25/04/56
Pipette Theory and Maintenance
LTC Carmen A. Bell
Chief, Laboratory Operations
USAMC-AFRIMS
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1
References
Pharma Express:
http://pharma.financialexpress.com/2008063
0/biotech13.shtml
ThermoScientific:
http://www.thermoscientifc.com/finnpipette
Wisconsin State Laboratory of Hygiene
USAMC-AFRIMS SOP 2170, Rev 7
Biosafety in Microbiological and Biomedical
Laboratories, 5th Edition (CDC/NIH)
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References
Connors, M. and Curtis, R. H. Pipetting error:
A real problem with a simple solution. Part I,
American Laboratory News (1999) 31(13):1619
Fleming, D.O. 1995. Laboratory biosafety
practices. In Fleming, et al (eds.), Laboratory
Safety: Principles and Practices, 2nd edition.
ASM Press, Washington
WHO Laboratory Biosafety Manual, 2nd edition
revised
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Objectives
Upon completion of this course, you will be
able to:
State the function of a pipette
Identify pipette safety
Identify the three types of pipettes
List the parts of a pipette
Use a pipette correctly
Understand basics of pipette calibration
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Pipette Function
Instruments used to handle, measure and
dispense liquids
Uses a pressure gradient to draw a liquid
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Types of Pipettes (1
(1 of 8)
Six types
Volumetric
Measuring
Mohr or Serological
Glass/ Pasteur
Air Displacement
Positive Displacement
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Types of Pipettes (2
(2 of 8)
Air
Displacement
-Single channel
(above right)
-Multichannel
(below right)
Measuring
Volumetric
Positive
Displacement
-Single channel
(right)
-Multichannel
(left)
Mohr
Serological
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Types of Pipettes (3
(3 of 8)
Volumetric
◦ Transfers a specific volume
◦ Capacity of 1-100 ml
◦ Used when precision is not important
Measuring
◦ Straight tubes with a tapered end
◦ Capacity .1ml and 25 ml
◦ Clearly marked hash marks along one side of
tube so multiple amounts can be dispensed
◦ Not very precise due to imperfections in the
tube
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Types of Pipettes (4
(4 of 8 )
Mohr and Serological
◦ Mohr: the hash marks always end before the
pipette tip
◦ Serological: the hash marks continue down
into the tops and some are “blow-out”
pipettes
Blow-out pipettes are marked with a frosted band
and two thin rings around the neck
◦ Pipettes have an open top and the user uses
their thumb to create a vacuum and seals the
liquid in the pipette
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Types of Pipettes (5
(5 of 8)
Glass/ Pasteur
◦ Glass tubes with a bulb on one end
◦ Calibrated to deliver or contain a specific
amount, or have graduated markings to
measure the volume of liquid they contain
◦ *Single use
◦ Precision is not critical
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Types of Pipettes (6
(6 of 8)
Air Displacement
◦ Digital
◦ Piston Driven
◦ Contains a piston inside a cylinder that can be
positions to specify a needed volume
◦ Expel the air first, draw up the liquid needed, then
expel the liquid and the remaining air
◦ A volume of air is always held between the liquid
and piston
◦ Very accurate
◦ Atmospheric pressure and type of liquid can
affect the performance
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Types of Pipettes (7
(7 of 8)
Positive Displacement
◦ Similar to Air Displacement pipettes because they
have a piston and cylinder
◦ Piston is always in direct contact with the liquid
◦ When depress, the piston moves down to the tip
opening
◦ When released, the piston rises to the
appropriate volume creating a partial vacuum that
draws in the liquid
◦ Used with volatile and viscous liquids
◦ *Most accurate and costly
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Types of Pipettes (8
(8 or 8)
Parts
Plunger button
Plunger rod
Friction ring
Tip ejector
Digital Volume
Indicator
Piston
Springs
Seal, O-ring
Shaft
Shaft coupling
Tip ejector
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Pipette Safety (1
(1 of 4)
Never mouth pipette
◦ Use rubber pumpette (bulb)
Use plastic pipettes if available
◦ Safer than glass
Do not allow the solution to be drawn into
the bulb
◦ Leaks chemicals into the top where fingers may
touch
Do not insert the pipette into the bulb,
insert the bulb onto the pipette
Use proper ergomomics (body position)
http://www.youtube.com/watch?v=bqAsXMSs27s
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Pipette Safety (2
(2 of 4)
When working with infectious agents,
always use a Biological Safety Cabinet
(BSC), or acceptable primary barrier
Never expel infectious fluids
◦ Potential aerosols
◦ Mark to mark pipetting is best
Never cause air bubbles to mix infectious
fluids
Discharge fluids close to other fluids, agar
level or microwells
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Pipette Safety (3
(3 of 4)
Pipettes used for transferring infectious
materials should always be plugged with
cotton
Syringes fitted with hypodermic needles
MUST NOT be used for pipetting
Avoid dropping material from the pipette
on the work surface
◦ Place a disinfectant-dampened towel or other
absorbent material on the work surface
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Pipette Safety (4
(4 of 4)
Place contaminated pipettes horizontally
in an autoclave bag on a tray
◦ Vertical positioning may cause aerosoles
Discard contaminated Pasteur pipettes
and broken glass in a biohazardous sharps
container.
◦ Dispose of when 2/3 full
◦ Pans or sharps containers should be kept
inside the BSC
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General Guidelines (1
(1 of 2)
Check your pipette to ensure it is clear of
dust and dirt
◦ Wipe, if needed with 70% ethanol
Set the volume within the range specified
Hold the pipette so the “griippy finger
rest” rest on the index finger
For accuracy, the pipette, tip and liquid
should be at the same temperature
Use recommended tips
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General Guidelines(2
Guidelines(2 of 2)
Tips are single use
Avoid turning the pipette on its side to prevent
contamination
Avoid contamination to or from fingers by using the
tip ejector and gloves
Store pipettes in an upright position when not being
used
*Pre-rinse (1-3 times) the tip with liquid to improve
accuracy, especially with positive displacement tips
*Check calibration regularly, depending on the
frequency of use and on the application, but at least
once a year
◦ They should NOT BE cleaned and reused
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Maintenance
Keep pipette clean
◦ Free of dust particles
Wipe the surface with a sponge moistened with
disinfectant
Store in an upright position
Clean
◦ Piston, Piston Spring and O-rings (70%
ethanol)
Lubricate
◦ Piston, Piston Spring and O-rings (grease)
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Calibration (1
(1 of 3)
Three parts
◦ Operator
◦ Equipment
◦ Environment
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Calibration
The pipette is held in the
calibration room at least
2 hours before testing
Check for maximum and
minimum volume or 10%
of the maximum volume
(whichever is higher)
Perform a series of ten
pipettings of both
volumes
Calculate the accuracy
and precision
*Adjust pipette if needed
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Calibration
Accurate
and
Precise
Accurate
But not
Precise
Precise
But not
Accurate
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Calibration
Operator
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Pipetting Technique ((11 of 9)
Optimizing volume range
Micrometer setting
Tip immersion angle
Tip immersion depth
Pre-rinsing
Aspiration rate
Dispensing technique
Consistency
**Errors from
poor technique
can range from
0.1 % to 5% or
more
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Pipetting Technique ((22 of 9)
Optimizing volume range
◦ Assures accuracy and precision
Normal
Range
10-100µL
20-200µL
100-1000µL
Optimizing
Range
35-100µL
70-200µL
350-1000µL
Optimizing volume range typically
improves accuracy up to 1%
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Pipetting Technique ((33 of 9)
Micrometer Setting
◦ Ensure the volume is set correctly
*Correct settings improve accuracy up to 0.2%
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Pipetting Technique ((44 of 9)
Angle
◦ Aspirate at 90 degrees
◦ Dispense at 45 degrees
*Improves accuracy up to 0.5%
Incorrect immersion
angle
Correct immersion
angle
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Pipetting Technique ((55 of 9)
Tip Immersion Depth
*Improves accuracy up to 5%
Pipette size
Immersion depth
2 and 10 µL
1 mm
20 and 100 µL
2-3 mm
200 and 1000 µL
3-6 mm
5000 µL and 10 mL
6-10 mm
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Pipetting Technique ((66 of 9)
Pre-rinsing
◦ Pre-rinsing tip with same liquid that is being
dispensed
◦ Aspirate with tip, and then dispense back into
reservoir or to waste
◦ Provides identical contract surfaces for all
aliquots
* Two rinses provides up to 0.2 % greater
accuracy
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Pipetting Technique ((77 of 9)
Aspiration Rate Effects
◦ Maintain smooth, controlled aspiration rate
◦ Aspirating too quickly causes
Splashing into the piston and seal damage
Introduction of aerosols/ sample crosscontamination
* Controlled aspiration improve accuracy 0.2% -5%
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Pipetting Technique ((88 of 9)
Dispensing Technique
◦ Thin-wall tips provide maximum droplet
dispensing
◦ Along side-wall techniques
*Correct dispensing technique improves accuracy 0.5%-1%
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Pipetting Technique ((99 of 9)
Consistency
◦
◦
◦
◦
Pipetting rhythm
Pressure on plunger
Speed and smoothness
Use electronic pipettes for best consistency
Less user dependent
* Consistency in pipetting can improve accuracy up to 1%
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Calibration
Equipment
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Pipetting Direction Technique
(1 of 9)
Forward
Reverse
◦ Used for pipetting solutions with a high
viscosity or a tendency to foam
◦ Only possible with air displacement pipettes
Repetitive
◦ Used for repeated pipetting of the same
volume.
◦ Only used with electronic and repeator
pipettes
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Pipetting Direction Technique
(2 of 9)
Forward
◦ Press the button to the first stop
◦ Place the tip in the solution and slowly release
the button
◦ Dispense the liquid into the receiving vessel
or fluid (pressing to the first stop, then press
to the second stop)
◦ Release the button
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Pipetting Direction Technique
(3 of 9)
Forward Pipetting Technique
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Pipetting Direction Technique
(4 of 9)
Reverse
◦ Press the operating button
◦ Place the tip in the solution and slowly release
the button
◦ Dispense the liquid into the receiving vessel
or fluid. The volume is equal to the set
volume. Hold the button in this position.
Some liquid should remain
◦ The remaining liquid can be pipetted back into
the original solution or disposed of
◦ Release the operating button
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Pipetting Direction Technique
(5 of 9)
Reverse Pipetting Technique
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Pipetting Direction Technique
(6 of 9)
Repetitive
◦ Press the operating button to the second stop
◦ Place the tip onto the solution and slowly release
the button. Withdraw the tip from the liquid,
touching against the side of the resevoir
◦ Dispense the liquid into the receiving vessel by
pressing the operating button to the first stop.
Hold the button in position. Some liquid will
remain in the tip. Do not dispense it.
◦ Continue pipetting by repeating the above two
steps
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Pipetting Direction Technique
(7 of 9)
Repetitive Pipetting Technique
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Pipetting Direction Technique
(8 of 9)
Whole Blood
◦ Use first two steps of the forward technique to fill
the tip with blood. Wipe the top with a dry clean
cloth
◦ Place the tip into the blood and press the operating
button to the first stop. Make sure the tip is below
the surface
◦ Release the button slowly to fill the tip with blood
◦ Press the button to the first stop and release slowly.
Repeat this process until the 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 button
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Pipetting Direction Technique
(9 of 9)
Whole Blood Technique
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Pipetting Recommendations
Solution/
Compound
Examples
Pipette
Tip
Technique
Aqueous solution
Buffers, diluted
salt solution
Air Displacement
Standard
Forward
Viscous solution
Protein and
nucleic solutions,
glycerol,Tween
20/40/60/80
Air Displacement
Positive
Displacement
Filter
Positive
Displacement
Reverse
Pipette slowly to
avoid bubble
formation
Nucleotide
solutions
Genomic DNA,
PCR Products
Air Displacement
Positive
Displacement
Filter or wide
orifice
Positive
Displacement
Forward
For genomic
DNA wide orifice
should be used to
avoid mechanical
shearing
Radioactive
compounds
Carbonate,Ythymidine
Air Displacement
Positive
Displacement
Filter
Positive
Displacement
Forward
Acid/ Alkalis
H2So4, HCL,
NaOH
Air Displacement
Filter
Forward
Positive
Displacement
Positive
Displacement
Air Displacement
Positive
Displacement
Filter
Positive
Displacement
Toxic samples
Volatile
compounds
Methanol, Hexane
Comments
Forward
Pipette rapidly to
avoid evaporation.
Carbon filter tips
prevent vapors
from going into
the Pipette
44
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Contamination Prevention
Pipette-to-Sample (contaminated
pipette/tips)
◦ Use sterilized tips or sterilized filter tips
◦ Change tips after pipetting each sample
Sample-to-Pipette (samples or aerosole in
cone)
◦
◦
◦
◦
Keep the pipette vertical
Release the push-button slowly
Use filter tips or positive displacement pipettes
Store the pipette vertically
Sample-to-Sample (sample A mixed with B)
◦ Change the tip after each sample
◦ If you suspect contamination, autoclave or clean
the pipette
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Parts of the Pipette
Plunger button
Plunger rod
Friction ring
Tip ejector
Digital Volume
Indicator
Piston
Springs
Seal, O-ring
Shaft
Shaft coupling
Tip ejector
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Calibration
Environment
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Factors affecting the accuracy of Air
Displacement Pipettes (1
(1 of 2)
Temperature
◦ The most important factor in pipetting
accuracy is the liquid temperature
Density
◦ Density is the mass/volume ratio of the liquid.
It varies according to the temperature and air
pressure
Altitude
◦ Altitude affects the accuracy through the air
pressure
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Factors affecting the accuracy of Air
Displacement Pipettes (2
(2 of 2)
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Why/ When Calibrate?
Calibrate
◦ Determine the difference between the
dispensed volume and selected volume
◦ Occurs upon receipt and periodically during
the lifetime of use (at least annually)
◦ Part of Quality Control
Checks accuracy and precision
Ensures integrity of data
Produces quality liquid handling
Produces quality results
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Calibration
The pipette is held in the
calibration room at least
2 hours before testing
Check for maximum and
minimum volume or 10%
of the maximum volume
(whichever is higher)
Perform a series of ten
pipettings of both
volumes
Calculate the accuracy
and precision
*Adjust pipette if needed
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51
Calibration
Accurate
and
Precise
Accurate
But not
Precise
Precise
But not
Accurate
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Calibration Methods
Gravimetric
◦ Most commonly used
◦ Based on simple principle
Colorimetric (photometric)
◦ Uses light to measure volume of diluted dye
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Calibration Methods
Gravimetric
◦ Uses a certain mass of water with a known
specific gravity to predict the volume
◦ Uses an enclosed calibrated analytical balance
with an accuracy of 0.0001g
◦ Requires a stringently controlled environment
Air and
Water
Temperature
Air Pressure
(Barometer)
Humidity
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Calibration Methods
Gravimetric (con’t)
◦ Requires highly skilled technician
◦ Requires an understanding of statistics
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Calibration Methods
Colorimetric
◦ This method involves the analysis of volumes
of diluted dye in a cell of known path length
◦ Less prone to environmental influences
◦ Requires standardized consumables
Dye must be very carefully manufactured
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Calibration Methods
Adjustments
Manual Pipettes
◦ Adjustment is done at the lower volume
◦ Use the service tool provided with the pipette
◦ Turn the service tool clockwise to increase the
volume or counter clockwise to decrease the
volume
◦ After adjusting, recalibrate
Electronic Pipettes
◦ Follow specific manufacturers’ instructions
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Questions
29