1. Cell disruption Saeb Aliwaini Saeb Aliwaini

2. Cell disruption To extract a product from cells : - The cells are usually first separated from the culture liquid medium - To reduce secreted extracellular substances and unutilized media components How ? By microfiltration or centrifugation Saeb Aliwaini

3. Type of cells Gram positive bacterial cells Gram negative bacterial cells Yeast cell fungi Cultured mammalian cells Cultured plant cells Ground tissue Saeb Aliwaini

4. In gram positive bacteria - The cell wall is composed of peptidoglycan, Teichoicacid and polysaccharides and is about 0.02 to 0.04 microns thick. Can be destroyed by the antibacterial enzyme lysozyme. Saeb Aliwaini

5. But in Gram negative bacteria • These don't have distinct cell walls but instead have multilayered envelops. * The peptidoglycan layer is significantly thinner than in gram positive bacteria. * An external layer composed of lipopolysaccharides and proteins is usually present * The presence of the periplasm layers which are two liquid filled gaps, one between the plasma membrane and the peptidoglycan layer and the other between the Peptidoglycan layer and the external lipopolysaccharides Saeb Aliwaini

6. Periplasmic layers also exits in gram positive bacteria but these are significantly thinner than those in gram negative bacteria. • But in many cases we need this layer to be extracted Yeasts have thick cell walls, typically 0.1 to 0.2 microns in thickness From polysaccharides such as chitins Moulds are similar to yeast but multicellular Mammalian cells are easy to disrubt Saeb Aliwaini

7. Plant cells on the other hand have very thick cell walls mainly composed of cellulose and other polysaccharides. • Cell wall wherever present is the main barrier which needs to be disrupted to recover intracellular products. lysozyme is used to disrupt the cell wall of gram positive bacteria since it degrades peptidoglycan which is a key cell wall constituent. Saeb Aliwaini

8. In gram negative bacteria it is less susceptible to lysis by lysozymes in since it is shielded by a layer composed of lipopolysaccharide and protein. • The plasma membrane can be easily destabilized by detergents. Or even osmotic shock This can be achieved simply by transferring the cell fro m Isotonic medium to distilled water Saeb Aliwaini

9. Cell disruption methods • Physical methods • 1 .Disruption in bead mill • 2. Disruption using a rotor-stator mill • 3. Disruption using French press • 4. Disruption using ultrasonic vibrations • Chemical and physicochemical methods • 1 .Disruption using detergents • 2. Disruption using enzymes (lysozyme) • 3. Disruption using solvents • 4. Disruption using osmotic shock Saeb Aliwaini

10. Cell disruption using bead mill A tubular vessel made of metal or thick glass within which the cell suspension is placed along with small metal or Glass beads. The cell disruption takes place due to the grinding action of the rolling beads as well as the impact resulting from the cascading beads. At low temperatures as (liquid nitrogen into the vessel) Commonly used for disrupting yeast cells and for grinding animal tissue Saeb Aliwaini

11. Cell disruption involves particle size reduction and has certain Similarities with grinding. According to the Kick's law of grinding, the amount of energy required to reduce the size of material is proportional to the size reduction ratio: Saeb Aliwaini

12. Cell disruption using rotor-stator mill Truncated cone shaped rotating object called the rotor. Stationary block with a tapered cavity called the stator Typical rotation speeds are in the 10,000 to 50,000 rpm range The high rate of shear generated in the space between the rotor and the stator as well as the turbulence thus generated are responsible for cell disruption Saeb Aliwaini

13. These mills are more commonly used for disruption of plant and animal tissues based material and are operated in the multi pass mode ,i.e. the disrupted material is sent back into the device for more complete disruption Saeb Aliwaini

14. Cell disruption using French press . Consists of a cylinder • The cell suspension is placed with in the cylinder and pressurized using the plunger • Provided with an impact plate, where the jet impinges causing further cell disruption • For small –scale recovery of intracellular proteins and DNA from bacterial and plant cells. Saeb Aliwaini

15. Cell disruption using ultrasonic vibrations • Ultrasound emitting tips of various sizes are available A frequency of 25kHz is commonly used for cell disruption For bacterial cells such as E. coli, 30 to 60 Seconds maybe sufficient for small samples Saeb Aliwaini