Sonics VCX750 (Serial No."X or lower)" - Manual (Page 24)

24
SECTION IV - OPERATING SUGGESTIONS AND TECHNIQUES
DISRUPTING CELLS
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 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.
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.
Gram negative bacteria typically require 10 to 15 minutes of processing, while
staphylococcus requires 20 to 30 minutes.
Micro-organisms 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. Generally, animal
cells are more easily disintegrated that plant cells, and red blood cells are more readily
disintegrated than muscle cells because they lack a protective cell wall.
Ultrasonic processing will typically cause the temperature of the sample to increase
especially with small volumes. Since high temperatures inhibit cavitation, the sample
temperature should be kept as low as possible - preferably just above its freezing point.
This can be accomplished by immersing the sample vessel in an ice-salt-water bath.
Temperature elevation can also be minimized by using the pulser.
Increasing hydrostatic pressure (typically 15-60 psi) and viscosity can enhance cell
disruption. For micro-organisms, the addition of glass beads in the 0.05 to 0.5mm size
range promotes cell disruption. Beads are almost a prerequisite when working with
spores and yeast. A good ratio is one volume of beads to two volumes of liquid. Glass
beads are available from Cataphote, Inc. P.O. Box 2369, Jackson, Mississippi 39225-
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