DELIVERY OF LIQUIDS BY PIPET
©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
6/30/88, rvsd 5 July '93,
21 July 1996, 20 July '97, 17 July 98, 14 July 01
This page has been accessed
times since 18 July 2001.
Pasteur observed that once a medium is
sterilized, it remains sterile unless contaminated from an external source.
He used this observation to reject spontaneous generation but it also lays
the foundation for sterile (or aseptic) technique, a technique crucial
to microbiology and medicine. Several practices are important to the success
of this technique:
Mastering sterile technique is one of the
most important prerequisites to working in a microbiological lab. While
the steps may seem overly detailed in the following narrative, early care
in learning proper technique establishes good technique for the rest of
your life. Compare these detailed steps with the demonstration given by
the professor. Patience pays off. Go slowly at the beginning, and verbally
(not physically) assist your fellow students as they work
through the steps.
Sterilize the instruments and keep them
that way: keep instruments off possibly contaminated surfaces, and
flame them to incinerate loose material which might fall off. Never lie
pipets down on the surface of a workbench.
Sterilize the field where work is to
be performed with 70% EtOH.
Exclude airborne contamination: keep
containers covered as much as possible
Minimize drafts: no breathing, coughing,
etc over sterile materials.
USING THE PIPET:
Write out the procedure which you plan
to perform in your notebook. Indicate how the sample was prepared and volumes
to be transferred. Construct a table of plates to be spread, with
plate number, specimen, plate type, dilution factor, aliquot, and two empty
columns for colonies counted and final calculated numbers. Label tubes
or plates appropriately.
Loosen all caps, mentally review the steps
you will be performing and arrange materials to ensure they are available
and easily accessible.
Select the appropriate pipet for volume to
be delivered, and set the volume to be transferred by rotating the plunger.
Pick up a sterile tip, reclose the lid on the box of tips immediately.
Hold displacement pipetter so that the thumb
operates the plunger and you keep your little finger on the pipetting
hand free for grasping the cap of the vessel.
Pick up specimen vessel with other hand, grip
cap of vessel with little finger of pipet hand and twist the vessel and
pull it down away from the cap. Do not lay the cap down.
Flame lip of vessel.
Pass sterile pipet through flame. (Very briefly
so that the plastic tip is not melted...)
DRAW UP SPECIMEN: Immerse tip of pipet
1/2 cm below surface of liquid, brace in place by gently pressing pipet
against the lip of the tube while drawing the fluid up. To avoid bubbles,
draw the fluid slowly into the tip by slowly raising your thumb.
TRANSFER ALIQUOT: Touch off excess
fluid from tip of pipet, tilt pipet horizontally to prevent dribbling during
transfer. (The fluid will draw up slightly into the tip pipet.)
Reflame the lip of specimen vessel, screw
it back into the cap, set vessel down.
DELIVER ALIQUOT: Pick up receiving
vessel. Remove its cap and flame its lip (as in steps 5 and 6). Insert
pipet, return to vertical, deliver desired volume, remove pipet, flame
lip, replace cap, discard tip into used pipet container for sterilization.