PRINCIPLES, USE, MAINTENANCE AND VERIFICATION OF AUTOMATIC PIPETTES

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PRINCIPLES, USE, MAINTENANCE AND VERIFICATION OF
AUTOMATIC PIPETTES Dr André de Kock
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INTRODUCTION

• Millions of pipetting operations done every day
• Accurate measurement of sample and reagents are
  vital for correct results
• Pipette users do not follow correct procedures,
  either because of lack of training or by cutting
  corners
• This lecture is to remind people of the good way
  to use pipettes

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DESCRIPTION Automatic pipettes are continuously
adjustable, general purpose micropipettes for
sampling and dispensing accurate liquid
volumes. Automatic pipettes usually operate on an
air displacement principle. Volume ranges covered
are between 0.1 ?l and 10 000 ?l. Pipettes use
detachable, disposable polypropylene tips. The
tips are autoclavable.
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• PIPETTING PROCEDURES
• The operation of the pipette is controlled by the
  operating button. For the best possible
  accuracy
• Push and release the button slowly at all times,
  especially when working with liquids that have a
  high viscosity.
• Never allow the operation button to snap back.
• Ensure that a clean tip is firmly pushed on the
  tip cone of the pipette and there are no foreign
  particles in the tip itself.
• Wet the newly attached tip with the solution
  being pipetted before any actual pipetting takes
  place.
• This is done by filling and emptying the tip 2-3
  times.
• Hold the pipette vertically during liquid intake.

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• Never wipe the tip!!!!!If one really has to do
  this, only use non-blotting paper and never with
  volumes smaller than 100?l.
• Ensure that the desired delivery volume clicks
  into place and the digits for the volume are
  clearly visible on the handle display before the
  pipette is used.
• Do not set volumes outside the pipettes
  specified volume range!!!
• Using excessive force to turn the push button
  outside the range may jam the mechanism and cause
  defects.

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Pipetting Techniques

• Forward technique
• Everyday, single aliquot pipetting
• Reverse
• Pipetting viscous and foaming liquids
• Small volume pipetting
• Repetitive
• Rapid and simple procedure for repeated delivery
  of the same volume.

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Pipetting whole blood or viscous fluids Depress
the operating button to the first stop. Dip the
tip under the surface of the liquid and slowly
release the button. Withdraw the tip whilst
touching the side of the container. Immerse the
tip into the liquid of the delivery
container. Push the button to the first
stop. Release the button slowly. Repeat above
steps a few times until the interior wall of the
tip is clear. Depress the button to the second
stop.
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MAINTENANCE When the pipette is not in use make
sure it is safely stored in a vertical position.
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Short term Check the pipette before use for dust
and dirt on the outside of the pipette. Pay
special attention to the tip cone. Clean with 70
ethanol if necessary.
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Long term Pipettes should be serviced at least
twice a year or more often if used
frequently. Disassemble the pipette (Refer to
manual). Clean the piston, the piston spring and
o-ring with a dry cloth. Check the cylinder for
foreign particles. Grease the cleaned parts with
lubricant provided with the pipette. Reassemble
the parts. Verify that the correct volumes are
pipetted.
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Verification
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1) Set the volume of the pipette as desired.
2) Place a tip firmly onto the tipcone.
3) Pipette d water into a pre-weighed container
at least ten times. 4) Record the weight
accurately. 5) Do the following
calculations a) Average volume in ml ?
b) Standard deviation SD
?(x-?)2 n-1 c)
deviation accuracy (A - ?) X 100 (A
set volume) A d) CV precision
(SD/?) X 100
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6) The CV should not be more that
1-2. 7) Recalibrate the pipette if necessary and
check again (Refer to package insert for
calibration). 8) With volumes smaller than 100?l,
one could make use of a spectrophotometric-diluti
on method. a) Weigh ten samples of
distilled water. b) Make up a stock
solution of a dye. c) Add the same volume
of stock to all the samples. d) Determine
the OD of the stock and all the samples.
e) Use the following formula to determine the
volume added VOL added ODtest x
VOLstandard ODstandard f) Treat these
values as above.
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PICASSO
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Pipetting test

• 28 staff members (techs, student techs,
  scientists MDs)
• Same pipette and same type of tips
• 20 - 200 ul pipette
• Verification at 40, 50 and 100ul
• Mean, accuracy precision calculated

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Observations

• 84 series of 10 each pipetted
• 1st aliquot pipetted is less than mean in 70
  of series
• 15.5 of series dit not pass the validation
• Vol 40 ul 50 ul 100ul
• Low 35.52 44.97 94.79
• High 41.77 51.63 110.53

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General conclusions

• General bad pipetting technique
• Staff members working for longer than 10 years
  also have problems
• Staff doing a lot of pipetting - no better
• General errors
• tip wetting
• not wiping tip against side of container
• tip too deep in liquid
• too fast filling of tip (air bubbles)
• too slow ejection of sample

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Possible remedies

• Better induction training for beginners
• Periodic re-training of all staff members
• Periodic testing of pipetting
• Regular in-house training

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