EXERCISE 1

Using Micropipettors
Molecular biology laboratories make frequent use of a volumetric measuring device called a
micropipettor, which is made to deliver precise, accurate volumes in the range of 0.2 to 1000
microliters (µL) in the hands of a skilled operator. Hence, it is important to learn how to operate
this instrument properly, otherwise it will not deliver the correct volume.

Metric Conversions
Familiarize yourself with the metric units of measurement and their conversions. This
exercise uses volume measurements (base unit: liter), but the prefixes will also apply to mass (base
unit: gram) or linear measurements (base unit: meter).
1 mL = 0.001 L or 1/1,000 L 1,000 mL = 1 L
1 µL = 0.000001 L or 1/1,000,000 L 1,000,000 µL = 1 L
1 µL = 0.001 mL or 1/1,000 mL 1,000 µL = 1 mL

What Not to Do
1. Do not use without putting a tip on the pipettor. The barrel should never touch the
liquid; only the tip.
2. Do not lay down the pipettor when its tip contains liquid. Always keep it vertical!
3. Do not attempt to set the volume above or below the stated range of the device.
4. Do not let the plunger snap back after taking up or ejecting fluid – this damages the
piston. Maintain steady control of the plunger speed.

On Working with Micropipettors

1. Examine your set of micropipettors. We will be using four to five different pipettors,
which cover a range from 0.2 to 1,000 µL. These are color-coded by size.
• Clear or white tips – smallest volume (0.1 - 10 µL)
• Yellow tips – mid-range (20 – 200 µL)
• Blue tips – largest (100 – 1,000 µL)
2. Locate the working parts. Identify the (1) plunger on top
of the device, the (2) digital volume readout on the body,
the separate (3) tip ejector plunger, and the (4) shaft
that accepts the tip.
3. Insert a tip onto the shaft of the pipettor. Get the correct
tip to fit your pipettor. 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 the liquid.
4. Practice operating the plunger located at the top of the
device as follows:
a. Place your thumb on the plunger. Use the rest of your
fingers to grip the instrument with your index finger
resting below the pipettor hook.
b. With steady pressure, depress the plunger until you
Figure 1.1 Labelled
reach the ‘first stop’, that is until you can detect
parts of a micropipettor
resistance. After which, depress further, until you get
(Bio-Rad).
to the ‘second stop’. Source: http://www.bio-rad.com
 5. These pipettors are termed “variable volume” since it is possible to set the volume to a
value within the range of the instrument. Each pipettor has its particular range of
values.

Table 1.1 Maximum and minimum volume ranges of pipettors in the laboratory and
their corresponding tips.
Volume range (µL)
Pipettor Tip
minimum maximum
P2 0.2 2.0 colorless
P10 0.5 10.0 colorless
P20 2.0 20.0 yellow
P100 20.0 100.0 yellow
P1000 200.0 1000.0 blue

Locate the volume adjustor. It is knob located near the top of the pipettor. At times, this may
be the plunger itself. After which, 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.

Do not attempt to set the volume beyond the pipettor’s minimum or maximum value –
doing so damages the gears.

Figure 1.2 The digital display for Gilson Pipetman® micropipettors.

Forward and Reverse Pipetting Techniques

Depending on the type of liquid to be aspirated, one of the two pipetting techniques may be
used, forward or reverse.
A forward technique is used when working with liquids such as water or with aqueous/salt
solutions. I working with salt solutions, pre-wetting the tip with pure water before aspiration
increases accuracy.
Accurate volumes of volatile compounds like alcohol are also be drawn using the forward
technique, but should be pipetted rapidly to avoid evaporation. Acids and bases are also pipetted
using this technique, but may require filtered tips especially for the fuming ones.
Forward pipetting technique is done following the pattern shown in Figure 1.3. After
dispensing the liquid using the forward technique, the tip should be completely empty.
Reverse pipetting technique is used for pipetting liquids that are oily, with high viscosity or
a tendency to foam. Figure 1.4 shows the steps in reverse pipetting. After dispensing the liquid
using the reverse technique, a small amount of liquid should remain inside the tip. Dispose this
remaining liquid with the used tip.

Figure 1.3 Forward technique Figure 1.4 Reverse technique

I. Measure and weigh 1000 µL, 100 µL, and 20 µL of distilled water on a pre-weighed
microcentrifuge tube. Repeat three times, recording the mass on the table below.

Table 1.2 Comparison of the masses (g) of different volumes (µL) of distilled water.
Mass of drop in grams
Trial number
1,000 µL 100 µL 20 µL
1
2
3
AVERAGE

II. Measure and weigh 1000 µL, 100 µL, and 20 µL of mineral oil on a pre-weighed
microcentrifuge tube. Repeat three times, recording the mass on the table below.

Table 1.3 Comparison of the masses (g) of different volumes (µL) of glycerol.
Mass of drop in grams
Trial number
1,000 µL 100 µL 20 µL
1
2
3
AVERAGE

Questions

1. Compare your results from Table 1.2 with the other groups’ results. Are the micropipettors
used by the class accurate or precise?
2. What factors may affect the accuracy and precision of micropipettors?
3. What accounts for the differences in the average masses of 1,000 µL of distilled water and
1,000 µL of glycerol? 100 µL of distilled water and 100 µL of glycerol? 20 µL of distilled
water and 20 µL of glycerol?
4. How does pre-wetting the micropipette tip with pure water help increase the accuracy of
pipetting salt solutions?

References

AccuTek Laboratories. Guide to Pipetting [internet]. 2017. Ben-Gurion University of Negev IL: [cited
2019 November 6]. Available from:
https://lifewp.bgu.ac.il/wp/zarivach/wp-content/uploads/2017/11/GuideToPipetting.pdf
Additional exercises in using micropipettors [Internet]. 2009. Oklahoma City Community College
Biotechnology Program: [cited 2009 June 15]. Available from:
http://www.occc.edu/BBDiscovery/documents/Modules/Micropipetting_Exercise.htm.

Learning to use a micropipettor [Internet]. 2009. Oklahoma City Community College Biotechnology
Program: [cited 2009 June 15]. Available from:
http://www.occc.edu/BBDiscovery/documents/Modules/Micropipetting_Exercise.htm.

Skill building activity 1: Small volumes [Internet]. SF Base: [cited 2009 June 15]. Available from:
http://www.usc.edu/org/cosee-west/Jun07Resources/PipetteUsetraining.pdf.