To contain and To deliver (TC & TD): Manufacturer stamp TC or TD near the top of the pipet.

TC pipet holds or contains

a particular volume but does not dispense that exact volume, whereas TD pipet will dispense that exact volume indicated.

(Source: Bishop's Clinical chemistry, 5th Ed)

Blowout and self-draining: A blowout pipet has continuous etched ring or two small, close continuous rings located near
the top of the pipet. This means that the last drop of liquid should be expelled into the receiving vessel. Without these
markings, a pipet is self-draining, and content of pipet drain by gravity.

Transfer pipettes: It transfers a known volume of liquid without further subdivisions. They include both volumetric and
Ostwald-Folin pipettes. They consist of a cylindrical bulb joined at both ends to narrower glass tubing. A calibration mark is
etched around the upper suction tube. The bore of delivery orifice should be sufficiently narrow to prevent rapid outflow of
liquid and incomplete drainage.

A volumetric transfer pipett is calibrated to deliver accurately a fixed volume of a dilute

aqueous solution. Ostwald-Folin pipettes have bulb closer to the delivery tip unlike volumetric pipett which has at center.
These (OF) are used for accurate measurement of viscous fluids, such as blood or serum. In contrast to volumetric pipett,
an Ostwald-Folin pipett has an etched ring near the mount piece, indicating it is a blow out pipett. With the use of pipetting
bulb, liquid is blown out of the pipett only after blood or serum has drained to the last drop in delivery tip. Volumetric pipet
are self draining and is used in diluting standards, calibrators, or quality-control materials.

Measuring pipettes: These are graduated or measuring pipette and can dispense different volumes as these have many
graduations. It has uniform graduations along its length. Two kinds are available; the Mohr pipette has graduations ends
above the tip and serological pipette has graduated marks down to the tip. Mohr pipet is self draining. The serological
pipette must be blown out to deliver the entire volume of the pipette and has an etched ring (or pair of rings) near the bulb
end of the pipette signifying that it is a blow-out pipette. Serological pipettes have a larger orifice than do the Mohr
pipettes and thus drain faster. Measuring pipettes are used for the transferring and measurement of reagents and making
dilutions and are not considered sufficiently accurate for measuring samples and calibrators. Micropippet is also a
measuring pippet which can measure less than 1mL.
Micropipettes:

These are pipettes to measure micro liter of volume. Inner wall of pipette can be coated with liquid so most micropipettes
are calibrated to contain (TC) that stated volume rather than to deliver it.

Semiautomated and automatic pipettes: These are programmable pipets that can automatically take up the specified
volume and can dispense in multiple wells at same time. There are 3 types’ air-displacement, positive-displacement and
dispenser pipets.
Pipette terms:
Adjustment - altering the pipette so that the dispensed volume is within the specifications.
Air Displacement Pipettes - are meant for general use with aqueous solutions. In air displacement pipettes, a certain
volume of air remains between the piston and the liquid.
Aspirate - to draw up the sample.
Blow-out - to empty the tip completely.
Calibration check - checking the difference between the dispensed volume and the selected volume.
Dispense - to deliver the sample.
Positive Displacement Pipettes - are used for high viscosity and volatile liquids. In positive displacement pipettes, the
piston is in direct contact with the liquid.

Air Displacement Pipettes

Air Displacement Pipette, used for standard pipetting applications, is highly accurate.
However, conditions such as temperature, atmospheric pressure as well as the specific gravity and viscosity of the
solution may have an effect on the performance of air displacement pipettes.

How does an Air Displacement Pipette work?

 The piston moves to the appropriate position when the volume is set.
 When the operating button is pressed to the first stop, the piston expels the same volume of air as indicated on
the volume setting.
 After immersing the tip into the liquid, the operating button is released. This creates a partial vacuum and the
specified volume of liquid is aspirated into the tip.
 When the operating button is pressed to the first stop again, the air dispenses the liquid. To empty the tip
completely the operating button is pressed to the second stop (blow out).
Factors affecting the accuracy of Air Displacement Pipettes:
Temperature
The most important factor in pipetting accuracy is the liquid temperature. The figure below shows the change in volume
when the liquid has a different temperature than the pipette and air. If the temperature of the liquid, pipette and air is the
same, the accuracy is not significantly affected.
Density
Density is the mass/volume ratio of the liquid. The density varies according to the temperature and air pressure. Typically,
the density of water is 0.996 kg/dm3, for ethanol 0.79 kg/dm3 and for sulfuric acid (H2SO4) 1.85 kg/dm3.
Altitude
The geographic altitude affects the accuracy through the air pressure. The air pressure decreases at higher altitudes and
the conversion factor Z decreases as well. The boiling point of some liquids can also change to quite close to room
temperature, increasing the evaporation loss dramatically.

Positive Displacement Pipette

Positive Displacement Pipette, is used for applications like PCR and other DNA amplification techniques. The micro-
syringe tips used in positive displacement pipettes are disposable. This helps to avoid sample-to-sample cross-
contamination (also known as sample carry-over), and contamination due to the aerosol effect.
How does a positive displacement pipette work?
 The piston moves to the appropriate position when the volume is set.
 When the operating button is pressed to the stop, the piston descends to the tip opening.
 After the tip is immersed into the liquid, the operating button is released. The plunger is then raised and a partial
vacuum is created. This causes the liquid to enter the tip.
 When the operating button is pressed again, the piston descends, expelling the liquid from the tip.
*Pre-rinsing (1-3 times) the tip with the liquid to be pipetted may improves accuracy, especially when using positive
displacement tips.
Avoid turning the pipette on its side when there is liquid in the tip. Liquid might go to the interior of the pipette and
contaminate the pipette.
Avoid contamination to or from fingers by using the tip ejector and gloves.
Store pipettes in an upright position when not in use. Pipette stands are ideal for this purpose.
Check calibration regularly, depending on the frequency of use and on the application, but at least once a year. If used
daily, a three-month interval is recommended. Follow the instructions for recalibration in the manufacturer�s instruction
manual.
Pipetting whole blood
Use forward technique steps 1 and 2 to fill the tip with blood (do not pre-rinse the tip). Wipe the tip carefully with a dry
clean cloth.
 Dip the tip into the blood and press the operating button to the first stop. Make sure the tip is sufficiently below the
surface.
 Release the operating button slowly to the ready position. This action will fill the tip with blood. Do not remove the
tip from the solution.
 Press the operating button to the first stop and release slowly. Repeat this process until the interior wall of the tip
is clear.
 Press the operating button to the second stop and completely empty the tip. Remove the tip by sliding it along the
wall of the vessel.
 Release the operating button to the ready position.

Recommendations for Pipetting different Solutions / Compounds

Solution/Compound Examples Pipette Tip Technique Comments
Aqueous solution Buffers, diluted salt Air Displacement Standard Forward
solution
Viscous solution Protein and nucleic Air Displacement Standard wide Reverse Pipette slowly to avoid
solutions, glycerol, Pos. orifice Pos. bubble formation.
Tween 20/40/60/80 Displacement Displacement
Volatile compounds Methanol, Hexane Air Displacement Filter Pos. Forward Pipette rapidly to avoid
Pos. Displacement evaporation. Carbon filter
Displacement tips prevent vapors from
going into the Pipette.
Nucleotide solutions Genomic DNA, PCR Air Displacement Filter or wideForward For genomic DNA wide
Products Pos. orifice orifice should be used to
Displacement Pos. Displacement avoid mechanical shearing.
Radioactive Carbonate, H- Air Displacement Filter Pos. Forward
compounds thymidine Pos. Displacement
Displacement
Acid / Alkalis H2SO4, HCI, NaOH Air Displacement Filter Forward
Toxic samples Pos. Pos. Displacement Forward
Displacement
Pipetting guidelines for selected compounds
Body Fluids
Whole Blood
 Pipette + tip combination: choose an air displacement pipette and a standard or wide orifice tip.
 Technique: use the whole blood pipetting technique. Reverse pipetting should be used if high accuracy is needed.
 Notice: some blood can remain in the tip and on the outer surface. Wipe the tip against the edge of the vessel to
remove excess liquid outside the tip before dispensing.
Serum
 Pipette + tip combination: choose an air displacement pipette and a standard or wide orifice tip.
 Technique: use the whole blood pipetting technique. Reverse pipetting should be used if high accuracy is needed.
 Notice: Residual serum can sometimes be found on the outer surface of the tip. Wipe the tip against the edge of
the vessel to remove excess liquid outside the tip before dispensing.
Oily Fluids
Glycerol
 Pipette + tip combination: choose an air displacement pipette and a standard or wide orifice tip.
 Technique: For high accuracy of performance, use the reverse pipetting technique.
 Notice: Oily fluids are difficult to pipette because of formation of air bubbles. Filling must be done very slowly to
prevent air bubbles. Wipe the tip against the edge of the vessel to remove excess liquid outside the tip before
dispensing. The use of a positive displacement pipette and tip is also useful for pipetting glycerol.
Preventing cross-contamination
Pipette-to-sample
A contaminated pipette or contaminated tips can cause contamination of samples.
Prevention:
 Use sterilized tips or sterilized filter tips and if possible autoclave the pipette.
 Change the tip after pipetting of each sample.
Sample-to-pipette
Samples or aerosols from samples can enter the cone of the pipette.
Prevention:
 Keep the pipette vertical when pipetting in order to prevent liquid from running into the pipette body.
 Release the push-button slowly.
 To avoid aerosol contamination, use filter tips or use a positive displacement pipette and tips.
 Store the pipette vertically.
Sample-to-sample (carry-over)
The remains of sample A can mix with next sample B inside the tip and may cause a false test result. Prevention:
 Change the tip after each sample.
 If you suspect that your pipette is contaminated, autoclave or clean your pipette.
Calibration of Pipettes, officially means determining the difference between the dispensed volume and the selected
volume. Adjustment means altering the pipette so that the dispensed volume is within certain specifications. During factory
calibration, performance is checked with different weighings at both the maximum volumes of the range and at the
minimum or 10% of the maximum volume, whichever is higher. When you purchase a Pipette, you should choose one that
is designed to permit recalibration and adjustment for different temperatures and various viscous liquids.
Calibration of Pipettes in a Quality System
The main objective of pipette calibration in a quality system is to ensure that measurements are made with the intended
accuracy. Very often error limits are taken from the manufacturer�s specifications, while far less accuracy is required to
perform the work. If these limits are not easily obtained, or vary, another option is to set the limits according to accepted
standards (DIN 12650 or ISO 8655). However, if the laboratory work requires the highest accuracy, the manufacturer �s
limits should be used. Basically every user should define their own limits, according to the application used and the
ambient conditions.

Sources of pipette error:
 Volatile liquids evaporate. Use in saturated air.
 High density liquids sink. Change technique.
 Viscous liquids stick to tip. Change technique.
 Hydrophilic tips will retain liquid. Pre-wet tip.
 Immersed tips carry over liquid. Immerse 2-3mm.
 Angled pipettes take up more liquid. Keep vertical.
 Surface tension reduces accuracy. Change technique.
 Hot and cold liquids. Pipette liquids at room temp.
 The human factor. Practice and observe technique.

General liquid handling questions

Question:
What is the difference between air displacement and positive displacement pipettes?
Answer: Both types of pipettes have a piston that moves in a cylinder, or capillary. In air displacement pipettes, a specified
volume of air remains between the piston and the liquid. In positive displacement pipettes, the piston is in direct contact
with the liquid. This keeps air from entering the tip, preventing contamination effectively. Air displacement pipettes are
meant for general use with aqueous solutions. Positive displacement pipettes are used for high viscosity and volatile
liquids.
Question:
How can I pipette viscous liquids?
Answer:
You can do so using an air displacement pipette with standard or wide orifice tip (reverse pipetting, slowly). An alternative
to this is to use a positive displacement system.
Question:
How can I prevent liquid dropping out of the tip when pipetting volatile compounds?
Answer:
If you use air displacement pipettes, aspirate and dispense the liquid a few times keeping the tip in the liquid. By doing so,
the air inside the pipette will be saturated with vapor of the volatile compound. It is recommend using positive
displacement pipettes for highly volatile compounds, since the built-in piston tip is in direct contact with the liquid.
Question:
How accurately can I pipette warm or cold liquids.
Answer:
The pipettes are calibrated by weighing distilled or de-ionized water of 20 - 25C. With warm liquids, you will get a smaller
mass with a certain volume, with cold liquids, you will get a higher mass.
 Mohr, backward or drain-out pipettes have a 0 mL mark just above the end of the pipette. A volume is
pulled into the pipette, sometimes to the maximum volume, then the needed volume is aliquoted out.
 Serological, forward or blow-out pipettes have no 0ml mark as that corresponds to an empty pipette. The
volume needed is pulled into the pipette and then dispensed.