Cell disruption technique by repeated freezing and thawing

 

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 for one hour.
4. The same pair of test tube with distilled water and saline was  kept at room temperature for next 1 hour. 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