STUDY OF PIPETTES AND PIPETTING TECHNIQUE Dennis Bryan A.
Valdez
Clinical Chemistry I: Laboratory BMLS II -H
Pipets
A laboratory instrument used to transport a
measure volume of liquid.
Pipettes come in several designs for various
purposes with differing levels of accuracy
and precision, from single piece glass
pipettes to more complex adjustable or
electronic pipettes.
Many pipette types work by creating a
partial vacuum above the liquid-holding
chamber and selectively releasing this
vacuum to draw up and dispense liquid.
PIPET CLASSIFICATION
I. Design
A. To contain (TC)
B. To deliver (TD)
II. Drainage characteristics
A. Blowout
B. Self-draining
III. Type
A. Measuring or graduated
1. Serologic
2. Mohr
3. Bacteriologic
4. Ball, Kolmer, or Kahn
5. Micropipet
B. Transfer
1. Volumetric
2. Ostwald-Folin
3. Pasteur pipets
4. Automatic macropipets or
micropipets
TYPE ACCORDING TO FUNCTION
1. Transfer Pipette
Designed to deliver accurately a fixed
volume of liquid.
Consist of a cylindrical bulb joined at both
ends to narrower glass tubing.
Calibration mark is etched around the upper
suction tube, and lower delivery tube is
drawn out to a gradual taper.
The bore of the delivery orifice should be
sufficiently narrow so that rapid outflow of
liquid and incomplete drainage can cause
measurement errors beyond tolerances
specified.
These pipettes are allowed to drain by
gravity.
Most-accurate type of pipette.
a. Volumetric Pipette
Calibrated accurately to deliver a fixed
volume of dilute aqueous solution such as a
standard, a filtrate, or a non-viscous
sample.
These pipettes are allowed to drain by
gravity.
The last drop should not be blown out (self-
draining).
Reliability of the calibration of the volumetric
pipette decreases with a decrease in size.
b. Ostwald-Folin Pipette
Similar to volumetric pipettes but have their
bulb closer to the delivery tip, thereby
reducing the surface area in contact with the
liquid.
Used for accurate measurement of viscous
fluids such as blood or serum.
Etched ring near the mouthpiece, indicating
that it is a blowout pipette.
Controlled, slow drainage is required with all
viscous solutions so that no residual film is
left on the walls of the pipette.
T.D. pipette (etched ring-blow out)
The top of the meniscus must be read when
the pipette is filled with opaque fluids such
as blood.
In dispensing:
AQUEOUS material follow the
LOWER meniscus.
VISCOUS material follow the
TOP / UPPER meniscus.
2. Measuring or Graduated Pipette
Piece of glass (Borosilicate) tubing that is
drawn out to a tip and graduated uniformly
along its length.
Primarily used for the measurements of
reagents and are not generally considered
sufficiently accurate for measuring samples
abd calibrators.
Least accurate
a. Mohr Pipette
Does not have graduations to the tip.
Self-draining pipet, but the tip should not be
allowed to touch the vessel while the pipet
is draining.
Calibrated just before the tip.
Require a controlled delivery of the solution
between the calibration marks.
b. Serological Pipette
Calibration marks extend to the tip.
Blow-out to deliver the entire volume of the
liquid.
Has etched / pair of rings near the bulb end.
Has larger orifice than do the Mohr pipet,
thus drains faster.
STUDY OF PIPETTES AND PIPETTING TECHNIQUE Dennis Bryan A. Valdez
c. Pasteur Pipette
Pasteur pipets do not have calibration
marks.
Used to transfer solutions or biologic fluids
without consideration of a specific volume.
Should not be used in any quantitative
analytic techniques.
TYPE ACCORDING TO DESIGN
1. TD (To Deliver) Pipette
Some fluid remain on the tip.
DO NOT SHAKE!
Deliver all the volume indicated including
the tip by "blowing-out" to get the last drop
o Single solid band
2. TC (To Contain) Pipette
Holds or contains a particular volume but
does not dispense that exact volume
Deliver the volume including the tip by
blowing out to get the last drop.
calibrated to allow the entire tip to drain,
leaving a tiny bit in the tip that should not be
delivered.
o Double band.
Usually for small volume.
TYPE ACCORDING TO
DRAINAGE CHARACTERISTICS
1. Blowout Pipette
has a continuous etched ring or two small,
close, continuous rings located near the top
of the pipette.
This means that the last drop of liquid
should be expelled into the receiving vessel.
2. Self-Draining
The user allows the contents of the pipette
to drain by gravity.
The tip of the pipette should not be in
contact with the accumulating fluid in the
receiving vessel during drainage.
SELECTION OF PIPETTE
For accuracy, always select a higher
volume than the volume intended to be
dispensed.
Reasons:
1. Error of pipette is found on the tip.
2. Control of the flow of liquid.
AUTOMATIC PIPETTE TYPES
Most routinely used pipet in todays clinical
chemistry laboratory.
Have many advantages, including safety,
stability, ease of use, increased precision,
the ability to save time, and less cleaning
required as a result of the contaminated
portions of the
The pipette body contains a plunger, which
provides the suction to pull liquid into the tip
when the piston is compressed and
released.
The maximum displacement of the plunger
is set by a dial on the pipette body, allowing
the delivery volume to be changed.
a. MICROpipette
Used for measurement of micro liter
volumes.
Most are calibrated to TC (to contain) the
stated volume rather than to deliver it
because the remaining volume coats the
inner wall of the pipette causes notable
error.
Dispense between 1 and 1000 l (1mL)
Two types of micropipettes are generally
used:
o air-displacement pipettes
o positive-displacement pipettes.
b. MACROpipette
Dispense greater volume of liquid.
TYPES:
1. Fixed Volume
associated with only one volume
2. Variable Volume
models able to select different volumes
3. Multi-channel
Used for microplates
Fixed volume
======================================
Air-Displacement Pipet
Relies on a piston for suction creation to
draw the sample into a disposable tip that
must be changed after each use.
piston does not come in contact with the
liquid.
Positive-Displacement Pipet
Operates by moving the piston in the pipet
tip or barrel, much like a hypodermic
syringe.
It does not require a different tip for each
use. Because of carry-over concerns,
rinsing and blotting between samples may
be required.
Dispensers and Dilutor / Dispensers
Automatic pipets that obtain the liquid from
a common reservoir and dispense it
repeatedly.
The dispensing pipets may be bottle-top,
motorized, handheld, or attached to a
dilutor.
The dilutor often combines sampling and
dispensing functions.
STUDY OF PIPETTES AND PIPETTING TECHNIQUE Dennis Bryan A. Valdez
1. Depress the Plunger to the First Stop.
2. Immerse Tip in Sample.
3. Draw up the sample:
a. allow the pushbutton to return slowly
and smoothly to the fully extended
UP POSITION
4. Withdraw the Tip
5. Dispense the Sample from the pipette
a. Touch the tip end to the side wall of
the receiving vessel and
b) Depress the plunger to the FIRST
STOP.
d) Press the plunger to the SECOND
STOP (the second point, of greater
resistance, at the bottom of the
stroke) to expel any residual liquid in
the tip.
6. Withdraw the Pipette
7. Discard the Tip
OR:
(1) Set the volume
(2) Attach disposable tip
(3) Depress the plunger to the first stop
(4) Immerse tip in sample
(5) Draw up the sample
(6) Pause
(7) Withdraw the tip
(8) Dispense the sample
(9) Withdraw the pipette
(10) Release plunger
(11) Discard the tip
Pippetting Guidelines and Precautions
Consistent SPEED and SMOOTHNESS
when you press and release the PLUNGER
Consistent pressure on the PLUNGER at
the FIRST STOP
Consistent and sufficient IMMERSION
DEPTH
Nearly VERTICAL POSITIONING of pipette
AVOID ALL AIR BUBBLES: Since the
plastic pipette shaft can be damaged if
liquids are drawn beyond the tip into the
shaft
NEVER lay the pipette on its SIDE nor
INVERT the pipette if liquid is in the tip.
H E L P F U L H I N T S F O R ACC U R AT E P R E C I S E
M E A S U R E M E N T S
1. Pipettes should be stored in an upright position.
2. Pre-wet the pipette tip three times prior to
delivering the specified sample volume.
3. Always use the properly prescribed tip for the
selected pipette to ensure a good seal. Check
the tip of the pipette shaft for scratches, hairline
fractures or other damage that may
compromise a tight seal between the pipette
and the tip.
4. Develop a steady aspiration and dispensing
cadence that emphasizes skill and consistent
speed.
5. Never invert, tilt at a sharp angle, or lay the
pipette on its side while there is liquid in the tip.
6. Allow all sample fluids and equipment to reach
room temperature prior to pipetting.
NOTE: Most pipettes are calibrated for
liquids at room temperature and standard
atmospheric pressure. If you are pipetting
extremely cold or hot solutions, the volumes may
vary slightly due to the temperature. Do not pre-
rinse the tips, and aspirate and dispense liquids as
quickly as possible.
7. Do not use adjustable pipettes outside
recommended ranges.
8. Calibrate pipettes every six months, or more
frequently if the lab is either clinical or GMP in
nature. There are many competent pipette
services that will calibrate pipettes for a
reasonable fee.
9. Prime the piston by depressing the plunger a
few times prior to using the pipette for the first
time each day of use.
10. When pipetting liquids that are significantly
more dense or less dense than water (e.g.,
fluids like glycerol, ethanol, or methanol),
consider using positive displacement pipettes.
11. Leaks in the seal will adversely affect the
reliability of the pipette. To verify that there is no
leak, adjust the volume setting to the maximum
setting and then aspirate water into a pipette
tip. Keep the pipette in a vertical position for 20
seconds and look for a drop of water at the
bottom of the tip. If a drop appears then there is
a leak. If there is no drop, submerge the same
water-filled tip in water so that the surface level
of the water in the container is just below the
level of the water in the pipette tip. The level of
water within the tip should remain the same. If
the level drops, then there is a leak. Try other
pipette tips or have your pipettes serviced
immediately.
