Sonics VCX750 User Manual download pdf (Page 24)

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.

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Sonics VCX750 User Manual Page 24