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17 Ways to Stop Pipetting Errors From Ruining Your Experiments

If you work at the bench, accurate pipetting is crucial. Without accurate pipetting, your experiments would not be reproducible, your stock solutions would be inaccurate, and your assays would have such large errors that comparing them would be meaningless. But luckily, there’s no need to worry—your trusty, precision micropipettes take care of that for you.

Or do they?

Precision instruments they may be, but the accuracy of your micropipettes depends on you. You need to maintain your pipettes, practice good technique, and have an understanding of how they work. Only once you have mastered those aspects can you claim to have anything like precision instruments at your disposal. Here’s how:

Know How Your Pipette Works

1. You Use an Air Displacement Pipette, Most Likely

An air displacement pipette works a bit like a syringe, except that there is an air-filled cushion between the piston and the sample. The air cushion prevents the piston from coming into contact with the solutions. Keeping the solution and the pipette barrel apart is good, but it also puts some limitations on the pipette.

Temperature and pressure affects the volume of the air cushion, which affects pipetting accuracy. Also, volatile solvents can evaporate into the air cushion, which leads to an inaccurate and lower dispensed volume than what is displayed on the pipette. The barrel of air displacement pipettes is also vulnerable to contamination by the pipetted solution. If you are working with corrosives or bio-hazardous material, this can be a problem.

2. Don’t Use an Air Displacement Pipette (depending on your application!)

Most of the information in this article relates to air displacement pipettes, but in certain situations a positive displacement pipette may be a better option. Positive displacement pipettes also work like a syringe, but they don’t have an air cushion—unlike air displacement pipettes. This makes them more accurate for pipetting volatile solvents because there is no place (air cushion) for the solvent to evaporate. The lack of air cushion also decreases the chance of contamination when pipetting corrosives and bio-hazardous material, which makes positive displacement pipettes more suitable to working with those reagents. These pipettes are expensive because the barrel and the tip are a unit and both are replaced when pipetting. A cheaper alternative is to use an air displacement pipette with barrier tips, but these only address some of the problems.

This Gilson information guide provides further on how air displacement and positive displacement pipettes work.

Take Care of Your Pipette

3. Have Your Pipette Serviced Every 6-12 Months…

However, you made need to have it serviced more often depending on your requirements for accuracy. The service should include re-calibration, greasing of the moving parts, and replacement of any worn out seals or other parts. It’s best to have this done by an experienced pipette doctor. If you work in university or large company, there may be pipette clinics you can use. In other cases, you can send your pipettes off to companies who will do it for you.

4. Check Your Pipettes Daily for Damage

Examine the nose of the barrel (where the tip is fitted) for any obvious damage. If there is a problem, have it serviced—because it is unlikely to be fit for the job.

5. Clean Your Pipettes Each Day Before Use

Wiping your pipette with some 70% ethanol should do it.

6. Store Your Pipette Vertically, Using a Pipette Holder

Storing your pipette this way prevents any liquids that are in the pipette barrel from getting any further inside and causing corrosion.

7. Never Put Your Pipette on Its Side With Liquid in the Tip

There’s nothing preventing the liquid from rolling down and into the pipette barrel (remember only air separates the liquid from the pipette barrel). Any liquid inside the pipette barrel results in contamination at the least and can cause some serious corrosion damage.

8. Use Well-Fitting Tips

Poorly fitting tips allow air to escape when drawing up and dispensing liquid, leading to inaccurate results.

This technical note from Biohit goes into more depth on how poorly selected tips can cause major errors in accuracy.

Practice Good Pipetting Technique

9. Make Sure You Know How to Pipette Properly

For the basics, take a look at Gilson’s pipetting technique manual. The most important rules to follow are:

  • Pipette with a slow, smooth action
  • Hold the pipette vertically when drawing liquid in
  • Only immerse the tip slightly when drawing liquid in—otherwise you will coat the outside of the tip with liquid, which will be transferred along with the volume inside the pipette
  • When dispensing the liquid, hold the pipette vertically but keep the sidewall of the receiving vessel at 45 degrees. Pipette against the sidewall or into the liquid that’s already there.

10. Test Your Accuracy

Check the accuracy of your pipetting technique by dispensing 100 µL onto a fine balance. The mass of the droplet you make should be around 0.1 g. Now do the same thing 10 times and record the masses you obtain. If the variation is more than +/- 0.5%, then you either need to re-evaluate your pipetting technique or practice more!

Know the Tricks That Increase Your Accuracy

11. Pre-Wetting the Pipette Tip

When you dispense liquid from your pipette, a coating of the liquid is left on the tip and makes the dispensed volume slightly less than what is displayed on the pipette. Pre-wetting the tip before you pipette will help increase your accuracy. To pre-wet your tip, draw up the liquid into your pipette and then dispense back into the original vessel. The coating is on the tip. Now, when you draw up the liquid again and dispense it into the receiving vessel, none of it will be left on the tip and lost to wetting. This is only recommended for volumes greater than 10 µL.

12. Reverse Pipetting

Use this technique when pipetting viscous liquids or volatile solvents. Reverse pipetting also helps when pipetting ultra-micro samples of 0.5 µL or less. Push the piston down to the purge position (the second stop), then draw the liquid up. There is too much liquid in the tip at this point. However, when the liquid is dispensed by pushing the piston to the aspirate position (the first stop), the extra liquid is left inside the tip. Using this method, the tip is “automatically” pre-wetted. The extra liquid also helps when pipetting volatile solvents, because some of the solvent will tend to evaporate into the air cushion.

13. Take the Ambient Temperature Into Account

The person or company that calibrated your pipette likely did so at room temperature. If you are working at a different temperature (e.g., in a cold room), then your pipette will not dispense the displayed volumes.

14. Take the Sample Temperature Into Account

In a recent Nature Methods publication, Millet and Barthlen observed a strange phenomenon. When repeatedly pipetting cold samples, the first dispensed volume is always larger than expected, but subsequent pipetting with the same tip gave the correct volume. The same was true for hot samples, except that the first dispensed volume was smaller than expected. Their solution was simple—dispense the first volume back into the original vessel, then start pipetting.

15. Use a Sensible Pipette for the Volume You Want to Dispense

The accuracy of your pipette decreases as the dispensed volume approaches the minimum the pipette can handle. For example, if you are dispensing 15 µL, then a 1 mL pipette would be terrible, a 200 µL pipette not so good, and 20 µL pipette ideal.

16. Use the Largest Volume Possible

Large volumes are easier to pipette accurately than small. Say you are performing an assay where you have to accurately pipette 5 µL. Pipetting that small amount accurately is not easy and will likely contribute greatly to the statistical error in your results. On the other hand, you could dilute the stock solution 10 times and pipette 50 µL of the solution. You could easily and accurately pipette this amount, which would yield much tighter error bars.

17. And Finally, Here’s How to Get Maximum Accuracy

An analytical balance is more accurate than any pipette. For maximum accuracy, use your pipette to dispense the volume you need but do it into a tared container on a balance. Then, calculate the actual pipetted volume from the mass (using the formula volume=mass/density). Of course, this only works for solutions of known density—but for aqueous solutions that have a density of 1, this is not a problem.

Happy pipetting!

Written by Nick Oswald with Biotix. Originally published in 2008, republished in 2016.

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20 Comments

  1. Cristy on December 17, 2016 at 9:06 pm

    What about rinsing the tip after dispensing into diluent? Should you pre-wet and also post rinse?

    • Dr Amanda Welch on December 19, 2016 at 3:18 pm

      If you’re going to do just one, my suggestion is to pre-wet. Pre-wetting is for making sure that you’ll dispense the correct amount of diluent/reagent.

  2. mo on October 27, 2016 at 4:04 pm

    When pipetting a solution, would using one’s mouth to draw the sample up into the pipet be considered standard acceptable practice?

    • Dr Amanda Welch on October 28, 2016 at 2:46 pm

      No. Not in the slightest. This was common a few decades ago, but the risks to your health are not worth it.

  3. VAL on June 22, 2016 at 5:04 pm

    I’m having a problem with test readings. My readings always come out lower than my co-workers who is using the same controls and have also ran the same exact samples results are 2s’s higher than mine. We have went over the procedures and observed each other techniques and yet we come up with no solution to the dilemma. What are some suggestions to this issue?

    • Kiran on June 28, 2016 at 10:33 am

      Have you tried a different set of pipettes? Also, if you are working with very small volumes, accuracy is important. A small change can make a big difference!

    • Benjamin on December 16, 2016 at 2:37 pm

      Hi Val, myself and a co-worker had the same issue. turns out when going to the first pressure point on the displacement pipette my naturally firmer grip was picking up very slightly more liquid than her (the amount was consistent each time). The solution was simply to practice doing it so gently that eventually we were getting the same. Try practicing on scales until they are the same?

  4. HariMari on June 18, 2016 at 6:25 pm

    Why we put maximum volume after using pipette why???

  5. Shamitha on April 16, 2016 at 9:00 am

    Could you tell me how to prevent bubbles from occurring on pipetting detergent solutions/ protein containing media? Usually when I press the second stop the solution in the well is filled with bubbles because of the air being pumped into it!

    -Shamitha

  6. icrgeek on June 23, 2011 at 6:19 pm

    I love all of the tips but I would like to add a pet peeve and expand upon on topic.

    Keep your tips covered. Tip boxes come with covers to keep dust, skin, etc out. It is simply not good enough to open the box at 9:00 am and close it a 5:00 pm. Learn to leave at most a single row exposed at a time when doing extended experiments. In the end, your dust filled tips may ruin my experiment even if they won’t ruin yours.

    Expansion of rule number 7. Never put you pippette on its side EVER. If there a new tip on it the tip will end up contaminated. If there is no tip on it the barrel of the pippette will be contaminated. And the final blow – hanging the pippette tip over the edge of the counter will end up with a contaminated tip / barrel after the pipette slams to the ground from the height of the countertop. (In Need of recalibration as least.)

  7. Prem on February 2, 2010 at 6:35 pm

    Very helpful article. I was not sure which technique to use for pipetting organic solvent. I used the reverse pipetting technique and found that that is the most accurate technique for this.

  8. Kazi on September 7, 2008 at 12:49 pm

    Hey, I have a question for my lab experiment and have no clue on what to write. The statement is to Devise three additional ways to test out your pipetting skills. What are some suggestions. Please help by today.

  9. Tobi on February 5, 2008 at 9:36 am

    When adjusting the volume, make it a habit to always arrive at the desired endpoint from the same direction because of the mechanical imprecision. That is, when you arrive at 157 microliter coming from 200 you will get a somewhat larger volume than coming from 100 microliters. This should especially be considered during calibration.

  10. WTJ on February 1, 2008 at 2:16 pm

    this is a good post! I learned a lot from here.

  11. Nick on February 1, 2008 at 12:25 pm

    Hi Ian,

    I have tested in often the past and pipetting 50ul is definitely more accurate. But you are right about the fact that the error in the dilution may be large.

    I should have said that when I do this I normally do the dilution very accurately by weighing the liquid out where possible (as in point 17) and diluting in a volumetric flask, rather than relying on a pipette.

  12. Ian on February 1, 2008 at 12:15 pm

    Pipetting 5 microlitres accurately is not easy and will likely contribute greatly to the statistical error in your results. On the other hand, if you diluted the stock solution 10 times and pipetted 50 microlitres, this would be much more accurate, giving you much tighter error bars.

    I don’t think this is necessarily correct (have you tested it?) because there is error at each step of pipetting. By doing this you may have more accurately pipetted the 50 ul volume, but you then have error in your pipetting as you dilute it down, which multiplies your original error. It may or may not be more accurate than just taking 5 ul in the first place.

    • rich sportsman on February 24, 2016 at 10:17 pm

      Yes, Nick should have said “…this would be much more precise…”; rather than “…this would be much more accurate…” .
      The increase in accuracy is less assured than the increase in precision in this case. In many procedures, particularly in assays with multiple samples and standards, the accuracy of adding a reagent [i.e. something that is introduced into every analysis tube] is less important than the precision.

      • Amer El-Hage on October 12, 2016 at 11:06 pm

        Agree and good to see your post Rich.
        I think Nick like many blends the definition of precision and accuracy. If you look at point 10. He introduced the word variability when he was talking about accuracy. Instead, he should have said something like “if the error or delta of the mean weight is more than +/- 0.5 mg (from 0.1 gm) or 0.5% ” then it would have been more in line with accuracy definition. Even though as you said it is not usually the critical element as precision in use of pipettes.

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