Lab report 1:

Micropipetting procedure

By Xheni Kuqo

Biology 111
6 February 2023

Supervisor:
prof.dr: Ermira Hodo

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 Abstract
Micron-scale studies on emulsions have, to date, been
largely limited to the imaging of colloidal structures. In this
communication, micropipette techniques are introduced as
a progression beyond mere visualization: using small
suction pipettes, mechanical experiments are performed on
individual emulsion drops, from which interfacial
properties can be deduced. To demonstrate this technique,
the interfacial tension, emulsion stability and adsorption
characteristics are directly assessed at the surfaces of
micron-sized water droplets that are dispersed in crude oil.

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 1) What is the principle of micropipetting and
pipetting in general?
A micropipette is a common yet essential laboratory instrument used to accurately
and precisely transfer volumes of liquid in the microliter range. Micropipettes are
available in single-channel and multi-channel variants. While the single-channel
micropipettes are used in labs that perform research related to molecular biology,
microbiology, immunology, cell culture, analytical chemistry, biochemistry and
genetics, the multichannel micropipettes are recommended for ELISA (diagnostic test),
molecular screening, kinetic studies and DNA amplification.

2) What types of pipettes are there and why do we have different pipette usage in
laboratory procedures?
Micropipette Classification Based on Working Principle

1. Air Displacement Micropipette:

The air displacement principle used by micropipette is made up of a piston that can aspirate and disperse liquid
samples while moving an air pocket upwards, downwards, and back. The internal mechanism of the pipette is
not in direct contact with liquid or sample. To pull the liquid into and out of the pipette, an irreplaceable
pipette tip is used.

2. Positive Displacement Micropipette:

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The piston in these micropipettes is in direct contact with the sample or material. The disposable tip of a
positive displacement micropipette is an extremely small syringe that is made up of a capillary and a piston
(which is movable inside) that can directly move liquid.

Micropipettes are classified into two types based on their operating

mechanism

1. Mechanical Micropipette: A spring piston mechanism is used to

operate micropipettes by hand.

2. Electronic Micropipette/ Automatic Micropipette/ Digital

Micropipette:

Typically, the electronic micropipette is automated and hence can also be called as automatic micropipette or
digital micropipette. Single-touch buttons are used for liquid aspiration and dispensing, rather than manually
pressing and depressing the plunger. Electronic pipettes frequently allow users to create customized programs
for the device, allowing the pipettes to be adapted to various application requirements.

Micropipette classification based on the number of channels

Micropipettes are classified into two types based on the number of channels:

1. Single Channel Micropipette: A micropipette with a single channel is the one that can be used to
aspirate or disperse the liquid.

2. Multi-Channel Micropipette: This type of micropipette has several channels for aspiration or
dispersing liquid. The most commonly used multichannel micropipette models are the 12 channel, 8
channel, and 16 channel. When dealing with large quantities of samples, multichannel micropipettes
can reduce the work of a single channel micropipette.

Volume/Capacity: Micropipettes are classified into two types based on their volume/capacity.

1. Micropipette with Fixed Volume

The amount of liquid to be dissipated or aspirated in a fixed-volume micropipette is fixed. They are used when
the same volume of liquid needs to be dispersed several times.

2. Volume Variable Micropipette or adjustable Micropipette

The volume range of the micropipette is limited and maximum. The amount of liquid aspirated and dispensed
can be adjusted (within the volume range) to meet the needs of the individual.

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 Areas to where micropipettes can be used.

Micropipettes are commonly used in microbiology, chemistry, and medical labs to ensure accurate and precise
specimen transmission.

The following are the most important fields in which a micropipette can be widely used:

 Pharmaceuticals
 Wellness and healthcare
 Food and Drink
 Environmental Surveillance
 Institutes of Higher Learning and Research
 Used in kits for Diagnostic testing.
 Widely used in Life Sciences
3) What are the parts of micropipette? What is each used for?
Components of a micropipette:

Micropipettes are available in different

designs and sizes. However, there are
certain components that are basic and
common to all micropipettes. These
include the plunger, digital display, tip
cone, tip ejector and grippy. Certain
micropipettes are provided with a
calibration tool and a micropipette stand
as an accessory.

Plunger:
The plunger performs the following two functions-
Volume adjustment:
Rotate the plunger clockwise/ anticlockwise to decrease/ increase the volume setting. A distinct click
sound at every volume change ensures perfect volume setting and prevents any accidental volume change.
Liquid aspiration/ dispensing:
Press and depress the plunger to aspirate or dispense liquid.
Tip Ejector:
The internal mechanism of the micropipette does not come in direct contact of the sample/liquid. Instead,
a disposable micropipette tip is used to draw the liquid into and dispense from the micropipette. So in

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order to allow the safe, effortless and quick ejection of tips, micropipettes are provided with a tip ejection
system. The tips can be easily removed from the micropipette by pressing the tip ejector button.
Volume Display:
This shows the volume of the liquid to be aspirated or dispensed.
Tip Cone:
The tip cone provides fitment to the tips. A pipette with a universal tip cone is preferred as it enhances the
compatibility of the instrument with most of the standard tips.

4) Explain in order the how you do micropipetting as a procedure while taking liquid.

Position 1:In this, the micropipette is at rest position.

Press fit a tip to the micropipette without directly touching the tip.
Position 2: In this, the plunger is depressed till the first stop.
To aspirate the liquid in the tip, press the plunger to the first stop. Immerse the pipette tip vertically in the
liquid.
Release the plunger
Slowly release the plunger while the tip is immersed. The liquid will be aspirated into the pipette tip.
The liquid is filled in the tip as per the preset micropipette volume.
Position 3: To dispense the liquid, place the tip on the inner wall of the receiving vessel at a steep angle.
Slowly press the plunger to the first stop to dispense the liquid.
To empty the tip completely, press the plunger to the second stop.
Wipe the tip on the inner wall while taking the tip out of the vessel.

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5) Explain the possible errors while micropipetting.
 Dried out pipette tips. Pipette tips should be made wet before use by aspirating and dispensing a
liquid sample at least three times before beginning the pipetting procedure.
 Incorrect solution temperature. Some solutions, like a reagent or distilled water, are temperature
sensitive, and others are easily affected by temperature.
 Using the wrong pipette tips. Tips that aren’t compatible with your pipette can cause the solution to
leak into the pipette itself, resulting in liquid volume loss and damage to the pipette itself.
 Wiping or dragging the pipette tip. Friction between the tip and other surfaces results in loss of
liquid volume.
 Pipetting at incorrect angles. Your pipette should be vertical when aspirating.
 Working too quickly while pipetting is another common pipetting error. Let the liquid settle into
the pipette tip for a second or two after aspirating.

How is a micropipette calibrated?

Micropipette calibration is a very important part of any laboratory’s routine for accurate and precise
pipetting results. To ensure the accuracy of sampling, it is important to check the pipette calibration every
month or in a few months’ gap. Executing a proper micropipette calibration according to ISO 8655
standards requires special ambient conditions. Factors such as air pressure, humidity, temperature and
even altitude affect the pipetting results. The below text explains the required material or equipment and
all the steps that you need to perform and check the calibration of a micropipette.
Here is a list of things that you will need in order to calibrate a micropipette:
 Micropipette that needs to be calibrated & tips
 Distilled Water
 Clean Beaker
 Thermometer
 Distilled Water Density Chart with Temperature
 Semi-Micro Balance with a readability of at least 0.0001 g

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  Notebook to write down the results
 Calculator

How to calibrate a pipette using a balance:

1. Leave the distilled water out for 15-20 minutes to ensure a
consistent temperature.
2. Use the thermometer to measure the temperature of the distilled
water.
3. Place empty and clean the beaker on the balance.
4. Take the beaker’s weight.
5. Ensure the micropipette is clean and ready to function.
6. When filling the micropipette up to the volume that will be
calibrated (1ml, for example) with the distilled water, make sure
air bubbles are not created in the flask and in the micropipette.
7. Dispense the distilled water in the beaker using the micropipette.
8. Use the balance to measure the distilled water’s weight.
9. Repeat the process 5-10 times at least and note each measurement.
10. Use the formula V = W * Z to calculate the volume dispensed by the pipette.
W is the weight of the distilled water
Z is the “conversion factor based on the density of the water” and
V is the calculated volume of dispensed water
The temperature of the water determines its density, which is why it is important to keep the
water temperature as constant as possible. Skip if you have the software that automatically does
that.
11. Average the results. If you did 10 tests, divide the sum of the 10 tests by 10.
12. The average weight of the distilled water is the W in the equation. You can now solve the
equation manually, or use the software.
13. The next step is determining the pipette’s accuracy, manually or via software. If you have to do it
manually, use the formula A = 100 x Vavg/V0
A stands for Accuracy.
Vavg is the average calculated volume.
V0 is the value assigned for the pipette to dispense.
It is recommended that the value should be between 99 and 101%.
14. Record the results. If the micropipette is properly calibrated, you can use it. If the results are
consistently wrong, do not use the pipettes or fix them before using it again.

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How to sterilize a micropipette?
 The micropipettes can be sterilized by autoclaving them at 121°C (252°F) and 15 PSI for
a duration of 15-20 minutes.
 No special preparation is
needed.
 You may use steam sterilization
bags if needed. After autoclaving, the
micropipette must be cooled to room
temperature for at least two hours. Before
pipetting, make sure that the instrument is
dry.
 It is recommended to check the
calibration after every sterilization cycle to achieve the best possible precision and accuracy.

What are micropipette starter kits? How are they beneficial

during lab setup?
A micropipette starter kit is the most convenient and cost-effective collection of pipetting essentials. It
includes a set of micropipettes with different volume ranges, micropipette tips that can be used in given
micropipettes and some other accessories for an efficient sampling process. A micropipette starter kit is
beneficial as it saves the time of selecting the micropipette and the compatible tips which can be used
with it. Microlit Micropipette Starter Kit combines 4 single channel pipettes to cover the complete
pipetting range (0.5-10ul pipette, 10-100ul pipette, 100-1000ul pipette, 1-10ml pipette). The micropipette
kit also includes a sleek carousel stand and boxes of compatible tips for each pipette so that one does not
have to go looking for the right tips.

When is micropipette calibration required?

In order to maintain the quality of sampling results, the micropipettes must be in good condition and
properly calibrated. Normally, the interval at which a micropipette needs to be calibrated depends on
many factors:
1. Frequency of pipette use
2. Type of liquid dispensed by the pipettes
3. Handling & care of pipette
Pipette applications that require superior accuracy also demand more frequent calibration

Regulations and standards published by organizations such as the FDA and ASTM International provide
minimum requirements to ensure the quality of laboratory testing results. Regulations specify that all
laboratory instruments used in sampling & production including micropipettes must be routinely
calibrated at suitable intervals.
In the same series, the Clinical and laboratory standards institute has provided the guidelines for single
and multi-channel micropipettes that specify these micropipettes must be calibrated every 3 to 6 months.
And a minimum of two volumes must be tested with 10 replicas at both nominal and lowest settings.
Establishing an appropriate calibration frequency minimizes the chances of incorrect liquid delivery in the
laboratory and ensures traceability, accountability and confidence in the results.

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 Applications/fields where micropipettes can be used:
Micropipettes are generally used in microbiology, chemistry, and medical testing laboratories for the
accurate and precise transfer of samples. While the single-channel micropipettes are used in labs that
perform research related to molecular biology, microbiology, immunology, cell culture, analytical
chemistry, biochemistry and genetics, the multichannel micropipettes are recommended for ELISA
(diagnostic test), molecular screening, kinetic studies and DNA amplification.
Some of the major fields where micropipette is widely used are:
6) Pharmaceuticals
7) Health Care
8) Food & Beverage
9) Environmental Monitoring
10) Academic & Research
Institutes
11) Diagnostic Kits
12) Life Sciences

Benefits of using a
micropipette
A micropipette is a reliable device to dispense precise quantities of liquid. It increases the performance of
the testing efficiently. Some other benefits of using a micropipette are listed below:

 Time-Saving
 Ergonomics
 Accuracy & Precision
 Ease of Use
6) Explain any possible mistake you did while micropipetting and
how you resolved it.
I followed all the instructions, so I don't think I did anything wrong.

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