Chapter Five

Operation and Functional Verification

of Automated Micropipettes

1 Micropipette operation
 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

2 Micropipette operation
 Chapter Outline

 Introduction
 Operating procedure
 Pipetting techniques
 Pipette troubleshooting
 General working remarks
 Principles of inspection and calibration

3 Micropipette operation
 Introduction to Automated Micropipette
 A pipette, pipet, pipettor or chemical dropper is a
laboratory tool commonly used to transport a measured
volume of liquid.

 Pipettes come in several designs for various purposes

with differing levels of accuracy and precision, from
single piece glass pipettes to more complex adjustable or
electronic pipettes.

4 Micropipette operation
 Introduction to Automated Micropipette

 Many pipette types work by creating a partial

vacuum above the liquid-holding chamber and
selectively releasing this vacuum to draw up and
dispense liquid.

 Measurement accuracy varies greatly depending on

the style.

 Pipettes that dispense between 1 and 1000 μl are

distinguished as micropipettes, while
macropipettes dispense greater volumes.
5 Micropipette operation
 Introduction to Automated Micropipette

 Automatic pipettes are used to accurately transfer

small liquid volumes, they are both accurate and
precise for small volumes
 But glass pipettes are not highly accurate for small volumes

 These pipettes are called “automatic” because

 Don’t 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.

6 Micropipette operation
 Parts of Automatic Micropipette

7 Micropipette operation
 Operating the Micropipette
Step 1: Set the Volume

Volume Adjustment Knob:

Pipettors – 3 Volumes:

Digital Volume Indicator:

8 Micropipette operation
 Step 2: 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

9 Micropipette operation
 Setting Adjustable Volume

 Some micropipettes have two

settings (course and fine) to
adjust the volume.

 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

 For example the smaller outer

ring may be used to adjust
the pipette to 50 µl but the
larger inner ring is used to
adjust the 0.5 µl setting.

10 Micropipette operation
Micropipette operation
 Step 3: Attach the Disposable Tip

Example of tip sizes:

•Blue tips for 1000 µl capacity
• yellow tips for 250 µl capacity

Attaching the
disposable tip

11 Micropipette operation
 Step 4: Depress the Plunger to the First Stop

Step 5: Immerse Tip in Sample

Step 6: Draw up the sample

Step 7: 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

12
 Step 8: 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

13 Micropipette operation
 Step 9: Dispense the Sample

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 Step 10: Withdraw the Pipette

Step 11: Release the Plunger

Step 12: Discard the Tip

Press ejector button to discard tip.

15 Micropipette operation
 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
16 Micropipette operation
 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.

17 Micropipette operation
 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.

18 Micropipette operation
 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 replacement pipette
shaft
19 Micropipette operation
 Pipette: Troubleshooting, Cont’d..
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
20 Micropipette operation
Action
 Follow manufacturer’s
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

21 Micropipette operation
 General working Remarks , Cont’d..
 Maintain consistent angle of pipette when
aspirating (min. 30º)

 Discard contaminated tips in appropriate

container after completion of task

 Store in an appropriate rack or stand

22 Micropipette operation
 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

23 Micropipette operation
 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

24 Micropipette operation
 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

25 Micropipette operation
 Gravimetric Calibration

 The process of determining accuracy and

precision of water dispensed by the pipette
based on its mass is termed gravimetric
calibration.

 It is based on the theory that 1 µL of pure water

has a mass of 1 mg. (i.e. 1ml water weighs 1gm)

26 Micropipette operation
  This table shows the accuracy and precision of pure
water dispensed that is allowed for micropipettes based
on their volumes.

Volume Measurement Standard

tested Accuracy Deviation
(µl) (mg) (mg)

≥ 0.2-10 ≤ 0.001 ≤ 0.002

≥ 10-100 ≤ 0.01 ≤ 0.02

≥ 100-1000 ≤ 0.1 ≤ 0.2

≥ 1000 ≤ 0.1 ≤ 0.2

For example, pipettes that dispense 100 µl should be accurate to 0.1 mg with
a standard deviation of up to 0.2 mg of pure water weighed with an analytic
balance
27 Micropipette operation
 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 μl

(0.050ml) 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 it is below or above the limit, adjustment is required until the

required range is attained.

28 Micropipette operation
 Testing Pipetting Accuracy, Cont’d….
 The accuracy of pipetting should be evaluated periodically (i.e. every
6-12 months)

 The accuracy error (AE) is determined using the following formula:

AE = Expected Volume – Measured Mean Volume X 100

Expected Volume

 Retainthe 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

29 Micropipette operation
 Checking Pipetting Precision
 Set the pipette to 50 µL (0.050mL)

 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%

30 Micropipette operation
 Precision and Accuracy Calculation
 Case scenario: A lab professional 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 lab professional should take?

 Answer:

 Check the precision and accuracy of the pipette

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

0.0000 g
0.1499
0.0495
0.1001
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 Gravimetric Measurements

34 Micropipette operation
 Calculations

35 Micropipette operation
 Calculations

36 Micropipette operation
 Calculations

37 Micropipette operation
 Application 2:
Precision Calculation
In order to be precise for patient testing, the pipette is
required to have a %CV of <2% and accuracy was
required to have < 1% error rate.
A quick precision study is performed with the following
results:

Volumes obtained from gravimetric testing of distilled water

0.048 mL
0.045 mL
0.051 mL
0.049 mL

Micropipette operation
0.053 mL
 Application 2:
Precision Calculation

Questions: In order to be precise for

What is the mean? patient testing, the pipette
What is the standard deviation? was required to have a %CV
What is the coefficient of variation < 2% and accuracy was
(%C.V.)?
The accuracy error is determined
required to have a <1%
using the following formula. What error rate.
is the accuracy error of this Volumes obtained from gravimetric
pipette? testing of distilled water
Expected Volume – Measured Mean Volume X 100 0.048 mL
Expected Volume
0.045 mL
0.051 mL
Is this pipette acceptable to use?
Why or why not? 0.049 mL

39 Micropipette operation 0.053 mL

Calculations for Precision and Accuracy

 Arithmetic mean
x
 x
N
 x
2

 Standard deviation SD 
x 2
N
N 1

 Coefficient of variation %CV  SD 100

x

Micropipette operation
 Application 2 Debrief: Precision Calculation

Questions Answers
1. What is the mean? 0.0492 mL or 49.2 uL
2. What is the coefficient of 6.1%
variation (%C.V.)?

3. What is the standard deviation? 0.003 mL or 3 uL

4. What is the accuracy error of this [(.05-.0492)/.05] x 100 =
pipette? 1.6%

5. Why or why not is this pipette Not acceptable since

acceptable to use? accuracy error > 1% and
41 Micropipette operation
precision > 2%
 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
42 Micropipette operation