How to Use a Pipette
Objective:
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This document will guide you through proper use of automatic and glass pipette use. These
techniques will maximize your reproducibility, precision, and accuracy by minimizing avoidable
errors.
It is strongly encouraged that you inform yourself further by reading on-line literature on general
pipette use. (See end of document for suggested further reading.)
Relevant sections from Exploring Chemical Analysis. Harris, D.C. 4th ed.
Ch. 2-6 pp. 50-51.
Automatic Pipettes
The following diagrams are from the Fisher Finnpipette® Digital user manual.
Basic Features and Principles. The automatic pipettes we will be using
operate on the principle of air displacement through an internal adjustable
plunger. This type of pipette is intended for use with polypropylene tips. The
sample is loaded into the disposable polymer tip rather into the pipette shaft.
Never allow solutions to enter the pipette body as they will damage the piston
seals and render the device inoperable until serviced.
Figure 1. Image of an
automatic pipette used
in class.
Features of note are:
a volume display,
a plunger that also serves to adjust
the volume of the pipetter as well as
load and dispense the sample,
 Be sure to familiarize yourself with these features.
and a thumb
operated tip ejector.
Basic Operation.
The accuracy and precision of a pipette does not depend solely on factory specifications and the
volume displayed (Image 1). It depends to a considerable degree on the pipette position, tip
conditioning, plunger release, and dispensing technique. The first three of these factors will be
discussed below.
 Position: The pipette should always be held in a vertical
position when loading the tip with sample.
o When aspirating a sample be sure that the tip is
always 2-5 mm below the surface of the solution.
 Tip conditioning: Tips should generally be pre-wetted or
rinsed with the sample to be delivered. This is especially
critical when delivering multiple aliquots of the same volume
or volumes below 10 L.
o If a new volume is dialed in on a pipette, a new tip
should be used and conditioned.
o Be sure the tip is well sealed to the pipette by
inspecting the visible “o-ring” that forms where the tip
contacts the pipette.
 Plunger operation: The plunger should always be depressed
or released in a smooth manner. Abrupt movements should
be avoided.
Figure 2. Schematic of a
negative displacement pipette
o Inconsistencies in plunger depression or release
with proper alignment relative to
speed can affect reproducibility when performing
sample.
repetitive pipetting of the same volume.
(http://www.ebbep.org/docs/basics/r
o Never move the plunger at a rate faster than the flow
aininpipettetechniques.ppt)
of the sample into or out of the tip. This rate will vary
depending on the viscosity of the sample.
o Abrupt plunger release can cause liquid to splash up into the pipette, contaminating the
pipette and future samples.
o Abrupt plunger release can also cause some of the sample to adhere to the tip above
the meniscus affecting the accuracy in delivered volume.
There are two techniques for accurately and precisely delivering a given volume as well as a proper
method for repetitive pipetting. The choice of which technique to employ depends on the properties and
volume of the solution being dispensed.
Some general considerations:
 Never dial the pipette beyond the operating range. Change the volume setting smoothly and
gently. Violent or rapid rotation of the plunger dial can damage the micrometer and adversely
affect pipette performance.
 Always dial down to the final volume.
 After loading the tip with sample, remove any liquid droplets adhered to the outside of the tip by
lightly dragging the tip along the side of the container.
 There are three plunger positions described in all techniques: (A) fully released, (B) first stop –
be aware that this stop is a soft stop and can be overshot if too much pressure is applied, and
(C) second stop – a hard stop.
Technique I: Forward Technique. Used with samples having physical properties - density, viscosity,
and vapor pressure - similar to water. This is not the ideal method for aqueous surfactant and protein
solutions (refer to Technique II).
1) With the pipette tip out of the solution, press the plunger to position B.
2) Immerse the tip in the solution and slowly release the plunger to
position A.
3) Expel the desired volume of sample by immersing the tip into the
receiving solution (if performing a dilution) or touching the tip to the
side of the receiving container and
a. pressing the plunger to position B and waiting a second before
b. further pressing the plunger to position C.
c. If the tip was immersed in a solution remove the tip along the
side of the container.
4) AFTER removing the tip from the sample, return the plunger to position A
5) Eject the tip into an appropriate waste container.
Technique II: Reverse Technique. Used with viscous, foamy, and volatile samples. This is also the
preferred technique when dispensing volumes less than 10 L.
1) With the pipette tip out of the solution, press the plunger to position C.
2) Immerse the tip in the solution and slowly release the plunger to
position A.
3) Expel the desired volume of sample by pressing the plunger to
position B and waiting a second.
a. DO NOT go beyond plunger position B.
b. If the tip was immersed in a solution remove the tip along
the side of the container.
4) Clear the tip into a waste container.
5) Return the plunger to position A and eject the tip into an appropriate waste container.
Repetitive pipetting. This is the preferred technique when delivering multiple equal aliquots of a
sample.
1) With the pipette tip out of the solution, press the plunger to position C.
2) Immerse the tip in the solution and slowly release the plunger to
position A.
3) Expel the desired volume of sample by pressing the plunger to
position B and waiting a second.
a. Be sure that the tip is in contact with the wall of the
receiving container above the solution.
b. DO NOT go beyond plunger position B.
c. DO NOT immerse the tip in a solution other than the stock solution as this will cross
contaminate your stock.
4) Repeat steps 2 and 3 as necessary.
a. Using this method you will always have some sample left in the tip between each
round. Do not worry, this volume should never be included in the delivery volume.
b. Be sure to keep the plunger depressed at position B until the tip is returned to the
stock solution for the next round.
Technical Data.
Manual Pipettes
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Pipettes (volumetric and serological) can be calibrated “to
contain” (TC) or “to deliver” (TD) a given volume of water
at a certain temperature (typically 20°C).
o These pipettes operate with an acceptable accuracy ±
5°C from the calibration temperature.
Figure 3. Information inscribed on a
pipette.
Like any other piece of volumetric glassware the volume is read from the
bottom of the meniscus.
o Be sure to be looking perpendicular and level with the meniscus or
you will incorrectly assess its position.
o Precision and accuracy
You can deliver a given volume by two methods depending on the pipette you are using
(TD or TC and volumetric or serological).
o Differential pipetting applies only to serological pipettes. In this method you deliver the
desired amount as the difference between two volume graduations on the pipette barrel.
o If you are delivering the entire pipette volume (volumetric and serological pipettes) be
sure to follow the guidelines for a TD or TC pipette regarding blowing out the last bit of
solution in the pipette.
NOTE: Regardless of whether you use a pump or a bulb always check the seal between
the pipette and bulb/pump.
o This can be done easily by loading some solution into the pipette and observing if any
leaks out.
Roller piston pump use
1. Securely attach your pipette to the pump.
a. Be sure there is an even seal between the
pipette and the pump by gently rotating the
pipette a couple of turns.
b. The pump piston should be all the way down.
2. Insert the pipette approximately 1 cm below the
surface of the solution and maintain the pipette
perpendicular to the ground.
3. Load the pipette with solution by slowly rotating
the knurled knob in a downward direction.
a. Be sure NOT to overfill the pipette and allow
Figure 4. Various sizes of pipette pumps
liquid to enter the pump. IF this happens you
for different size pipettes.
will have assume the system is contaminated
and rinse the pump. These pumps are not fun to clean as they are difficult to dry once
rinsed.
b. Fill the pipette slightly above the desired volume.
4. Slowly rotate the knurled knob in an upward direction to set the meniscus to the desired
mark.
5. Remove the pipette from the solution and briefly drag it along the side of the container to
remove any liquid adhering to the outside of the pipette.
a. If necessary, you may use a Kim-Wipe to clean the outside of the pipette (not a sterile
technique!). Be sure not to touch the open end of the tip.
6. Transfer the pipette to the receiving container and touch the tip to the side just above the
solution (if performing a dilution).
7. Slowly rotate the knurled knob in an upward direction and deliver the desired volume.
Bulb use
1. Securely attach your pipette to the bulb.
a. Be sure there is an even seal between the
pipette and the bulb by gently rotating the
pipette a couple of turns.
2. Press A button and squeeze bulb.
a. Once bulb is fully decompressed, release A
button.
b. This creates a negative pressure differential
between inside the bulb and ambient pressure
thus generating your liquid aspiration
capability.
3. Insert the pipette approximately 1 cm below the
surface of the solution maintain the pipette
perpendicular to the ground.
4. Load the pipette with solution by gently pressing
Figure 5. Valved pipette bulb.
the S button.
a. Be sure NOT to overfill the pipette and allow liquid to enter the bulb. IF this happens
you will have to assume the system is contaminated and rinse the bulb. These bulbs
are not fun to clean as they are difficult to dry once rinsed.
b. Fill the pipette slightly above the desired volume.
c. Release the S button to stop sample aspiration.
5. You can slowly release the solution from the pipette by gently pressing the E button.
a. Keep a careful eye on the meniscus and release the E button once it has reached the
desired volume.
6. Remove the pipette from the solution and briefly drag it along the side of the container to
remove any liquid adhering to the outside of the pipette.
a. If necessary, you may use a Kim-Wipe to clean the outside of the pipette (not a sterile
technique!). Be sure not to touch the open end of the tip.
7. Transfer the pipette to the receiving container and touch the tip to the side just above the
solution (if performing a dilution).
8. Press the E button and deliver the desired volume.