Sonics VC750 (Serial No. "Y through "AB")" - Manual (Page 22)

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Single-cell organisms (micro-organisms) consist of a semipermeable, tough, rigid
outer cell wall surrounding the protoplasmic membrane and cytoplasm. The
cytoplasm is made up of nucleic acid, protein, carbohydrates, lipids, enzymes,
inorganic ions, vitamins, pigments, inclusion bodies, and about 80% water. In order
to isolate and extract any of these substances from inside the cell, it is necessary to
break the cell wall and protoplasmic membrane. In some cases the cell may excrete
the desired substance without assistance, but in most cases, the cells must be lysed
in order for these intracellular substances to be released. Breaking cell membranes
and releasing the contents present significant challenges. The process must be fast
and thorough to maximize the protein yield. Because the energy applied must be great
enough to break the cell membranes or walls, yet gentle enough to avoid physically
or chemically damaging cell content, the Vibra-Cell with its variable intensity capability
is ideally suited for this application.
Microorganisms differ greatly in their sensitivity to ultrasonic disintegration. For
example, the most readily disintegrated are the rod-like forms (bacilli), while the
spherical organisms (cocci) are much more resistant. The group Mycobacteria, to
which the tuberculosis organism belongs, is particularly difficult to disrupt.
Yeast, gram-positive bacteria, and to a lesser extent, gram-negative bacteria have
considerably harder cell walls in comparison to animal cells, and require relatively high
power for cell disruption.
Bacteria 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. Typically, glass beads and lysis solutions are added
to a bacterial cell pellet and the sample is sonicated for a few minutes. It is possible
to lyse some Gram negative bacteria by sonication in lysis solution alone. Bacteria
cell walls can be digested with lysozyme to form spheroplasts. Gram positive bacteria
usually require more rigorous digestion (increased incubation time, increased
incubation temperature, etc.) than Gram negative organisms. The spheroplasts are
then easily lysed in a GITC lysis buffer with ultrasonics.
Gram negative bacteria typically require 10 to 15 minutes of processing, while
staphylococcus requires 20 to 30 minutes.
The level of intensity that should be used is application dependent. For example high
intensity might be recommended for the break up of cells, but should never be used
when the release of intracellular components might be objectionable e.g., Organelle
isolation.