Chapter 3

Operation and Functional

Verification of Automated
Micropipettes
Learning Objectives
At the end of this topic students will be able to;
Describe the key components of a micropipettes
Discuss operation feature of micropipette
Describe different pipetting techniques
Describe methods used to verify pipette calibration
Calculate statistical data to verify pipette accuracy and
precision
Chapter Outline

1. Introduction
2. Operating procedure
3. Pipetting techniques
4. Pipette troubleshooting
5. General working remarks
6. Principles of inspection and calibration
Introduction to Automated
Micropipette
Automatic pipettes are used to accurately transfer small
liquid volumes
Glass pipettes are not highly accurate < 1 milliliter (1
mL), but the automatic pipettes are both accurate and
precise for small volumes
These pipettes are called automatic because
Dont require a suction bulb or adjusting the volume required by
viewing the meniscus.
The volume is automatically drawn up and dispensed using a
plunger button.
Introduction, Contd..
Parts of Automatic Micropipettor
Operating the Micropipette
Step 1: Set the Volume
Volume Adjustment Knob:
Pipettors 3 Volumes:

Digital Volume Indicator:

 Operating the Micropipette, Contd.
Step 1: Read the Volume
How to Read the Volume Indicator:

(a): P-20 Model (b): P-200 Model (c): P-1000 Model

6.86 m l = 0.00686 132.4 m l = 0.1324 262 m l= 0.262
or 6.86 x 10-3 ml or 1.324 x 10-1 ml or 2.62 x 10-1 ml
Operating the Micropipette, Contd.
Setting Adjustable Volume

Course and fine setting

of the pipette line.
Because the course and
fine settings are
separate, it is not
necessary to "wind" the
pipette as would be
done on a screw-type
adjustment
Operating the Micropipette, Contd.
Step 2: Attach the Disposable Tip

Example of tip sizes:

Blue tips for 1000 uL capacity
and yellow tips for 250 uL
capacity

Attaching the
disposable tip
Operating the Micropipette, Contd..
Step 3: Depress the Plunger to the First Stop
Step 4: Immerse Tip in Sample
Step 5: Draw up the sample
Step 6: Pause
Procedural Remarks
Hold the pipette vertically
when aspirating
Place tip just below the
sample
Avoid air bubbles
Wait a few seconds to
ensure that the full volume
of sample is drawn into the
plastic tip
Operating the Micropipette, Cont.
Step 7: Withdraw the Tip
Remove the tip from the sample liquid. No liquid should
remain on the OUTSIDE of the tip. Wipe away any droplets
on the outside of the tip with a lint-free tissue, but only wipe
droplets from the side of the tip. NEVER TOUCH THE TIP
OPENING or you may absorb part of your sample.

Proper Droplet Removal WRONG Droplet Removal

Operating the Micropipette, contd.
Step 8: Dispense the Sample
Operating the Micropipette

Step 9: Withdraw the Pipette

Step 10: Release the Plunger
Step 11: Discard the Tip

Press ejector button to discard tip.

Pipetting Techniques
Commonly, there are two types of pipetting techniques;
Forward pipetting
Reverse pipetting
Forward pipetting is recommended for aqueous solutions
like buffers, diluted acids or alkalis
Reverse pipetting is recommended for small volumes
Avoids the risk of splashing, foam or bubble formation
Is used for solutions with a high viscosity or a
tendency to foam
Pipetting Techniques, contd..
Forward pipetting

1. Press the operating button to the first stop.

2. Dip the tip into the liquid to a depth of 1 cm, and slowly
release the operating button. Withdraw the tip from the
liquid, touching it against the edge of the reservoir to
remove excess liquid.
3. Dispense the liquid into the receiving vessel by gently
pressing the operating button to the first stop. After
one second, press the operating button to the second
stop. Remove the tip from the vessel, sliding it along
the wall of the vessel.
4. Release the operating button to the ready position.
Pipetting Techniques, contd..
Reversing Pipetting

1. Press the operating button to the second stop.

2. Dip the tip into the liquid, and slowly release the operating button
[wait 1-2 seconds to reach equilibrium in the tip and withdraw the
tip from the liquid, touching it against the edge of the reservoir to
remove excess liquid].
3. Dispense the liquid into the receiving vessels by gently and
steadily pressing the operating button to the first stop
4. Remove the tip from the receiving vessel without blowing out. The
liquid remaining in the tip can be pipetted back into the original
solution or thrown away with the tip.
5. Release the operating button to the ready position.
 Pipette Troubleshooting

Problem Potential Cause Action

Leakage Tip(s) incorrectly Attach firmly
attached
Foreign articles Clean tip cones
between the tip and
cone
O-ring damaged Change the O-ring
Scratches or hairline Contact distributor; use
fractures on the shaft replacement pipette
Pipette: Troubleshooting, Contd..
Problem Potential Cause
Inaccurate Incorrect operation
dispensing
Tip incorrectly attached
Pipette calibrated at
room temperature, used
with cold liquid
Tip immersed to deep
Tip at an angle
while aspirating
Action
Follow manufacturers
instructions carefully
Firmly attach tip
Calibrate and use at same
temperature
Immerse only 2-3 mm
Hold vertically
General working Remarks

Avoid air bubbles in the tip during aspiration

Keep consistent pickup/dispense rhythm during
the whole pipetting cycle
Keep consistent speed and smoothness of the
pushbutton move
General working Remarks ,
Contd..
Maintain consistent angle of pipette when
aspirating (min. 30)
Discard contaminated tips in appropriate
container after completion of task
Store in an appropriate rack or stand
Principles of Preventive Maintenance

Select the appropriate pipette for the volume

required
Always use your pipette with a proper tip on
Ensure that the pipette, tips, and specimen are
at the same temperature
Principles of Preventive, Contd.
Routine Maintenance

Check at the beginning of each workday for dust

and dirt on the outside surfaces. Pay particular
attention to the tip cone
In addition to routine calibration, pipettes should
be cleaned and lubricated as recommended by
the manufacturer
Inspection and Calibration of
Pipettes
Inspect the pipette for scratches or other damage to the
nose cone
Check for leaks by aspirating the maximum listed volume
of distilled water
If the pipette is dropped or you suspect any type of
damage, do calibration
Calibrate at least once or twice a year
Inspection and Calibration, Contd.
Testing Pipetting Accuracy
The following procedure can be used to test how accurately the
pipette measures volume (water is used because the weight of
water is 1g/mL):
Using the correct pipetting technique, pipette 10 replicates of 50
l of distilled water and record the weight to the nearest 0.0000
g (using four digits after decimal point)
Calculate the mean weight
The mean weight should fall within [0.0490 and 0.0510]
If is below or above the limit, adjustment is required until the
required range is attained.
Testing Pipetting Accuracy, Contd.
The accuracy of pipetting should be evaluated
periodically (i.e. every 6-12 months)
Retain the evaluation records for each pipette for quality
management purposes
Using the pipetting technique, pipette 10 replicates of
distilled water and record the weights
Select a volume normally used in the performance of
the assay
Checking Pipetting Precision

Set the pipette to 50 L

Using pipetting technique, pipette 5 replicates of distilled
water and record the weights
Calculate the mean, standard deviation and coefficient of
variation (CV)
The CV for replicates should be < 0.3%
Application 1 Debrief: Precision
and Accuracy Calculation
A technologist observed that when using the
pipette method for analysis, the volume of sample
dispensed into each tube seemed to look different.

Question:

What is the next step the technologist should take?

Answer:

Check the precision and accuracy of the pipette

Example Data and Calculations,
The following data were obtaine after 10 replicate of
volume measurments was done. Calculate the mean,
sd and %cv of the data.
Weight
0.0495 Mean: ?
0.0506 s: ?
0.0498 %CV: ?
0.0501
0.0506
0.0492
0.0498
0.0507
0.0502
0.0491
Pipetting

0.0000 g
0.1499
0.0495
0.1001
Review Questions
1. Discuss the advantage of using volumetric equipment
when preparing standard solutions, diluting controls
and patient samples
2. Discuss the need to verify pipette calibration
periodically
3. Discuss methods used to verify pipette calibration
4. Explain the appropriate operation of automatic/
micropipettes