Sonics VC70T - Manual (Page 13)

or Sigmund Lindner GmbH. P.O. Box 29. D-95483 Warmensteinach, Germany. Phone
(49) 0 92 77 9 94 10, FAX (49) 0 92 77 9 94 99.
When processing difficult cells, pretreatment with an enzyme such as lysozyme or
byaluronidase might be beneficial. Glycosidase has been used successfully with yeast,
lysostaphin with staphylococcus, collagenase with skin and cartilage, and trypsin
hyaluronidase with liver and kidney.
If enzymes cannot be used, the following procedures should be considered: Freezing the
sample at -70
°C overnight, then thawing it in water immediately prior to ultrasonic
processing.
Whenever possible, the tissues should be diced very small to permit movement within the
liquid. Tough tissues such as skin and muscle should be macerated first in a blender or
the like for about 10 seconds, and confined to a small vessel during ultrasonic treatment.
Freezing followed by powdering could also be resorted to if this procedure is not
detrimental. If sub-cellular particles are desired intact, the amplitude should be kept low,
and the processing time increased.
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 duration’s, 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 hydrogen gas, or dropping a small pellet of dry ice in
the sample, will also inhibit free radical formation.
The problem of oxidation is a serious one particularly where the study of sulpdhydril
enzymes is concerned. This may be partially controlled using free radical traps such as
cysteine, reduced gluthathione or comparable substances, or by processing in the
presence of an inert atmosphere. 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. e.g. Forcing helium or nitrogen through the
sample will also reduce aerobic oxidation.
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
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