(Compiled) Reviewer: Cell and Molecular Biology Laboratory

Exercise 1: Use of Micropipette

Biology involves measurement Uses:
 Different equipment/instruments/tools 1. Plunger – Used to take up and expel liquid
2. Eject button – Pushes the metal bar down which
MICROPIPETTES pushes the pipette tip off
 Precision instruments that are designed to 3. Friction Ring
accurately and precisely measure/transfer very small 4. Micrometer
volumes of reagents in the microliter range, as small 5. Shaft
as 10-4 mL or 0.10μL. 6. Volume Adjustment – Rotate to increase and
 Several brands of micropipettors are in the market decrease the set volume
o LabMate® 7. Volume Window
o HTL - Displays set volume
 Most used in chemistry, biology, forensic, - Place values shown depend on pipette size
pharmaceutical, and drug discovery labs, among
others.
 Manipulation of this instrument is basically the same
for all brands except that the ones that will be used
in the laboratory and discussion will be primarily for
the said brand to avoid confusion.
 Expensive and must be handled with care!

Classified according to USE

1. Fixed volume – Can be used only for a given volume
2. Variable – Can be used for a certain range of
volumes

Classified according to the number of channels 8. Pipette Barrel

- With the pipette tip, a tube filled with air
1. Single
- Pushing the plunger forces out a volume of air
2. Multichannel/repeat – Can be used with 8- or 12-
(this volume is set by the user)
channels at a time.
- Releasing the plunger lets that air back in
Parts UNLESS the pipette tip is in a liquid
>> takes up that volume of liquid instead of air
- Pushing the plunger forces out the liquid
9. Pipette Tip
- Sized to fit specific pipette
- Change tips every time there’s a risk of
contamination
- Must only be used once, discard after use

* Microcentrifuge tubes/microfuge
- Little plastic tubes which are essential to any lab
that handles small volume of liquid
- Used to spin small (2 ml or less) liquid samples at
high speeds (generally tens of thousands times g-
force)
- Used for all sorts of applications from sample
storage to running reactions and spinning

pg. 1 (Dumaguit, 2020)


CALIBRATION OF MICROPIPETTES
 Calibrated to deliver volumes within a certain
range
o P-2: Red & ranges from 0.2µL-2 µL
o P-20: Yellow & ranges frosm 2µL-20 µL
o P-200: Green & ranges from 20µL-200 µL
o P-1000: Blue & ranges from 100µL-1000 µL
D. Pipette different volumes of red dye onto the
 Always select the smallest volume pipette that will
transfer the volume
HOW TO USE A MICROPIPETTE
Before using the micropipette
 Sterilize the tips
 Autoclave – 121C, 15PSI, 15-20mins
Simulation Protocol
A. Set the volume on the P20 micropipette
1. Select the P20 micropipette
2. Select the volume setting to set the volume to 20
uL and then select “Save volume”
3. Select the P20 tip box to open it.
4. Move the P20 micropipette onto the P20 tip box
to attach a tip
5. Select the P20 tip box to close it
B. Draw up the red dye into the micropipette
1. Select the red dye solution tube to open it.
2. Select the micropipette and place it in the red
dye solution.
3. Draw up the red dye by first pushing down the
plunger until it reaches the first stop and slowly
releasing the plunger.
4. Close the red dye solution tube.
C. Pipette the red dye onto the blotting paper
1. Move the micropipette to circle A on the blotting
paper to dispense the red dye.
2. Dispense the red dye by holding down the
plunger until it reaches the second stop and
slowly releasing it.
3. Move the micropipette over the trash can and
press on the eject mechanism to eject the tip.
4. Select the P20 micropipette and place it back
into the rack.
blotting paper
1. In circle B on the blotting paper, follow the same
process as before to dispense 15 uL of red dye.
2. In circle C on the blotting paper, follow the same
process as before to dispense 7.5 uL of red dye.
3. In circle D on the blotting paper, follow the same
process as before to dispense 2uL of red dye.
PowerPoint Protocol
1. Rotate the volume adjustor to set the desired
setting.
 Caution: NEVER turn the indicator dial beyond
the upper or lower volume limits of the
micropipette! This could damage the piston
2. Firmly seat a proper-sized tip on the end of the
micropipettes.
3. Always hold the tube firmly between your thumb
and forefinger. Hold the tube nearly at eye level
to observe the change in the fluid level in the pipet
tip.
4. Micropipettes work by air displacement.
 For best control,
o grasp the micropipette in your palm
o wrap your fingers around the barrel
o work the plunger (piston)with the thumb.
 Depressing to the first stop
o measures the desired volume.
 Depressing to the second stop
o introduces an additional volume of air to
blow out any solution remaining in the
tip.
o “last drop’
a. Air displacement (Moving the piston displaces
air which moves the liquid.)
pg. 2 (Dumaguit, 2020)
 `
5. Drawing a sample to the tip
1) Positive displacement (Plunger directly
displaces the fluid)  Check if there is no air space at the very end
of the tip to avoid future pipetting errors; Held
 Each tip contains a disposable plunger like
vertically or keep the angle less than 20⁰
a syringe
o Caution: The most common -and serious
 The plunger directly displaces the fluid with
no error -operator error is depressing the plunger
to the second stop before filling the
 These are the most accurate type of
micropipette tip.
pipette
6. Dispensing a sample from the tip
 They are also the most expensive
 Slide the pipet out of the reagent tube with
the measurement plunger depressed, to avoid
sucking any liquid back into the tip.
7. Ejecting tip
 Tips are not reusable

pg. 3 (Dumaguit, 2020)

 PIPETTING TECHNIQUES
1. Forward pipetting technique

Materials/equipment
2. Repetitive Pipetting Technique  Micropipettors and tips
 Analytical balance
 Distilled water
 Parafilm or aluminum foil
Accuracy
3. Reverse Pipetting Technique  Refers to the performance of the micropipette
relative to a standard (the intended) value.
 Procedure
1) Turn analytical balance on.
2) Place parafilm or aluminum foil on the
4. Pipetting whole blood technique weighing balance. Press TARE.
3) Pipet 50L of dH2O into the parafilm. Record
weight up to 4 decimal places.
4) Press TARE again and repeat the procedure 5
times
5) Compute for average weight and percent error
(% error) using the following formula.

6) Repeat entire procedure using 100L and 200L

dH2O
Precision
 Provides information about reproducibility,
ACCURACY AND PRECISION without any reference to a standard. It reflects
 Micropipettes are precision instruments that are random errors that can never be entirely
designed to accurately and precisely transfer eliminated from a procedure.
volumes in the microliter range.  Procedure
 Manufacturers 1) This task will be performed 3 times.
o Determine the accuracy and precision of 2) Turn analytical balance on.
micropipettes by using them to transfer defined 3) Place parafilm or aluminum foil on the
volumes of distilled water to a drop that is then weighing balance. Press TARE.
weighed on an analytical balance. 4) Pipet 75uL of dH2O into the parafilm. Record
weight.
5) Press TARE again and repeat procedure.
6) Compute for average weight and standard
deviation.
Summary of Results
1) Collect the data.
pg. 4 (Dumaguit, 2020)
 2) Record results of accuracy in a table. Use average
values only.
3) Record results of precision in a table and show in
bar graph. Indicate error bars using the standard
error of the mean (S.E.) may be computed using
the following formula:
SD
SE=
√N
TIPS
 Never set a micropipette to a volume above or
below the maximum or minimum specified volume.
This may negatively affect the calibration of the
micropipette, leading to less accuracy when
transferring liquids.
 Always try and place the tip in the top layer of the
solution, about 2-5mm below the surface of the
liquid.
 Micropipettes should always be used within the
specified volume range to avoid negatively affecting
the calibration of the micropipette.
 Close the tip box to avoid contamination
 Always close the lids on the tubes to avoid
contamination.
 Always place the micropipette back on the rack
when you are finished using it.
 Never put the micropipette with liquid drawn up
down on the bench
 To remove the liquid from a micropipette, press the
plunger down to the second stop
(YOUTUBE) HOW TO PIPETTE CORRECTLY
2-button Pipette
 (1) Control button with two stops
 (2) Ejector button
2 Factors Influencing Accurate Liquid Uptake
1. Aspiration Angle
 Should be vertical, otherwise too much liquid
is aspirated
2. Immersion Depth
 The depth of the pipette tip inside the liquid
should be large as necessary but as small as
possible
 If the tip is immersed too deeply, additional
liquid can be easily aspirated due to the
capillary effect.
Achieve accurate and reliable pipetting results by:
a. Soft tip attachment
b. Vertical aspiration
c. Low immersion depth
d. Pre-wetting
e. Dispensing angle 20-45º to the vessel wall
Steps
1. Attach the tip
Note: Apply light pressure to insert the pipette
into the tip. Too much force or movement
strains the arm and the pipette.
2. Press the control button down to the first stop and
immerse the tip into the liquid.
3. Once the aspiration angle and immersion depth is
checked, slowly release the control button.
4. Press the control button down to the first stop
again, and aspirate once more.
5. Repeat 2-3 times to saturate the air in the tip (pre-
wet)
6. Dispense all liquid before aspirating the sample
liquid.
Note: The dispensing angle of 20-45º with
contact to vessel wall: this guarantees optimal
flowout of the liquid into the target vessel.
7. Press the control button down to the first stop,
then perform the blowout by pressing down to the
second stop.
Note: Blowout ensures that the tip is emptied
completely.
8. Ejection of the tip to a garbage bin by pressing the
ejector button.
(YOUTUBE) PIPETTE’S ACCURACY AND PRECISION
Mechanical pipette
 Almost the most important device inside any
laboratory procedure that deals with liquid transfer
 Used to transfer small volumes of liquid
Gravimetric Method
 A procedure to ensure the accuracy of the pipette
is a must in order to make sure that your pipette is
actually drawing and dispensing the right volumes
for accurate and reliable results
 Provided by ISO (International Organization for
Standardization)
 Involves three levels
pg. 5 (Dumaguit, 2020)
 a. 100%  Viscous, foaming, high-vapor-pressure, and
b. 50% other “problematic” liquids
c. 10%
of the maximum volume. WORKSHEET QUESTION WITH ANSWERS

List of Materials: Question 1. Indicate the parts of the micropipette

1. Containers labeled with numbers. This will serve as your review of
2. Balance the different parts of the micropipette.
3. Suitable Tips
4. Distilled Water
5. Thermometer
6. Barometer
Technical procedure:
1. Determine the correction factor (Z factor): the
correction factor using the temperature and the
atmospheric pressure of the laboratory.
2. Use ISO’s table to locate the Z factor (Ex. 23ºC, 95
kPa: Z= 1.0034)
3. Set up the balance.
4. Put the flask on the balance
Note: It is recommended to have water in the
flask prior starting the procedure
5. Blank to prevent flask weight from interfering with
your measurements
6. Put on a tip to a pipette to be calibrated
7. Draw the liquid solution using a pipette
Note: Recommended to draw and dispense
Question 2. What are the functions of each of the
multiple times to wet the tip before using it
labeled parts in the micropipette shown in Q1?
8. Start with 100% volume: dispense in the flask in the
balance AND THEN measure the weight. (Repeat 1. Plunger button - A button that is pressed to expel a
the procedure 9 more times) solution and is depressed to take up a solution.
9. Repeat the same procedure with 50% and 10% of 2. Tip Ejector button - A button which is pushed when
the maximum volume.
you want to remove a tip which then signals the tip
10. Multiply each single measurement by the
ejector to eject the disposable tip
correction factor (Z factor).
11. Identify the mean of each of the three maximum 3. Volume Adjuster - A mechanism that is rotated to
value increase or decrease the volume of the solution
12. Calculate the systemic and random error: that will be displaced .
4. Tip Ejector - The metal bar which pushes the
disposable pipette tip off when the tip ejector
button is triggered.

Question 3-8. Identify the measured volume on the

13. Determine the maximum permissible error for the micropipette.
pipette, given by ISO.

(YOUTUBE) AIR VS. POSITIVE DISPLACEMENT

Air Displacement
 Air pressure fails to expel viscous material
 Results in POOR accuracy and precision
Positive Displacement
 All material is expelled
pg. 6 (Dumaguit, 2020)

Question 11. Write your interpretation of the results
you got in levels 1, 2, and 3 of the simulation.
The actual and ideal results obtained from levels 1, 2
and 3 of the simulation were precise and accurate for
the reason that the researchers have followed the basic
guidelines and contexts of the procedure. This includes
selecting the appropriate micropipette for the given
volume (P2, P20, P200 and P1000), closing the tip box
and cover of the red dye solution every after use,
properly disposing the micropipette tip and replacing it
with a new one before proceeding to the next step, and
most importantly, proper handling of the plunger by
pressing it until the first stop for drawing up the liquid
and pressing it until the second stop in order to expel
the liquid. On the other hand, the predicted and actual
results had shown different values considering the fact
that no specific micropipette was used in order to blot
the liquid and only a reference sample was given for
estimating the size of the liquid in the predictions tab,
while in the actual experiment, the researchers have
utilized specific micropipettes which gave more
accurate and precise results.
Question 12. If the pipettor use is color blue, what is the
range of volume of liquid that may be delivered? if
yellow?
Blue = 100 - 1000 uL
Yellow = 2 - 20 uL
Question 13. Always place the tip in the top layer of the
solution, about 5-7 mm below the surface of the liquid.
False, it should be 2-5mm below the surface
Question 14. You may use the tip twice in
micropipetting a solution.
False, you cannot reuse a DISPOSABLE tip
Question 15. When are you more prone to commit
mistakes, when pipetting small volumes or larger
volumes? Why?
Scientists are more prone to commit mistakes when
pipetting small volumes (~0.2µL to 5µL) than larger
volumes for according to the ISO-8655 definition, the
maximum error limits are higher for small-volume
pipettes than large-volume pipettes (Krishnan, K. U.,
Adisesh, M., Navaneethakrishnan, L., & Manjunathan,
R., 2019). In the case of micropipettes, more
precautions are needed in order to control the
movement of the piston. When pipetting smaller
volumes, there is usually insufficient stream velocity to
overcome the effects of surface tension and the
associated capillary effect or the ability of any liquid to
flow out in narrow spaces at the dispensing tip opening
(Astle, 1998). Moreover, considering that the microliter
(µL) unit is 10−6 liters, small adjustments with regards
to factors such as temperature and the pipetting
technique used may also lead to a massive increase in
systematic and random error values (“How to Pipette
Small Volumes with Handheld Manual Pipettes”, 2018;
Depalma, 2017).
Question 16. Why is it important to push the plunger to
the first stop when drawing up liquid?
This is to ensure the correct volume of liquid is obtained
in the tip, and also to make sure that there are no extra
air pockets in the micropipette before submerging the
tip in the sample liquid, preventing contamination. If
the plunger was pushed to the second stop when
drawing up liquid, this would lead to the tip drawing up
too much liquid than the intended volume.
Question 17. What is the main purpose of disposable
tips on a micropipette?
Disposable Tips are used so that the micropipette can
dispense liquids in various volumes (e.g. 2uL, 20uL,
200uL), giving micropipettes use for various
experiments. Compared to serological pipettes, which
must be clean before and after use, the disposable
element of these tips prevent the need for constant
washing in between pipetting, allowing for a series of
dispensing while limiting the contamination risk of the
experiment's variables.
pg. 7 (Dumaguit, 2020)
Question 18. In doing the simulation, what are the
sources of errors that you encountered? Enumerate at
least 3.
1. Not double checking which micropipette is needed,
it is always advisable to use the smallest volume
pipette in relation to the amount of volume
needed.
2. Setting the incorrect volume of the micropipette,
which would lead to less or more samples that is
needed for an experiment.
3. Forgetting to dispose the used tip, which would
lead to contamination and would compromise the
results.
These are some of the sources of errors and the
probable consequences a person might encounter when
using micropipettes to transfer liquid samples.
Question 19. How will you know if the micropipette is
accurate? If precise? Support your answer. Use proper
citations.
One of the most known ways to measure micropipette
precision and accuracy is by testing it in controlled
environments. The following is the Rainin Instrument
Co. Inc factory-approved method by using gravimetric
analysis in testing micropipette accuracy and precision
with some notes from Lemon (2016). Before using the
micropipette, the following are important: (1.) Tips must
be sterilized before use; (2.) A special container is
commonly used when calibrating micropipettes, which
control evaporation, and; (3.) The micropipette shaft
must not be held throughout the procedure. One
method described is the "2 Volumes X 4 Weighing
Factory Method." On a calibrated balance (which must
be free from nuisances such as wall shaking, footsteps,
etc.), the container will be placed. Two volume sets will
be tested for any model of Rainin Micropipette, 10% of
the nominal capacity, and 100% of the nominal capacity
(e.g. if it is a P200 micropipette, it will be tested with
20uL and 200uL). Both of these will be done four times
each (hence 2 Volumes X 4 Weighing); this is to test
precision. Afterwards, the mean volume and standard
deviation of each volume setting will be calculated to
determine accuracy of the micropipette based on
standardized specifications (see Rainin p. 13). By using
this method, we will be able to determine if: (1.) The
micropipette delivers the same volumes consistently
and; (2.) If those volumes delivered are near the
accepted standard values.
EXTRA QUESTIONS
1. What do you think is the main purpose of this
simulation?
 To learn the correct way to handle a
micropipette in a laboratory.
2. Why is it important to press the plunger down to
the second stop when expelling liquid from a
micropipette?
 To ensure all the liquid in the tip is expelled.
3. Why is it important to push the plunger to the first
stop when drawing up liquid?
 If you go to the second stop, you will measure
too much liquid.
4. If the window on a P20 micropipette displays the
number “1 5 5” from to bottom, what volume will
be measured?
 15.5 microliters
5. If you do not change tips between solutions, what
may occur?
 You may cross contaminate the liquids you are
measuring.
6. At the end of a micropipetting procedure, a student
ended up with less than the volume they intended
to measure. What might have gone wrong?
 They may have drawn up the sample too quickly
and had air bubbles in the liquid
7. Micropipettes are commonly used in biotechnology
labs to
 Measure small volumes of liquid
8. Despite the different appearances, which part do all
micropipettes have in common? Select all that
apply.
 Plunger
 Barrel
 Volume adjustment knob
 Tip ejector
9. What volume is being measured by the pipette
shown below?
pg. 8 (Dumaguit, 2020)
  2.00 microliters
10. Which reasoning best explains your choice?
 The zeroes in the window are a different color
from the two, indicating that they come after a
decimal place
11. Which micropipette should you use to measure a
volume of 25 microliters?
 P200
12. Which of the reasons best justifies your answer?
 Micropipettes are named for the highest
volume that they can measure.

pg. 9 (Dumaguit, 2020)