Correct pipetting – how does it actually work? Depending on the type of pipette, different procedures are possible. The good thing is that the differences are quite small and they are easy to do with good preparation. We have briefly summarized in this blog post which different pipettes are available and what you should pay attention to when pipetting correctly in the laboratory.
Not all pipettes are the same – depending on the quantity and type of medium to be worked with, different types of pipettes may be used. A first distinction can be made with regard to the principle of operation. For example, you can pipette correctly with the function of the bulb pipette, but also with the function of the air cushion pipette.
- Solid pipette: In so-called solid pipettes or direct displacement pipettes, the liquid to be aspirated is drawn in directly through the piston stroke. As a result, it comes into direct contact with the piston and cylinder. Pipettes of the direct displacement principle are usually used for non-aqueous and viscous liquids (glycerol or acetone come to mind). They have a fixed volume marked by a line.
- Air cushion pipette: In contrast, air cushion pipettes are used more for correct pipetting in the milliliter and microliter range. With this functional principle, the sample is not aspirated directly into the pipette, but into a plastic pipette tip to be attached.
|Do you still have questions? Our experts will also be happy to explain to you in detail how to use the volumetric pipette correctly.|
In the following, some types of pipettes are presented that function either according to the solid pipette principle or the air cushion principle:
Dropping pipettes, which can also be found under the name “Pasteur pipettes”, belong to the solid pipettes. They are mainly used when small quantities of liquid are to be transferred from one container to another without direct volume specification. They consist of a glass tube with a diameter of about 5 mm and a rubber suction cup at the upper end. To use such a volumetric pipette correctly, the rubber suction cup must be pressed.
Microliter and milliliter pipettes
For particularly small volumes (from 5000 µL to 0.1 µL), microliter or milliliter pipettes are used, which work more accurately than glass pipettes. They are similar in design to piston-stroke pipettes and were formerly also known as “Marburg pipettes”. It is especially important to use this pipette correctly, as the slightest deviations can easily happen.
If there is a volume scale on the pipette, it can be referred to as a graduated pipette – this differs from the volumetric pipette. Therefore, these models can be used for variable fluid volumes, providing more flexibility. However, the danger of working somewhat imprecisely lurks. To make it much easier to pipette correctly, the volumetric pipette is suitable: this makes it much easier to produce a repeatable result.
Multipettes or hand dispensers also have an air cushion, but can absorb liquids through a direct-displacement reservoir. Thus, larger volumes can be pipetted simultaneously, which are not dispensed “in one”, but in several small steps of the same volume. It can also be used to fill microplates more quickly.
Open pipettes are now obsolete and are hardly ever used. This is also because it is more difficult to use this pipette correctly: First, the liquid is aspirated with the mouth through the open pipette and released by letting go at the destination. However, with this direct displacement principle, contamination or even health concerns from vapors or contact quickly occur.
This type of pipette belongs to the air cushion pipettes and has a movable piston that moves an underlying column of air. In the upward movement, a vacuum is created under the air, drawing liquid into the attached pipette tip. Since only this part comes into contact with the sample, it is made of plastic and can be discarded afterwards. These pipettes occur both with a fixed volume and with a variable volume.
Incidentally, direct-displacement piston-stroke pipettes represent a special form in which the air cushion is eliminated by a built-in piston with direct contact. This allows highly viscous solutions to be pipetted correctly without coming into contact with aerosols from the air cushion.
Multichannel pipettes also belong to the piston-stroke pipettes and can be considered a special type. They have eight to 12 channels that can be operated simultaneously. These are arranged in a grid so that several plates can be processed simultaneously – saving time in the lab.
Structure of an Eppendorf pipette
Probably one of the best-known dispensing systems is the Eppendorf pipette, which is named after the manufacturer and is nowadays a must in every laboratory. It belongs to the air cushion pipettes. To pipette correctly with an Eppendorf, the setup should be known. This is divided into the following components:
- Control button: This is located at the very top of the pipette and must be pressed down to aspirate liquid. This causes the piston to move downward and displace the air volume.
- Totalizer: The totalizer serves as a volume display, with the set volume being read from top to bottom. It can be adjusted for variable pipettes via an adjustment ring under the dispensing knob.
- Piston: The piston is usually made of ceramic and is located inside the Eppendorf pipette. It is surrounded by a seal and sits on the air cushion.
- Tip ejector: The tip ejector is the elongated part in the lower section of the pipette. It is located under the piston.
- Pipette cone: Finally, there is the tip cone onto which the plastic pipette tip is placed. It optimizes the force when picking up and delivering the tip.
Since the design of these pipettes is basically no different from other air cushion pipettes, it is quite easy to pipette correctly with an Eppendorf as well.
8 tips for correct pipetting
Fortunately, proper pipetting is one of the easier tasks in the laboratory, as long as proper procedures are followed. This includes, for example, avoiding some common beginner mistakes. Checking the pipette beforehand should also be part of the fixed work steps. The following tips can help simplify the process even more.
To avoid incorrect volumes or even substance reactions, attention should be paid to the temperatures. This includes the pipette and tips as well as the liquids being worked with. Ideally, these should be adjusted to theroom temperature, if this is possible.
2. Immersion Angle
Another important factor is the immersion angle. This should be approximately the same for each sampling and should not exceed 20°. This ensures that only the liquid is actually absorbed – and no unwanted air pockets pass through.
3. Immersion Depth
Not only the angle, but also the immersion depth plays an elementary role in correct pipetting. In particular, pipettes with plastic tips should not be able to enter the liquid so deeply that areas outside the tip come into direct contact. Usually, an immersion depth of two to three millimeters is quite sufficient for aspiration. In addition, any droplet formation at the pipette tip is prevented.
4. Strip Properly
After dispensing, the pipette tip should also be wiped off on the vessel to prevent carryover of drops. This keeps the work surface around the vessel clean and dry – and also prevents unwanted amounts of liquid from being transported outside the pipette volume. Either the sidewall, the liquid surface or a dispersion in the liquid itselfcan be used for stripping.
5. Adjust Calibration
When working with material whosedensity is significantly higherthan that of water, most pipettes must be calibrated before proper pipetting can be performed. Otherwise, there is a risk that the volume display will not be accurate enough. In order to be able to use the pipette correctly later, the calibration should be performed according to the liquid density.
6. Prefill Tips
After a new plastic tip is attached, the liquid should bedispensed two to three times before the actual collection. For example, temperature differences are compensated and the air space inside the pipette tip is moistened. This helps to prevent evaporation during the first dispersant.
7. Observe Volume Range
Speaking of the first dispersant, both the first and last dispersant should be discarded for multiple dispersals. Statistically, these two pipetting procedures contain the largest volume deviations, which can have a negative effect on the overall result.
8. Do not use all dispensers
Each graduated pipette is equipped with a volume range which should definitely be taken into account when pipetting. Ideal results are achieved only when the amount of fluid absorbed is between 35% and 100% of the volume range. Then user-related pipetting errors are reduced to a minimum.
9. Pipette forward and reverse
In addition, two pipetting procedures can be differentiated. These are the following:
Forward pipetting: In this method, the operating button is pressed in to the first pressure point and then released to aspirate the liquid. To finally release it, the control knob is pressed in again – this time as far as it will go.
Backward pipetting: If, on the other hand, highly viscous liquids are to be pipetted, there is a risk with the forward method that the set volume will not be completely absorbed by the first pressure point. Therefore, when pipetting backwards, the liquid is immediately pushed in as far as it will go and then blown out again by letting go.
Learn to pipette correctly in just a few steps
If the previously mentioned tips are followed, the correct use of a pipette is quite simple. The individual steps of the process then look as follows:
1. Observe pipetting volumes
The first step is to set the pipetting volume. This is done quite simply on the counter. It is important to observe the maximum pipetting volume.
2. Put on pipette tip
The pipette tip is attached by pressing the pipette onto the tip with a little pressure. For this purpose, it should not be picked up by hand, but should remain in a vertical position on the table stand or shelf holder.
3. Volume recording/recording medium
The liquid is then aspirated using the forward or reverse method. To do this, the control knob must be pressed either to the first stop or completely. The pipette must be held vertically in the liquid for about two to three millimeters.
4. Volume delivery / discharge medium
The tip is then carefully wiped off and taken to the target medium. There, the control knob must finally be pressed in completely to the second pressure pointin order to empty the liquid.
5. Eject pipette tip
To eject the plastic tip after use, simply press the corresponding eject button. This is best done directly above the discard tray as the tips used will be discarded. Cleaning glass pipettes is also essential and should therefore not be neglected under any circumstances.
Correct pipetting has to be learned
Correct pipetting is one of the basic skills in every laboratory and is quite simple in principle, but of course it still needs to be learned correctly. If you follow the tips and best practices above, you’ll be on the safe side. The rule is: avoiding mistakes saves time and practice makes perfect.
A graduated pipette is a type of pipette with a volume scale. This means that different volumes of liquid can be aspirated – in contrast to proper pipetting with volumetric pipettes.
A distinction can be made between various types of pipettes that function according to the direct displacement principle or the air cushion principle. Common representatives are, for example, bulb pipettes, dropper pipettes, piston-stroke pipettes and mulit pipettes.
The Peleusball belongs to the pipetting aids and functions like a large rubber ball. First, the valve at the top is opened and the ball is compressed before the valve is closed. The vacuum allows liquids to be aspirated via the second valve. Finally, the third valve is used for draining.
While micropipettes are used for volumes between 0.1 µl and 5000 µl, bulb pipettes are mostly suitable for 5 to 10 ml. Pasteur pipettes are usually 2 ml. In principle, most graduated pipettes can be set to a certain volume range, which depends on the size of the pipette.
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