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Basics of Pipetting
Used to Transfer Liquids Accurately and Precisely
DSI: Dolphin Science Investigators
Midwest Research Institute
Biotech Fridays
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What is a pipette? A pipette (or pipet) is a laboratory instrument used to
transport a measured volume of liquid.
Pipettes are commonly used in chemistry and
molecular biology research as well as medical
testing.
Pipettes can come in several different designs for
various purposes as well as both manual and
electronic types.
A pipette works by creating a vacuum above the
liquid-handling chamber (tip) and selectively
releasing the vacuum to draw and dispense liquid.
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Types of Pipettes
Air Displacement Pipettes:recommended for aqueoussolutions and generallaboratory work.
The piston is part of the pipetteand moves a cushion of airbetween itself and the sample inorder to aspirate (draw up) anddispense the liquid.
The volume of air displaced isequivalent to the volume of liquidaspirated.
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Types of Pipettes
Positive DisplacementPipettes: better suitedfor use with viscous
(thick) and corrosive(damaging) liquids The piston is part of the
disposable pipette tip andforms a seal in the pipette
tip so that the sample is indirect contact with thepiston (no air cushon).
Performs in a mannersimilar to a syringe.
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We will use air displacement pipets.
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Single Channel vs. Multi-Channel
Single channel pipettes are for pipetting from
one sample container at a time
Multi-channel pipettes are for high throughput
applications and pipette from multiple sample
wells at once.
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Bulb Pipettes Bulb pipettes are a less
advanced option for pipetting
but may be all that is available.
The plastic bulb is attached to
the top of a disposable
serological pipet. Aspiration is
accomplished by squeezing the
bulb, then placing the pipet into
the liquid to aspirate and slowly
releasing pressure on the bulb
until the correct volume has
been drawn up.
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Bulb Pipetting
4.49 mL reading 4.88 mL reading 5.00 mL reading
Reading the Volume
Determine the volume of solution in a pipet by reading the bottom
of the meniscus at eye level. Record the volume using all certain
digits and one uncertain digit. Certain digits are obtained from
calibration marks. Uncertain digits (the last digit in the number) are
estimated between calibration marks. Some examples are shown
below:
meniscus
This method is not very accurate since the volume aspirated is controlled
visually by the user instead of a mechanical set point. It should only be used
when highly accurate volumes are not required.
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Variable vs. Fixed Volume
Variable volume pipettes are the most common
found in laboratories. This pipette can cover a
specified range of volumes and is set by turning
the volume adjustment knob. Fixed volume pipettes can be used when the
same volume is being aspirated and dispensed
many times. The fixed volume pipette will only
aspirate the specified volume but usually has abetter accuracy and precision for that volume
than a variable volume pipette might.
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Manual vs. Electronic
In manual pipettes the piston action is
accomplished by the user manually depressingand releasing the plunger with their thumb.
Electronic pipettes do the work for you the
piston action is controlled by the pipette itselfthe user pushes a button to aspirate and
dispense the liquid. Electronic pipettes reduce
the force needed by the users thumb and are
helpful when repetitive pipetting is required.Electronic pipettes often have multiple options
included such as mixing and multi-dispensing
of the sample.
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Examples of Manual and Electronic
Manual
single
channel
Manual
multi-
channel
Electronic
single
channel
Electronic
multi-
channel
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Parts of a pipette
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Our DSI Pipets
The pipetting devices we will use measure
microliters (abbreviated mL).
Sometimes, for convenience, it is typed ul,
but this is not technically correct.
A microliter is 1/1000th of a milliliter, or
1/1,000,000 of a liter.
In other words
SMALL!
http://www.earthzine.org/2009/01/06/c-more-researchers-in-
hawaii-probe-marine-microbes-train-a-new-cadre-of-scientists/
http://www.earthzine.org/wp-content/uploads/2008/12/doran-2.jpg7/30/2019 DSI Basics of Pipetting (1)
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You have to look very carefully to see
what you are pipetting.
http://www.yale.edu/caccone/ecosave/images/pipetting.jpg
It could be
even less than
this!
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Accuracy and Precision
Accuracy is when multiple sample deliveries at a set volume
average to the expected volume.
Precision is when multiple sample deliveries at a set volume are
close to each other.
These specifications are independent of one another.
The goal is to be both accurate and precise in your sampledeliveries. With proper technique, conditions, and care of the
pipettes this can be achieved.
low accuracy
high precisionhigh accuracy
low precision
high accuracy
high precision
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Environmental Factors Affecting
Pipetting Air pressure
Temperature of air, pipette, tips, andliquids
Evaporation rates
Surface tension of liquids Relative Humidity
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Ways to optimize pipette performance
Choose the right pipette for the job. Check for leaks or any other pipette malfunctions. If the
pipette does not appear to be functioning properly ensure
that the tip is properly fitted on the pipette and that it is set
to the correct volume. If it is still not functioning remove it
from use and notify the lab manager to have it serviced.
Choose the correct pipette tip
Correct size
Correct style Have pipettes calibrated and serviced regularly.
Read the Manufacturers Manual to understand the pipette
being used.
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Tips to minimize pipetting errors Choose the correct pipette volume for best accuracy
and precision the volume being pipetted should be within
35% - 100% of the pipettes range.
Allow all liquids and equipment to equilibrate to ambient
temperature before beginning work.
Pre-rinse the pipette tip by aspirating and dispensing the
sample liquid at least 3 times before aspirating a samplefor delivery.
Immerse the tip vertically into the sample liquid well clear
of the container walls and bottom and at a depth of
approximately 2 5mm below the meniscus.
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Tips to minimize pipetting errors
Aspirate using a consistent speed, rhythm, and plunger
pressure. Hold the tip in the sample for 1 second after aspiration
and withdraw the tip slowly and smoothly without
touching the sides of the container.
To dispense touch the pipette tip to the sidewall of thecontainer where sample is to be delivered just above the
liquid the sample is being dispensed into. Use consistent
speed, rhythm, and plunger pressure to dispense.
Put the pipette in its stand between pipetting cycles toavoid warming the pipette in your hand this can affect
the volume of liquid dispensed.
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References
www.rainin.com
www.artel-usa.com
http://www.gilson.com Practical Pipette Calibration Presented
by Larry Newman of Anmar Metrology,
Inc.
http://www.rainin.com/http://www.artel-usa.com/http://www.gilson.com/http://www.gilson.com/http://www.artel-usa.com/http://www.artel-usa.com/http://www.artel-usa.com/http://www.rainin.com/