such as hydrogen peroxide will suffice.
Important! Do not use alcohols or aprotic solvents. These will precipitate and this will complicate their removal.
Important! Do not expose your pipette to UV light to remove DNA. This will quickly degrade all types of plastic and shorten the life of your pipette.

Calibrating a Micropipette in Accordance with ISO 8655

Your pipette should be calibrated every twelve months, and checked every three months (called an as-found calibration). In the interest of keeping this article at a manageable length, please refer to this pamphlet by Gilson. It is the most well-written literature available: thoroughly explaining current best practices that meet or exceed ISO 8655.

Disassembly, Reassembly, and Calibration Specifics by Model

Instructions for the disassembly, reassembly, and calibration vary from one model to another. Instruction manuals are provided below for the most common pipette models.

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