Sonics VC500 - Manual (Page 19)

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zymolase, glucalase and / or lyticase to produce spheroplasts that are readily lysed may
also be useful.
To disrupt filamentous fungi, scrape the mycelial mat into a cold mortar, add liquid
nitrogen and grind to a fine powder with a pestle. The powder can then be thoroughly
sonicated in lysis buffer to solubilize completely. As fungi may also be rich in
polysaccharides, pretreatment with polyvinylpyrrolidone (PVP) may be beneficial.
Bacteria, like plants, are extremely diverse; therefore, it is difficult to make one
recommendation for all bacteria. Ultrasonic processing will lyse most Gram positive and
Gram negative bacteria, including mycobacteria. Although it is recommended that glass
beads and lysis solution be used; it is possible to lyse some Gram negative bacteria by
sonicating in lysis solution without beads. Bacteria cell walls can be digested with
lysozyme to form spheroplasts. Gram positive bacteria usually require more rigorous
digestion and longer processing time. The spheroplasts are then lysed with sonication in
GITC lysis buffer.
Disruption of cells found in soil and sediments is accomplished one of two ways. One
technique isolates the bacterial cells from the material prior to the RNA isolation
procedure. This is accomplished by homogenization of wet soil in a mechanical blender
followed by a slow speed centrifugation to remove fungal biomass and soil debris. The
supernatant is centrifuged again at a higher speed to pellet the bacteria cells. Cells can
then be lysed as described above for bacteria. Other techniques describe RNA isolation
from the soil or sediment directly. For example, one method requires soil to be added to a
diatomaceous earth and lysis buffer, and then sonicated. The sample is then centrifuged
to remove solid debris.
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.