Aim
To demonstrate the extent of cell disruption by
freezing and thawing.
Principle
Microorganisms are protected by extremely tough
cell walls. In order to release their cellular contents during downstream
processing a number of methods are available for cell disruption. Cell
disruption can be done by physical or chemical methods.
Repeated freezing and thawing is a physical cell
disruption method used to break cells and release intracellular materials. The
technique works on the principle that ice crystals are formed during freezing
and their melting during thawing damage the cell membrane and cell wall,
leading to cell lysis. During
freezing treatment, water inside cells forms ice crystals which expand and
puncture cellular structures during thawing. Rapid melting causes osmotic shock
and membrane rupture. Repeated cycles
increase the extent of cell disruption. The intracellular contents comes out which can be
easily separated by centrifugation. By plating microbial suspension after
centrifugation the number of viable cells can be obtained.
It is crucial that cell
disruption methods do not denature labile materials present in the cell.
Repeated freezing and thawing is a simple, inexpensive, and chemical-free cell
disruption technique that efficiently releases intracellular biomolecules while
preserving many heat-sensitive and biologically active compounds. It is commonly used for laboratory-scale
extraction of biomolecules from microbial and animal cells.
Materials required
1. Overnight grown culture of E.coli
2. Sterile distilled water
3. Sterile saline
4. Nutrient Agar
5.Routine Microbiological facilities
Procedure:
1.10 ml of distilled water and saline were taken
in sterile test tubes.
2. Test tubes were inoculated with 0.5 ml of
overnight broth culture of E coli.
3. One test tube each
of distilled water and saline were kept at 5°C.
4. The second pair of test tube with distilled
water and saline was kept at room temperature for
next 1hour. This process was repeated for 4 times
5. 0.1 ml sample were taken from each tube and
proper dilution was plated.
6. The plates were incubated at 37 °C
overnight and colonies were counted.
Result
A fall
in cell count was obtained in tubes subjected to freezing and thawing
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