Frequently Asked Questions

Frequently Asked Questions

36 Articles
1 2

The pipet tip tends to drip when liquids like ethanol, acetone or chloroform are used. The cause of the dripping is the high vapor pressure of the liquid that leads to an expansion of the air cushion inside manual pipettes. One way to avoid dripping is pre-wetting the pipet tip. You have to aspirate and dispense the liquid 2-3 times prior to transferring the liquid into your destination vessel. Another option is to use a different pipetting system, the so called positive displacement system. It is without an air cushion and therefore no dripping occurs.

Pipetting viscous liquids like glycerol, oil or blood with an air cushion pipette is difficult. You have to use a very slow aspiration speed, wait for several seconds until the liquid retightened and then use a very slow dispensing speed. But still the delivered volume will not be accurate. One better way to pipette viscous liquids is reverse pipetting. Ideally you choose a positive displacement system for this task, since no air is present inside the tip and aspirating as well as dispensing of viscous liquids is easy.

No, cutting the pipet tip is the worst thing to do when transferring glycerol. The pipetting result will neither be precise, nor accurate. The pipet tip is deformed and the orifice will be frayed so that plastic residues might also fall into the sample. It is better to use a full pipet tip and reverse pipetting technique or a positive displacement system.

Some liquids contain a large amount of protein, like BSA-solution or cell culture medium. These tend to build up foam during pipetting, especially in the moment of blow-out when air is introduced into the sample. To avoid this phenomenon one should apply reverse pipetting technique with air cushion pipettes. Another option is the usage of positive displacement systems. These lack the air cushion and therefore no foam can build up during dispensing of such liquids.

Pipetting detergents such as Triton X-100 or Tween 20 lower the surface tension of aqueous solutions. Furthermore they tend to stick to the inside of a pipet tip after dispensing. That means that never all the liquid inside the pipet tip is dispensed, leading to sample loss, inaccurate volume delivery and in the long run higher reagent costs. Efficient pipetting of detergents is only possible with pipet tips specifically developed for these liquids or a positive displacement system.

Aerosols can be formed by almost every liquid. If the air humidity inside the pipette is below liquid humidity small microdrops evaporate upwards into the pipet tip and up into the pipette cone. If the liquids used are warm e.g., cell culture medium, the evaporation increases. The inside of the pipette cone can suffer from corrosion over time. But even worse is the risk of contamination brought into samples via aerosols. If an infectious liquid or bacterial culture evaporates into the pipette cone you will dispense the infected and contaminated aerosols with the blow-out. It is possible that you cross-contaminate multiple samples. Preventing aerosols is necessary and can be achieved by using high quality two-layered filter tips that block fine aerosols, bacteria and viruses. If you are using highly infectious liquids a positive displacement system might be more advantageous because the whole sample is enclosed in the tip without any contact to the liquid handling instrument.

When using a two-button pipette you have to aspirate and dispense the liquid with one button. Then you switch over to a second button for tip ejection. Liquid aerosols ascend up into the pipet tip and even higher into the pipette cone during aspiration. Then the liquid is dispensed and you aspirate air again when releasing the operation button. Thereby you may aspirate the aerosols again, prior to tip ejection via the second button. With a one-button operation pipette you aspirate and dispense the liquid and then eject the tip in the same downwards  movement. So you do not aspirate aerosols back into your pipette.

7 things are important for an accurate pipetting result:

1. Hold the pipette vertical during aspiration
2. Immerse the pipet tip only as much as needed into the liquid (~ 2-3 mm)
3. Release the operation button slow and calm
4. Rest the pipet tip ~ 2 sec in the liquid before pulling the pipette up
5. Dispense at a 20-45° angle with contact to the vessel wall
6. Dispense at a calm and moderate speed
7. Perform the blow-out

A pipette calibration is necessary to reassure that it is working correctly in terms of accuracy and precision. The standard for pipette calibration is called ISO 8655 and includes all permitted error values of different pipette types. Air-cushion and positive displacement pipettes, as well as dispensers and bottle top dispensers are respected. The ISO 8655 states a calibration once a year. If you are working under regulated conditions, such as pharmaceutics, it might be needed to calibrate your pipettes every 3 months.

Liquids with a high density such as sulfuric acid or phosphoric acid have a tendency to pull down the air-cushion inside the pipette. Thereby the air-cushion is elongated which leads to lower volume aspiration than set on the pipette. The pipetting result is always inaccurate without having the pipette adjusted to the higher density. After adjustment the pipette aspirates a little bit more liquid due to a smaller air-cushion. The pipetting result is correct then.

An air-cushion pipette is the most known pipette type. It is used together with pipet tips. Inside the pipette and the pipet tip is air of the surrounding area, the so called air-cushion. This air is moved downwards when pressing the operation button and the sample liquid is aspirated due to the partial vaccuum inside the pipette. The sample liquid is then dispensed by the air pressure when the operation button is pressed down again. The blow-out at the end of a pipetting event is a little amount of extra air that is blown out to deliver all liquid out of the pipet tip.

A positive displacement system is mainly used for dispensers, but also some pipettes exist that work according to this principle. Since the instruments themselves do not have a piston, a special tip with an integrated piston is needed to complete the system. When the tip is inserted into the instrument the piston is coupled and gets pulled upwards during usage. The sample liquid is aspirated directly without any air between the tip's piston and the liquid. With this system some difficult liquids like glycerol, ethanol, blood or cell culture medium can be easily aspirated and dispensed.

Reverse pipetting is a technique used especially to transfer viscous liquids (e.g., glycerol) or to load samples on agarose gels. Thereby the operation button is pressed down completely to the last stop as if a blow-out was performed. Then the liquid is aspirated and during dispensing the operation buton is only pressed down to the first stop. Like this always a little residue rests in the tip. Glycerol can be pipetted more acurately using this technique. In the case of loading samples on agarose gels one can prepare small drops (~1 µL) of loading dye on a flat surface. Then a pipette is set to 5 µL, and DNA is aspirated and dispensed on the drop of loading dye. After mixing a couple of times reverse pipetting is performed and the operation button is pressed down completely. Now the sample plus the loading dye can be aspirated at once without setting the pipette to 6 µL. The whole sample is then loaded on a gel and the operation button is pressed down to second stop again to secure that all sample is dispensed into the gel pocket.

A positive displacement system can be used for any type of sample. But it is recommended especially for viscous, volatile, dense, detergent- and protein-containing liquids. Since it lacks the air-cushion, more precise results can be achieved than with classic pipettes. Often positive displacement systems are so called dispensers. So when working with plates it is advantageous to use a dispenser. With this instrument the whole plate can be filled with only one aspiration, but multiple dispensing steps.

Reverse pipetting means uptake of liquid including the blow-out volume and dispensing of liquid without blow-out volume. Reverse pipetting is therefore recommended to reduce the error, resulting in more volume being aspirated than necessary. The excess aspirated solution remains in the tip and is discarded. 

Regardless of the volume, the air-cushion should be wetted by multiple filling and emptying of the tip before dispensing. This saturates the air cushion and leads to more accurate and reproducible results.

For viscous liquids we recommend positive-displacement instruments like Biomaster, Multipette and Varipette or an air-cushion pipette (Research plus, Reference 2) in combination with reverse pipetting technique (see userguide 20 and 21 for details).

Polyethylene glycol is very viscous. Therefore, instruments with positive displacement principle can be used. These systems have an integrated piston in the tip and do not have an air-cushion. Thereby, they are capable of dealing with problematic liquids.

For chloroform we recommend positive-displacement instruments or an air-cushion pipette in combination with pre-wetting and reverse pipetting technique (see userguide 20 and 21 for details or download the poster "Pipetting techniques").

Diethyl ether has such a high vapor pressure that it would leak out of the tip immediately using an  air-cushion pipette. For this reason, it can only be pipetted using a positive-displacement pipette.

36 Articles
1 2

Become a pipetting expert and explore more topics here: