Never let the plunger “snap back” because this can damage the piston.

Learning to Use a Micropipettor
Biotechnology laboratories make frequent use of a volumetric measuring device called a micropipettor, which
is made to deliver precise, accurate volumes in the range of 1 to 1000 microliters (mL) in the hands of a skilled
operator. Hence, it is important to learn how to operate this instrument properly because otherwise it will not
deliver the correct volume. Moreover, each device costs about $200 or more, and if misused, costly repairs or
replacement will be required. So take some time to learn how to do it right!
Materials: a set of micropipettors with adjustable volumes and colored liquid in microcentrifuge tubes.
Look over this checklist of DON’Ts.
BIG NO-NOs:
-Do NOT use without putting a tip on the pipettor; the barrel should never touch the liquid, only the tip.
-Do NOT lay down the pipettor when its tip contains liquid. Keep it vertical!
-Do NOT attempt to set the volume above or below the stated range of the device. Pay careful attention to the
volume scale and the range!
-Do NOT let the plunger snap back after taking up or ejecting liquid as this damages the piston. Maintain
steady control of the plunger speed.
Becoming Familiar with the Micropipettors
1. Examine your set of micropipettors. Typically, you will have three, but you may have four, and they should
cover the range from 0.5 or 1.0 to 1000 microliters (mL). Sometimes the pipettors (and also the accompanying
tips) are color-coded by size, clear or white for smallest, yellow for mid-range, blue for largest volume, but this
is not always the case. Look at your set for the presence or absence of color-coding (circle one).
2. Locate working parts. Pick up any one micropipettor. Identify (but do not manipulate yet) the plunger on
the top of the device, the digital volume readout on the body, the separate tip ejector plunger, and the shaft
that accepts the tip.
3. Insert a tip onto the shaft of the pipettor.
1. Get the correct tip to fit your pipettor. Color coding may help but is not always present (e.g. yellow tip
to pipettor with yellow top).
2. Press firmly to create an airtight seal between the barrel of the shaft and the tip so the instrument
can do its work in drawing up the precise volume of liquid.
Never insert a pipettor into liquid without a tip and this would ruin the piston that measures the precise
volume.
4. Practice operating the plunger on the top of the device as follows: (we are not involving liquid yet)
1. Place your thumb on the plunger. Use the rest of your fingers to grip the instrument with your
index finger resting in the slot.
2. Gently, with steady pressure, depress the plunger till you reach the “first stop” that is, till you can
detect resistance. Then depress further, till you get to the “second stop.” (One version of a
micropipettor might have a “third“ which serves to eject the tip, but most versions have only
the two stops with a separate tip ejector.)
3. Repeat a few times to get the feel of the stops and to practice steady control of plunger speed.
Never let the plunger “snap back” because this can damage the piston.
5. Setting the Volume. These pipettors are termed “variable volume” because you can set the volume to a
value within the range of the instrument. Each pipettor has its particular range of values. Examine your pipettor
set and jot down the values for the volume range of each pipettor:
1. smallest: _________________
2. mid-range: _________________
3. largest: _________________
Locate the volume adjustor: sometimes this is the plunger itself or sometimes it is a knob near the top of the
pipettor.
Next, look at the volume readout window; different brands of pipettors have different approaches to the readout
scale. Your pipettor may have a direct digital readout with the decimal place clearly indicated for ease of use or
it may have a micrometer readout setting that varies with the size of the pipettor (the Daiggers, for example). A
direct readout is easy, no need to explain, the decimal points are shown. But the micrometer readouts require
more attention. See appendix A for detailed directions if you are using one of these pipettor sets; tricky!
Do not attempt to set the volume beyond the pipette’s minimum or maximum value and doing so
damages the gears. That is why it is so important to know how to read the range and volume readout of each
kind of pipettor.
6. Working with Sample Liquid.
First arrange your materials on your workspace for easy access, and sit at a height so that you can rest your
elbow on the work surface.
•
•
•
•
Pick up a micropipettor with a tip in place. If it is the small-volume pipettor, set the volume to 4 mL. If it
is the mid-range pipettor, set the volume to 75 mL. If the largest volume pipettor, set the volume to 350
mL.
Obtain a microcentrifuge tube containing colored liquid sample. Hold the opened tube firmly in one
hand at nearly eye level to observe the movement of the liquid as you operate the pipettor.
Hold the micropipettor in a vertical position over the tube.
To withdraw sample from the tube:
•
depress the plunger to the first stop and hold. (This action displaces air equal to set volume from the tip
and hence, the term an air-displacement pipettor.)
•
Immerse the tip of the pipettor into the sample liquid. Do not push the tip into the very bottom of the
tube as this may restrict withdrawal of the sample liquid.
•
Slowly release the plunger to draw up the liquid into the tip.
•
Pause for a second or two to allow full intake of liquid into tip.
•
Remove tip from sample liquid. (Some recommend sliding the tip along the inside of the tube, others
say to pull the tip straight out).
•
Examine the liquid in the tip to be sure that there is no air space at the end of the tip. (There will be, of
course, an air space on top where the tip meets the pipettor shaft. This cushion of air protects the
pipettor itself from being contaminated by the sample.)
•
If there is an air space on bottom, eject liquid sample and repeat sample uptake steps till there is no
visible air space at the bottom of the tip.
•
To assure maximum accuracy, you can examine the outside of the tip and wipe off liquid droplets that
cling to outside, taking care to stay away from tip outlet to avoid removing liquid from the tip interior.
Always keep the pipettor in an upright position while there is liquid in the tip, so that the liquid does
not contaminate and damage the shaft of the pipettor.
•
•
•
•
•
•
•
To deliver sample into a new tube:
Insert the tip of the pipettor into the inside wall of the new tube. This creates a capillary effect to help
remove sample completely from the tip.
Slowly depress the plunger to the first stop, then on to the second stop to release the last bit of sample
liquid. Pause for a second with plunger in depressed position to allow time for full release.
Keep the plunger depressed while you remove the tip from the new tube out of the liquid (this avoids
withdrawing liquid back into the tip!)
Next, Release plunger gradually to resting position.
If you touch the tip into a liquid mixture, be sure to use a fresh tip for the next sample delivery to avoid
cross-contamination of reagents. Eject tip into discard container for later disposal.
Practice this technique with all three (or four) pipettors in your set to get the feel of each pipettor and get a
visual picture of volumes in the pipettor tips.
Credits: This activity was compiled from various sources, including DNA Science by Freyer and Micklos,
Basic Laboratory Methods for Biotechnology by Seidman and Moore, a USC website
www.usc.edu/org/cosee-west/Jun07Resources/PipetteUsetraining.pdf, and the FAO of U.N. website
www.fao.org/docrep/005/ac802e/ac802e05.htm .
Appendix A: Using the Daigger pipettors with micrometer volume readouts
Smallest: often 0.5-10 mL
Window shows:
0
1
1
1
0
0
Read 1.1 mL 10.0 mL
Mid-Range: 10-100 mL or 20-100 mL
Window shows:
0
2
6
1
0
0
Read 26 mL 100 mL
Highest Range: typically 100-1000 mL (still only 3 places)
Window shows:
0
1
1
1
0
0
Read 110 mL 1000 mL
0
1
1
So note this window on the left when shown on the smallest range pipettor will measure 1.1 mL while the same
window on the largest range pipettor will measure 110 mL. Tricky!
Moreover, on these Daigger pipettors, the bottom number in the window has hairline division marks which
represent fractions of that value and if your window on the largest pipettor looks like below, the volume is 224
mL while on the smallest pipettor that same window would indicate a volume of 2.24 mL !!! What would it be on
the mid-range? ______
0
2
|||2|||

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