Pipettes

Nadia Rasheed
Lecturer
Chemical Pathology Department
UHS Lahore
 Learning Objectives
Student should know about
• Definition of pipettes
• Classification of pipettes
• Pipette selection
• Pipette specifications
• Pipette use
• Pipette calibration
 What are pipettes?
Pipettes are glass or plastic
mechanical devices which are used
to transfer specific amounts of liquid
from one container to another.
 Classification
1.Based on designs
• TC
• TD
2.Based on drainage characteristics
• Blow out
• Self draining
 Classification
3.Based on measurements
• Serological
• Mohr
• Bacteriological
• Ball,kolmar,kahn
• Micropipette
 Classification
4.BASED ON TRANSFER
CHARACTERISTICS
 Volumetric
 Ostwald folin

 Pasteur pipette

 Automatic micro or macro pipette

5.Manual and Automatic Pipettes

 To contain pipette

 A To Contain pipette holds a

particular volume but does not
dispense the exact volume
 To Deliver

 To deliver pipette will dispense the

volume indicated
 Blow out pipette

If a serological pipette has a frosted band or two thin

rings around the neck, then it is a blow-out pipette.

This means that after all the liquid has been allowed
to drain out by gravity, the last drop remaining in the
tip is also forced out.
 Blow out pipette

The frosted band should

not be confused with
thicker colored rings or
colored dots, which are a
manufacturer’s code for
the maximum volume
of the pipette.

Remember, only blow-

out a serological pipette
if it has a frosted band
or two thin rings.
 Self draining pipette
 When the marker are absent it is self
draining pipette

 The user allows the contents of the

pipette to drain by gravity
 Measuring pipette
 These pipettes are
capable of dispensing
several different
volumes
 Graduation lines are
indicated on the
pipette
 A 5ml pipette can be
used for measuring
4ml,3ml,2ml or 1ml
Specification on a Measuring Pipette

Printed on the neck of the pipette are the specifications

that indicate:

• the maximum volume of liquid that can be transferred

• the size of the divisions on the pipette
• the temperature at which calibrations were made
• if the pipette is a “to deliver”(TD) or “to contain”(TC) pipette.
 Mohr pipet
 Uniform diameter
 Tapered delivery tip

 Graduations incised on stem only

 Self draining

 Long tips
 Serological pipette
 Long glass or plastic tube
 Uniform diameter

 Tapered tips

 Graduated till tip

 Blow out
 Bacteriologic pipette
 Glass or polypropylene
 Use for transport of bacterial

specimen esp. pus

 Sealed by flame and cotton plug

 Provide maximum sterility

 Volumetric pipette

 Deliver a single specific volume of liquid

 Shaped like rolling pins with a large belly, one blunt end, the neck, and
one tapering end, the tip.

 Accurate and precise

 Used in diluting standards,calibrators,QC material

 Specification on a volumetric
pipette
On a volumetric pipette, the specifications indicate
 how much liquid will be transferred if the liquid
is drawn up to the calibration line on the neck
 the temperature at which the calibration was
made
 whether it is a TD or TC pipette
 Ostwald folin pipette
 Mainly a blow out pipette

 Used with biological fluids viscosity

greater then water
 Pasteur pipette
 Also called dropper or dropping
pipette
 Do not have calibration marks

 Used to transfer solutions or fluids

without consideration of specific

volume
 Automatic pipettes

Types:-
1.Micropipette
2.Macropipette
 Micropipette

Micropipette measures the volume less then 1ml

 Macropipette
 Macropipette measures the volume
greater then 1ml
 Advantages of automatic pipettes
1.Time saving
2.Safely used
3.Ease of used
4.Stability
5.Precision
6.Lack of required cleaning
7.Disposable tips
 Types
1.Air displacement
2.Positive displacement
3.Dispensers
 Air displacement pipette

 Relies on piston to create suction to

draw sample into disposable tip
 Piston does not come in contact with

the liquid
 Forward pipetting

 Reverse pipetting
 Forward Pipetting
• Hold in vertical position.
• Depress the plunger to first stop position
• Immerse tip in the liquid.
• move plunger smoothly to the rest position.
• Place tip at an angle (10 to 45°) against the
inside wall of the receiving vessel.
• Depress plunger to the first stop position
• depress the plunger to the second stop position
(“blow-out” )
• Allow the plunger to move up to the rest
position.
 Reverse Pipetting
• Hold vertical position.
• Depress the plunger to the second stop position.
• Immerse the pipette tip in the liquid.
• Allow the plunger to move up smoothly to the rest
position.
• Place tip at an angle (10 to 45°) against the
inside wall of the receiving vessel.
• Depress the plunger to the first stop position.
Wait one second.
• If the pipette tip is not to be re-used, depress the
plunger to purge position over an appropriate
waste container and then eject the tip.
 Positive displacement pipette
 Operates by moving the piston in the
pipette tip
 Does not require different tips for

each use
 Dispensers

 Automatic pipette obtain liquid from

common reservoir and dispense it
repeatedly
 Calibration
 Types

 Gravimetric
 Spectrophotometric
 Gravimetric method
 Gravimetric measurement of dispense
aliquot of water with density correction
 Procedure
1. Water, weighing vial & pipet at temp room.
2. Record temp of pure water.
3. Weigh empty stoppered vial. (w )
v

4. Dispense sample of water in weighed vial.

5. Re-stoppered, reweigh and record.(w )
f
 • Refer to table “true capacity of glass vessels from the
weight of contained water when weighed in air” to obtain
correction factor(Ft)
• Calculate volume delivered(VD) as
VD, ml =(w –w )xFt
f v

 Example for 10ml pipet:

 w =31.9961g
f

 w =22.391g
v

 T = 24°C

 Ft = 1.003771

VD = 31.9961-22.0391x1.003771
= 9.9945 mL
Deviation or error
(10-9.9945) x 100 = 0.055%
10
0.1% error is acceptable.
 Spectrophotometric method
 Alternative to gravimetric
 Use compound that absorb light

 p-nitrophenol
 Procedure for calibration of 10µL
pipette
 Reagents requirement
• Na0H 0.01 mol/L
• p-nitriphenol 105mg/dL
Preparation of reagent
Dissolve 105mg of p-nitriphenol in 100mL deionized water
Preparation of dilutions
1. Reference dilution
fill three 250mL volumetric flasks with 0.01 mol/L NaOH.
Than add to each 1.0mL p-nitriphenol using different
pipet each time.
2. Test dilution
arrange 5 test tubes, using calibrated pipet add 2.5mL
NaOH to each.
to each add 10µL p-nitriphenol using test micropipet.
read absorbance of each reference and test dilution at
401nm in 10mm cuvet.
 p-nitriphenol in NaOH give reading 0.550
 Average the readings of three reference dilution
(A1) should be equal to 0.550
 Average the five test dilutions (A2)
 Calculation
VD (µL) = A2 x D x V
A1
Where
D is dilution of test dilution (1/251 here)
V is final volume in microliters of test
dilution (2510µL)

Error allowed= 0.5%-1%.

 Pipetting techniques
 Holds vertically
 Read at eye level

 Unrestricted flow

 Blow out after drain

 Lower part of meniscus at line level

 Precautions

 NEVER mouth pipette.

 Chipped and cracked

pipettes should be
replaced as they are
unsafe and may affect the
accuracy of
measurements.

 Hold the pipette by the

upper third of the tube
and keep the tip from
touching anything.
 Precautions
 Note upper meniscus in coloured
solution
 And lower meniscus in colouress

solutions
 Cleaning
Cleaning
 Clean dirty pipettes by
placing in soapy water
solution in a tray.
 Place disposable pipettes in a
cardboard holder.
 Do not leave pipettes on
counters or sinks. If you are
working with radioactive
materials be sure to dispose
pipettes in a properly marked
container.
 Handling of sterile pipettes

When using sterile

pipettes, be sure to use
proper sanitary techniques.
If you have a sterile
package of disposable
pipettes, tear only a small
corner of the package open
and push one pipette out of
this opening, then
immediately close the
package to prevent
contamination.
 If you are using
sterile pipettes in a
pipette canister,
place the canister on
its side, slide off the
cover, pull out one
pipette and replace
the cover
immediately.
 Transferring a precise volume of
liquid
A pipette bulb is used
to draw liquid up into
the pipette. There are
many types of pipette
bulbs.
3 are used routinely.
The most commonly
used is a rubber bulb.
 Other pipette bulbs

Other pipette
bulbs that are
often used
include the
Vedosa pipette
filler, seen on the
left, and the
Pipette Pumper,
on the right.
THANKS