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World Precision Instruments
the pipette. However, a 0.1–0.2 PSI pressure imbalance will not cause a significant
problem if the tip is small enough. The gravity of the fluid and the flow resistance
caused by friction from the glass wall will both help to stop the solution flow at this
pressure level. If the lowest pressure setting still can’t stop the fluid from oozing
out, try to switch off the hold pressure to see if the gravity and friction are sufficient
to counterbalance the backfill. In addition, resetting the regulator could allow the
regulator to perform much better in this range (see the “Troubleshooting The Hold
Pressure” on page 16).
The capillary action can also be reduced by adding a hydrophobic fluid (such as
silicone oil) behind the hydrophilic saline solution. It can be completely eliminated
by silanizing the shank of the pipette (silanization increases the
θ
in the LaPlace
equation to 90º).
Micropipette Manufacture
The volume of fluid ejected is markedly dependent on the micropipette tip size.
When using micron-sized tips a reduction in tip-size of a few percent may give an
order of magnitude difference in the flow rate. With tip sizes less than 1µm, pressure
ejection becomes increasingly difficult and special steps must be taken.
The most important of these steps is cleaning the glass. Small amounts of dust or
grease can easily clog micron-sized tips. Cleaning with chromic acid solutions before
pulling the electrode is commonly performed, but care must be taken to thoroughly
rinse the pipettes to remove all traces of the chromic acid, which has some affinity
for glass. Some researchers prefer hydrochloric or nitric acid.
Silanization of the glass is also recommended for small tips. With 1µm and smaller
tips, capillary action becomes prohibitively large, and the hydrophilic surface of
the glass greatly limits the flow of fluid through the tip. Silanization decreases the
surface tension and allows the fluid to flow smoothly through the tip. For similar
reasons, use of a capillary with an internal filament is contraindicated. Some of the
many papers on the art of silanization are listed in the bibliography.
When using the vacuum line to hold and manipulate individual cells a large tip
(about 10–20% of cell size) is recommended. To prevent damage to the cell this tip
should be fire-polished.
Volume Calibration
For ejected volumes greater than 1nL visual inspection using a microscope can
be an accurate gauge of volume. A single pulse deposits a drop of fluid on the tip
of the micropipette. The volume of this drop may be calculated by measuring the
radius of the drop and assuming the drop to be spherical. Fig. 11 may be helpful
in determining the volume for a given radius. See “Appendix” on page 19 for a
comparison of spherical and cubical volumes.
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