Practical pipetting: a guide

The forward method of operating a mechanical pipette.

Published 12th February 2017 by Andy Connelly. Last updated 8th May 2017 by Andy Connelly.

Introduction

The overlap between cooking and science is huge. So, when I heard the key ability of a chef being described as: “Perfect and fast. Fast and perfect” it rang some bells; particularly for the time consuming laboratory process of transferring liquids. We may use different words such as “accurate and precise” but the principle is the same. Get it wrong and your experiment may be ruined, take too long and you’ll be there forever.

DISCLAIMER: I am not an expert on pipetting. The content of this blog is what I have discovered through my efforts to understand the subject. I have done my best to make the information here in as accurate as possible. If you spot any errors or admissions, or have any comments, please let me know.

Science and cooking.
Figure 1: Science and cooking.

Transferring small volumes of liquid accurately, and without contamination, is such an important process that a huge variety of devices have been developed to help the process (see Table 1). This list is just a few of the most common devices:

  1. Graduated pipettes: Usually made of glass, with graduations to allow variable volume pipetting. They are the pipette equivalent of a measuring cylinder.
  2. Volumetric pipettes: Usually made of glass with a single graduation allow repeated pipetting of the same volume. They are the pipette equivalent of a volumetric flask.
  3. Positive displacement pipette: a calibrated pipette with disposable plastic syringe like tips which allow variable volume pipetting. Allows pipetting with minimum possibility of contamination.
  4. Air displacement (micro)pipettes: a calibrated pipette with plastic disposable tips which allow variable volume pipetting.
  5. Pasteur pipette: A small glass tube which has been drawn to a point. Usually treated as disposable but with a rubber bulb which is reused as it does not come into contact with the liquid.
  6. Transfer pipettes: Like a Pasteur pipettes but are made from a single piece of plastic and have guideline graduations (not calibrated). The bulb can serve as the liquid-holding chamber.

Of the above options the air displacement pipette has become the go-to technology for speed, accuracy, and precision. Air displacement pipettes were invented in the 1950s but were not available for sale until the 1960s when Eppendorf introduced the microlter system [6]. Gilson, Finnpipette, and other companies followed suit in the 1970s and now there are pipettes available from many manufacturers which can pipette volumes from 10ml down to a few microlitres.

Types of pipette
Table 1: Types of pipette and details about their use.

How the air displacement pipette works

The basic principle behind the air displacement pipette is simple (see Figure 2). On depression of the plunger button the piston is depressed and air is displaced from the inside of the pipette. On release of the button the spring pushes the plunger back up and so a pressure drop is produced inside the body and tip of the pipette. The allows liquid to be pushed into the pipette by atmospheric pressure. This liquid can then be dispensed out into a different vessel by depressing the plunger button.

Labelled diagram of an air displacement pipette.
Figure 2: Labelled diagram of an air displacement pipette.

The pipette is calibrated such that the volume of liquid that enters the pipette can be specified depending on the volume of air displaced (how far the piston moves). However, this simplicity means that small changes to the environment can have a large effect on the volume pipetted.

  • Pressure: changes in atmospheric pressure due to altitude can affect the accuracy of the delivery (around 0.2% per kilometre) and so recalibration may be required [1]
  • Temperature: because the volume of air is highly dependent on temperature ideally all liquids and equipment should be allowed to equilibrate to lab temperature. You should also avoid excessive handling of the pipette barrel and tip as this may warm the air inside. If you are pipetting cold liquids it is recommended to avoid pre-wetting and use each tip only once to minimise contact with the cold liquid [1].
  • Depth: hydrostatic pressure is created by the height of water/solution above the tip. If the tip is placed too deep into the liquid this will change the hydrostatic pressure and so will alter the amount of liquid in the tip. You should use the same depth that was used to calibrate the pipette (see Table 2). A sensible depth also avoids droplets forming on the outside of the tip (too deep) or sucking up air with your sample (too shallow) (see [1 & 7] for more details).
  • Tilt: It is not recommended to tilt the pipettor during pipetting. It can produce significant inaccuracies. It is also difficult to reproduce the same angle each time so precision can be affected. For example, a pipettor calibrated to 1000 ml at vertical position may provide 1003 ml at an angle of 45° [1].
immersion_depth
Table 2: Immersion depth for pipetting. Depth may depend on manufacturer [7].

Water – a useful liquid

Water’s unusual set of physical properties allows life in the oceans to exist; it allows moderation of the Earth’s climate; and it allows us to easily transfer aqueous solutions in the laboratory. The surface tension of water is sufficient to allow us to hold it in the tip of a pipette and the viscosity such that it can be ejected effectively. However, we still need to give nature a helping hand.

The plastic pipette tips are hydrophobic on manufacture. As such, to properly take up the correct (accurate) amount of liquid they must be pre-wetted. This is done by taking in (aspirating) and ejecting (expelling) an amount of the sample liquid 3 times before aspirating a sample for delivery.

Other liquids we might want to pipette are not so forgiving as water. Table 1 suggests various options for pipettes depending on the liquid you wish to pipette. If you do wish to pipette very viscous liquids, organic solvents, or heterogeneous liquids (e.g. blood) using an air displacement pipette then you may need to adjust your pipetting method (see below) and the type of tip you use.

Types of tip

Most manufacturers will produce a standard pipette tip which will be ideal for pipetting aqueous solutions using their pipettes or be a “universal” tip suitable for many different pipettes. There are various other options including [5]:

  • Wide bore tips which enables dispensing viscous liquids more accurately.
  • Filter tip: these have a filter within the tip which helps reduce cross contamination; useful for for DNA, RNA, and protein samples. The filter prevents liquid from splashing accidentally inside the pipette and aerosols from penetrating into the pipette tip cone during pipetting.
  • Sterile tips are free of human DNA, DNase,RNase and endotoxins. These are usually sterilised using gamma radiation.

How to pipette

There are 4 main pipetting methods [5]:

  • Forward: Most commonly used method. Ideal for aqueous solutions such as dilute reagents, buffers, diluted acids or alkalis. However, not good for bubble forming liquids or viscous liquids.
  • Reverse: This method avoids bubble formation and is ideal for viscous solutions and protein rich liquids [4].
  • Repetitive: A variation on the reverse technique this method is used for repeated pipetting of the same volume.
  • Heterogeneous: When pre-rinsing the tip is not possible. This technique is used for pipetting heterogeneous samples, such as blood or serum.

Here I will only present the first two of these methods (see [5] for description of other methods). Whichever method you use there are certain processes to go through before you start:

Prior to use

  • Check the pipettor for dirt and damage:
    • If dirty on the outside use ethanol to clean,
    • If the pipette has a nozzle protection filter this should be check for cleanliness and replaced, or cleaned, if necessary
    • If the pipette is damaged it should not be used and may require servicing
  • Make sure the pipette tips are appropriate for the pipette you are using. They need to match the volume of the pipette AND the brand of pipette.
  • Allow liquids and equipment to equilibrate to ambient temperature before pipetting.
  • Do not lay the pipette down, or invert the pipette, when a filled pipette tip is attached as it can contaminate the inside of the pipette.
  • Pre-wet the pipette tip by aspirating and ejecting an amount of the sample liquid 3 times before aspirating a sample for delivery. Unless manual for pipette instructs otherwise.
  • Ensure you have a comfortable pipetting position – repeated pipetting can cause physical stress [2 & 3].
  • Check your risk and COSHH assessment for any hazards in your experiment and take appropriate precautions.

Method 1: Forward (‘normal’) mode pipetting

This is the standard method used for the majority of samples (see Figure 3).

  1. Hold the pipette vertically; depress the plunger button to the first stop (A).
  2. Place the tip just under the surface of the liquid (see Table 2)
  3. Smoothly release the plunger button (B) keeping the tip at a constant depth.
  4. Carefully withdraw the tip from the liquid, touching against the edge of the container to remove excess.
  5. To dispense the liquid, hold the tip at an angle of around 30-45º against the wall of the receiving container. Depress the plunger button to the first stop (C) and hold for one second.
  6. Push the pipette to the second stop (D) while sliding the pipette tip against the walls of the container.

Release the plunger button at this point to return it to the uppermost position (E).

The forward method of operating a mechanical pipette.
Figure 3: The forward method of operating a mechanical pipette.

Method 2: Reverse mode pipetting

The reverse mode is used when working with viscous and volatile liquids (see Figure 4).

  1. Hold the pipette vertically; depress the plunger button to the first stop (A) and then second stop (B).
  2. Place the tip just under the surface of the liquid (see Table 2)
  3. Release the plunger button smoothly to the upper stop (D). This may take a little time when using viscous liquids. If your procedure allows, wipe excess from the outer surface of the tip or touch against the edge of the container to remove excess.
  4. To dispense the liquid, depress the plunger button to the first stop only (E).
  5. Hold the plunger button at the first stop.
  6. The liquid that remains in the tip should not be included in the delivery.
  7. To ensure that the correct volume is delivered the liquid remaining in the tip should be discarded with the tip. Releasing the plunger button at this point returns it to its uppermost position (F).
The reverse method of operating a mechanical pipette.
Figure 4: The reverse method of operating a mechanical pipette.

Summary

Pipetting is a vital part of laboratory work which can make or break your experiments. Good and appropriate pipetting technique is often neglected but is vitally important if you want to be “Perfect and fast, fast and perfect”.

This is only a brief summary of the facets of pipetting – are there any habits that people in your laboratory have picked up that drive you nuts? Any key aspects you think I’ve missed out? Please, let me know.

 Glossary

  • Accuracy: How close to the “true” value a pipette dispenses. The “true” value is usually taken as the volume set on the pipette (e.g. 900µl).
  • Aspirate: drawing liquid up into a pipette tip
  • Blow-out: discharging the residual liquid from the tip
  • Calibration check: to check the the accuracy and precision of the dispensed volume.
  • Dispense: discharging liquid from the tip in a controlled manner
  • Precision: the amount of variation between repeated dispensing of the same volume of water.

References

  1. BIOHIT, Liquid Handling Application Notes, 5. Major sources of error of air displacement pipettes
  2. BIOHIT, Liquid Handling Application Notes, 9. The design of pipettors can prevent Pipetting Related Upper Limb Disor)ders (PRULD)
  3. BIOHIT, Liquid Handling Application Notes, 8. Safety in pipetting.
  4. Dispense Liquids Containing Proteins More Reliably with Reverse Pipe, Application
    Note: AN-HP-REVPIPET-0909
  5. Thermo Scientific, Good Laboratory Pipetting guide, BRHPGLPGuide0058 1209, (2010)
  6. http://corporate.eppendorf.com/en/company/history/
  7. Learning the basics – how to work with volumetric instruments, Volumetric Measurement in the Laboratory, BRAND.

Appendix A – Testing your pipetting ability

The accuracy and precision of pipettes may drift over time. For most laboratories an annual service to clean and calibrate the pipette is acceptable. However, certain labs, such as accredited laboratories, may require more regular maintenance. This can be done ‘in-house’ but is not simple especially without an appropriately calibrated balance.

If you are concerned about the accuracy and precision of your pipette you can, very easily, check. This is also a great way to test your skills! You will need a balance of appropriate precision (4 or 5 place) which should also have been calibrated!

  1. Pipette a volume of liquid on to a weight boat and weigh (e.g. 900µl deionised water)
  2. Record mass and repeat 5 times
  3. Use the equation below to calculate the percentage accuracy (i.e. how close to the desired value it is)
  4. Using the equation below calculate the percentage precision (i.e. how “repeatable” the pipetting is).
  5. Compare these values to the those in Table 3.

See below for example. As you can see from Table 3 one should always try to use pipettes towards the top of their range. In principle this will minimise the percentage error. However, a well calibrated pipette will reach much better percentage precision and accuracy than the values below.

Results

For results of 1000µl pipette set to 900µl I got:

  • 910µl, 887µl, , 882µl, 902µl, and 921µl.
  • Mean=900.4 µl
  • Sample standard deviation = 16

Accuracy:

Calculating the accuracy of a pipette.
Equation 1: Calculating the accuracy of a pipette [5].
Comparing this to Table 3 this is well within calibration limits.

Precision:

Calculating the precision of a pipette.
Equation 2: Calculating the precision of a pipette [5].
This is above the calibration limits shown in Table 3 so this pipette should not be used as the results are very variable between pipettings. However, it could also be poor pipetting technique! So, it may be worth checking with someone else.

Calibration guidelines for the listed pipettes from EN ISO 8655
Table 3: These values are the calibration guidelines for the listed pipettes from EN ISO 8655. A well calibrated pipette will give better accuracy and precision than these limits but these values represent a ‘worst case scenario’ for a calibrated pipette. (See EN ISO 8655 also [5])
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3 Comments

  1. Hi Andy, your figures for forward and reverse pipetting are great. Do you give permission for use of your figures and/or text? If so, what are your conditions?

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