14
OPERATING SUGGESTIONS AND TECHNIQUES
DISRUPTING CELLS
The disruption of cells is an important stage in the isolation and preparation of
intracellular products. From research levels through to production, many areas of
biotechnology, particularly recombinant technology, necessitate the use of ultrasonics for
cell disruption. Although some biological products are secreted from the cell or released
during autolysis, many others require sonication to release intracellular material. Cell
disruption focuses on obtaining the desired product from within the cell, and it is the cell
wall that must be disrupted to allow cell contents extraction.
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 and sonicated 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
contents, 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.
The ability to control the amplitude at the probe tip is a prerequisite for process
optimization. And because each application requires its own set of processing parameters,
due to variation in volume and composition, the optimum amplitude can only be
determined empirically. When processing a new sample, it is recommended that the
amplitude be set first at 50% (30% with a microtip) and then increased of decreased as
required.
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.
Gram negative bacteria typically require 10 to 15 minutes of processing, while
staphylococcus requires 20 to 30 minutes.
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