Sonics VC5040PB - Manual (Page 17)

14
When processing a sample with ultrasonics, always immerse the probe deep enough
below the surface of the sample to inhibit aerosoling or foaming, foaming substantially
reduces cavitation. Processing at a lower power setting without foam is much more
effective than processing at a higher power setting with foam. Decreasing the power,
increasing processing time and lowering the temperature of the sample will usually
prevent aerosoling and foaming. Do not use any antifoaming agents or surfactants.
During cavitation, free radicals are formed which, if they are allowed to accumulate,
can greatly affect the biological integrity of the sample by reacting with proteins,
polysaccharides, or nucleic acids. Although during short periods of processing their
formation is not normally considered a problem; for longer durations, the addition of
free radical scavengers such as, carbon dioxide, N
2
O, cysteine, reduced glutahione,
dithiothreitol or other SH compounds, might be beneficial. Saturating the sample with
a protective atmosphere of helium or nitrogen gas, or dropping a small pellet of dry
ice in the sample, will also inhibit free radical formation. Whereas it is true that gas is
required for effective cellular disruption, it is not necessary that the vapor phase be
oxygen or air since any gas except carbon dioxide will work just as well.
Following ultrasonic processing, the cell debris can be centrifuged at 15,000 rpm for
10 minutes.
Since the greatest concentration of energy is beneath the probe, it is imperative that
the sample be kept as close to the tip as possible, liquids are easily processed
because the free moving cells circulate repeatedly below the probe. Solid materials
however have a tendency to be repelled by the ultrasonic, and should be processed
in a vessel large enough to accommodate the probe, yet small enough to restrict
sample movement. For small samples, conical shaped test tubes are recommended.
Although plastic tubes work well, glass and stainless steel tubes usually work better
than plastic ones.
Make sure that the probe is not touching the bottom of the vessel. Allowing the probe
to contact the vessel will decrease the power output, and cause minute grey glass
particles to migrate into the sample. Although these glass particles will not adversely
affect the chemical composition of the sample, they will form a thin grey layer on
centrifuging. If the probe has to come in contact with a solid sample, use a standard
20mm (3/4”) diameter stainless steel centrifuge tube cut to 70mm (3") length. Do not
use a glass tube. Microtips must never come in contact with anything but the liquid,
because the stress resulting at the point of contact with a hard surface will cause the
microtip to fracture. Although larger probes will not fracture if they come in contact
with a glass vessel, they may cause the vessel to fracture.