Automatic Micropipettes

Successful completion of this learning module
requires that you be able to do the following:
1. Select the proper automatic micropipette to transfer a
specified volume of sample.
2. Set a specified volume on the pipette volume
indicator using the volume adjustment knob.
3. Read a digital volume setting in both microliter (μL)
and milliliter (mL) units.
4. Demonstrate the correct technique to accurately
transfer a sample of a stock solution to another vessel.
Introduction
• Automatic micropipettes are used in biological,
biochemical, and microbiological teaching and
research laboratories to accurately transfer small
liquid volumes.
• Glass pipettes, which are used in chemical
laboratories, are not highly accurate for volumes
less than 1 milliliter (1 mL).
Introduction
• The most common brand of automatic micropipette
encountered in UM-D laboratories is the
“Pipetman”
– It’s a continuously
adjustable digital
pipette.
– It can be set to
transfer any volume
within the individual
pipette’s volume range.
Pipetman Models Available
The complete set of seven Pipetman models covers
the volume range from 0.1 μL to 5,000 μL (5 mL).
Pipetman Model
Range Possible
(μL)
Recommended Range
(μL)
Smallest μL
increment
P-2
0 to 2
0.1 to 2
0.002
P-10
0 to 10
0.5 to 10
0.02
P-20*
0 to 20
2 to 20
0.02
P-100
0 to 100
10 to 100
0.2
P-200*
0 to 200
50 to 200
0.2
P-1000*
0 to 1000
100 to 1000
2.0
P-5000
0 to 5000
500 to 5000
2.0
* Most common models in teaching labs
You will most often use the P-20, P-200, and P-1000
models which are recommended for volume transfers of
2 to 20 μL, 50 to 200 μL, and 100 to 1,000 μL (1 mL),
respectively.
What is a microliter (μL)?
• This is a good point at which to review the units
for small volumes of liquids
– A microliter (μL) is one-millionth of a liter
– Remember that a liter is a larger volume, such as half
the volume of a typical 2-liter bottle of pop
1 microliter (μL)
= one-millionth of a liter
= 0.000001 liter (L)
= 1 x 10-6 L
What is a milliliter?
• Milliliters (mL) are one-thousandth of a liter.
Most glass pipettes transfer volumes in milliliters.
1 milliliter (mL)
= one-thousandth of a liter
= 0.001 liter (L)
= 1 x 10-3 liter (L)
• A milliliter (mL) contains 1,000 microliters (μL)
• Given the example volume relationships below,
confirm your understanding by completing the
missing volumes in Problem Set on the next
slide. Answers are at the end of this learning
module.
1 milliliter (mL)
= 1000 microliters (μL)
0.15 mL
2.37 mL
= 150 μL
= 2,370 μL
Problem Set:
?? ml
= 5.26 μL
?? mL
= 91.5 μL
3.58 mL
= ?? μL
0.069 mL
= ?? μL
Pipette Operation
The automatic pipette operates via a
stainless steel piston which displaces air to
create a vacuum (analogous to an
eyedropper) that will draw a liquid sample
into the disposable plastic tip.
Pipette Operation
When you press down on the plunger button,
the piston is moved down, which forces air out
of the pipette. When the plunger button is
allowed to go back up, the vacuum created in
the pipette draws liquid up.
Pipette Operation
• The plunger button should
be depressed using your
thumb.
• The pipette then draws up
the liquid sample when the
plunger button is slowly
released.
• The plunger button can then
be depressed again to
dispense the liquid sample
into a receptacle.
Pipette Operation
• When operating the plunger, two points of
resistance are met.
– The first stop is the point when the piston reaches the
calibrated volume on the dial.
– The second stop is used to fully expel the sample
volume into a receiving vessel (equivalent to blowing
out a glass pipette).
Start Depressing
Plunger
First Stop
Second Stop
• Polypropylene
disposable tips fit
snugly on the end of
the plastic shaft.
• Using the tip ejector
button, tips may be
removed from the
pipette without
having to touch any
dangerous materials
on the tips.
• The tip ejector button
engages the tip ejector
arm which pushes the
tip off the end of the
pipette.
• The volume adjustment
knob includes a stainless
steel micrometer to dial
in the exact amount of
liquid to be transferred.
• That ‘dialed in’ volume
is displayed in a side
window, the digital
volume indicator (not
evident in this photo).
Setting the Volume
Setting the automatic micropipette volume involves:
1. Selecting the proper pipette to use.
 You must choose the pipette whose
capacity is closest to, but more than, the
desired volume.
 The desired volume should be within the
recommended range for that pipette, and
ideally at the mid- to high- end of the
pipette’s recommended range (most
accurate).
Setting the Volume
Setting the automatic micropipette volume involves:
1. Selecting the proper pipette to use.
For instance, to transfer 2.33 μL of a
liquid, the P-10 Pipetman is the best
choice since 2.33 μL is included in its
recommended volume range and 2.33 μL
is in the middle of the pipette’s
recommended range.
Setting the Volume
Setting the automatic micropipette volume involves:
1. Selecting the proper micropipette to use.
2. Rotating the volume adjustment knob until the digital
volume indicator reads the desired volume.
Volume Adjustment Knob
Digital
Volume
Indicator
Principle Underlying Volume
Adjustment in the Micropipette:
Volume Adjustment Knob
Digital
Volume
Indicator
• Turning the volume
adjustment knob
adjusts the piston
displacement to
draw up the exact
volume of liquid.
1. To set a volume, hold the pipette in one hand and turn the
volume adjustment knob with the other hand until the
correct volume shows on the digital indicator.
2. There is a friction ring on the knob which "locks" the
volume.
3. Proper adjustment involves turning the knob 1/3 revolution
beyond the desired setting and then slowly back to the
exact setting to prevent reduced accuracy from
"mechanical backlash".
a. If you pass the desired setting by mistake, turn the dial 1/3
revolution above the desired setting and reset the volume
slowly.
• The volume indicator (the window on the pipette) consists
of three numbered dials and is read from top to bottom.
The three digits indicate the microliter volumes selected
and are colored black or red.
– All black digits indicate WHOLE microliter amounts.
• Examine the volume indicators on the pipettes
– On the P-20 pipette, the red digits show tenths or hundreds of
a microliter.
– On the P-1000 pipette, the red digits are in the top box; and
they read either 0 (for all volumes less than 1000 μL) or 1 (for
volumes approaching the maximum 1000 μL).
P-20
P-200
P-1000
• Significant digits vary for each of the pipettes.
– The bottom dial includes vernier markings
• The P-20 can record to the nearest hundredth of a
microliter (0.01 μl)
• The P-200, to the nearest tenth (0.1 μl)
• The P-1000, to the nearest microliter (1 μl)
P-20
P-200
P-1000
Setting and reading the volume indicator is
often confusing to students. For instance,
notice the volumes for the following dial
settings:
P-20
Top
Middle
Bottom
μL
mL
1
2
5
12.5
0
9
8
9.8
P-200
1
2
5
125
P-1000
0
9
8
98
0
2
5
250
0.250
0
9
8
980
0.980
Study and practice dialing in the volumes below
to give you confidence in handling and reading
automatic micropipettes.
P-20 = 6.86 μL
P-200 = 132.4 μL
P-1000 = 262 μL
Step-by-Step Recap of Liquid Transfer
Using An Automatic Micropipette
1. Set the volume
a. Hold the pipette in one hand while turning the volume
adjustment knob
b. Turn 1/3 revolution above the desired volume setting
and then slowly back down to the desired volume
setting for optimal accuracy
Step-by-Step Recap of Liquid Transfer
Using An Automatic Micropipette
1. Set the volume
2. Attach a disposable tip
a. Press a disposable tip of proper size firmly to the pipette
shaft
Step-by-Step Recap of Liquid Transfer
Using An Automatic Micropipette
1. Set the volume
2. Attach a disposable tip
3. Depress the plunger to the
first stop
a. Using your thumb, depress the
plunger gently and smoothly
until resistance is felt. That
point of resistance is the first
stop.
Step-by-Step Recap of Liquid Transfer
Using An Automatic Micropipette
1.
2.
3.
4.
Set the volume
Attach a disposable tip
Depress the plunger to the first stop
Immerse tip in sample & Draw up
sample
a. Immerse the disposable tip into the
sample liquid to a depth of 3 – 4 mm
below the liquid surface.
b. Allow the plunger button to return
slowly & smoothly to the fully
extended up position.
Step-by-Step Recap of Liquid Transfer
Using An Automatic Micropipette
1.
2.
3.
4.
Set the volume
Attach a disposable tip
Depress the plunger to the first stop
Immerse tip in sample & Draw up
sample
a. Immerse the disposable tip into the
sample liquid to a depth of 3 – 4 mm
below the liquid surface.
b. Allow the plunger button to return
slowly & smoothly to the fully
extended up position.
NEVER
LET THE
PLUNGER
SNAP
BACK UP!!
Step-by-Step Recap of Liquid Transfer
Using An Automatic Micropipette
1.
2.
3.
4.
5.
Set the volume
Attach a disposable tip
Depress the plunger to the first stop
Immerse tip in sample & Draw up sample
Pause
a. Wait a few seconds to ensure that the full volume of
sample is drawn into the plastic tip
Step-by-Step Recap of Liquid Transfer
Using An Automatic Micropipette
1.
2.
3.
4.
5.
6.
Set the volume
Attach a disposable tip
Depress the plunger to the first stop
Immerse tip in sample & Draw up sample
Pause
Withdraw the pipette from the sample
a. No liquid should remain on the outside of the tip.
b. Wipe away outside droplets with a Kimwipe, but never
touch the end of the tip or you may absorb part of
your sample!
7. Dispense the sample
a. Touch the tip to the side wall of
the receiving vessel
b. Depress the plunger to the
FIRST STOP
c. Pause for at least one second
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
Step-by-Step Recap of Liquid Transfer
Using An Automatic Micropipette
7. Dispense the sample
8. Withdraw the pipette from the receiving vessel
a. With the plunger fully depressed, withdraw the pipette
from the receiving vessel carefully sliding the tip along
the wall of the vessel
Step-by-Step Recap of Liquid Transfer
Using An Automatic Micropipette
7. Dispense the sample
8. Withdraw the pipette
9. Release plunger
a. Gently allow the
plunger to return to the
up position. Do not
allow the plunger to
spring back up!
Step-by-Step Recap of Liquid Transfer
Using An Automatic Micropipette
10. Discard the tip
a. Discard the tip by depressing the tip ejector button
Do you know all the steps?
1. Set the volume
2. Attach a disposable tip
3. Depress the plunger to the first stop
4. Immerse the tip in the sample and draw up sample
5. Pause
6. Withdraw the pipette from the sample
7. Dispense the sample into the receiving vessel
8. Withdraw the pipette from the receiving vessel
9. Release the plunger
10. Discard the tip
PIPETTING CONSIDERATIONS
• Accuracy and Precision:
– Accuracy means the closeness with which the
dispensed volume approximates the volume set on
the pipette.
– Precision is the "scatter" or reproducibility of
individual measurements of the same volume.
• The table below shows both absolute and relative (absolute as
a percent of the actual measurement) accuracy and precision
of the most common Pipetman models:
Pipette Model
Volume
(μL)
Relative
Accuracy
(%)
Absolute
Accuracy
(μL)
Relative
Precision
(%)
Absolute
Precision
(μL)
P-20
2
5
0.1
2
0.04
10
1
0.1
0.5
0.05
20
1
0.2
0.3
0.06
50
1
0.5
0.4
0.2
100
0.8
0.8
0.25
0.25
200
0.8
1.6
0.15
0.3
100
3
3
0.6
0.6
500
0.8
4
0.2
1
1000
0.8
8
0.13
1.3
P-200
P-1000
PIPETTING CONSIDERATIONS
• Accuracy and Precision:
– Relative accuracies are generally about 1% or less
– Relative precision is less than 0.5% except when
transferring the smallest recommended volume for a
given pipette model
– Using the pipettes to transfer volumes which are below
the recommended range will introduce large errors
PIPETTING CONSIDERATIONS
• Accuracy and Precision:
– Air bubbles in the tip can greatly reduce
pipetting accuracy
• If an air bubble is trapped within the tip
during intake, dispense the sample into the
original vessel, check the tip immersion
depth, and pipette more slowly.
• If an air bubble appears a second time,
discard the tip and use a new one.
PIPETTING CONSIDERATIONS
• Accuracy and Precision:
– Never lay the pipette on its side or invert the pipette
when liquid is in the tip
PIPETTING CONSIDERATIONS
• Accuracy and Precision:
– Use consistent speed and smoothness when you press
and release the plunger
– Use consistent pressure on the plunger at the first stop
– Use consistent and sufficient immersion depth for the
pipette tip
• Accuracy and Precision:
– Maintain the pipette in nearly vertical positioning (less
than 20o) throughout use
PIPETTING CONSIDERATIONS
• Pre-Rinsing Recommended:
– When pipetting a solution (e.g., serum or other proteincontaining solutions, organic solvents), a significant film
may be retained on the inside of the wall of the disposable
tip, resulting in an excessive volume error
– Since this film remains relatively constant in successive
pipettings with the same tip, excellent precision may be
obtained by discarding the first filling of the tip and
refilling the tip a second time or more times to use as the
sample(s)
– This procedure is recommended when critical
reproducibility (precision) is required
PIPETTING CONSIDERATIONS
• Pipetting Liquids of Varying Density:
– The Pipetman is calibrated using water. If a solution is used whose density is
far different from water's, transferred volumes will be incorrect.
• For example, the P-20 will deliver only 8.5 mL of a dense CsCl solution when set to
deliver 10 mL.
– You can compensate for solutions of higher or lower density by setting the
volume adjustment higher or lower, respectively, than the required volume.
– However, the amount of compensation must be determined empirically. This
involves changing the volume setting, weighing the dispensed sample, and
calculating the volume of sample from its experimentally measured density
(volume of sample = mass/density).
• Very dense or viscous liquids, or liquids with a high vapor pressure, may
not be suitable for the Pipetman which operates using an air displacement
(vacuum) method.
– Instead, positive displacement pipets (MICROMAN or EPPENDORF brands)
are recommended.
PIPETTING CONSIDERATIONS
• Temperature Considerations:
– You can measure warm or cold liquids with good
precision by using a consistent pipetting rhythm
• A consistent rhythm will help to minimize any differences in
heating or cooling effects within the pipette
• Do not pause between the aspirating and dispensing phases
of sample transfer, and do not take the time to pre-rinse
Once you feel comfortable doing the following:
1. Select the proper automatic micropipette to transfer a
specified volume of sample.
2. Set a specified volume on the pipette volume
indicator using the volume adjustment knob.
3. Read a digital volume setting in both microliter (μL)
and milliliter (mL) units.
4. Demonstrate the correct technique to accurately
transfer a sample of a stock solution to another vessel.
Obtain and complete a posttest from a Science
Learning Center Student Assistant. Good luck!
PROBLEM SET ANSWERS:
0.00526 mL
0.0914 mL
3.58 mL
0.069 mL
= 5.26 μL
= 91.5 μL
= 3,580 μL
= 69 μL