Source: BiochemFFA_8_1.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
To separate compounds from cellular environments, one must first break open (lyse) the cells. Cells are broken open, in buffered solutions, to obtain a lysate. There are several ways of accomplishing this.
- Osmotic shock and enzymes: One way to lyse cells is by lowering the ionic strength of the medium the cells are in. This can cause cells to swell and burst. Mild surfactants may be used to disrupt membranes. Most bacteria, yeast, and plant tissues are resistant to osmotic shocks, because of the presence of cell walls, and stronger disruption techniques are usually required. Enzymes may be useful in helping to degrade the cell walls. Lysozyme, for example, is very useful for breaking down bacterial walls. Other enzymes commonly employed include cellulase (plants), proteases, mannases, and others.
- Mechanical disruption: Mechanical agitation may be employed in the form of beads that are shaken with a mixture of cells. In this method, cells are bombarded with tiny, glass beads that break the cells open. Sonication (20-50 kHz sound waves) provides an alternative type of agitation that can be effective. The method is noisy, however, and generates heat that can be problematic for heat-sensitive compounds.
- Pressure disruption: Another means of disrupting cells involves using a “cell bomb”. In this method, cells are placed under very high pressure (up to 25,000 psi) and then the pressure is rapidly released. The rapid pressure change causes dissolved gases in cells to be released as bubbles which, in turn, break open cells.
- Cryopulverization: Cryopulverization is often employed for samples having a tough extracellular matrix, such as connective tissue, seed, and cartilage. In this technique, tissues are frozen using liquid nitrogen and then impact pulverization (typically, grinding, using a mortar and pestle or a powerful electric grinder) is performed. The powder so obtained is then suspended in the appropriate buffer.
Figure 8.1 - A sonicator in use. Wikipedia
Whatever method is employed to create a lysate, crude fractions obtained from it must be further processed via fractionation.