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Cell disruption plunger

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					             Cell disruption
Biological products:
1. Extracellular
2. Intracellular
3. Periplasmic
Cells
• Gram positive bacterial cells
• Gram negative bacterial cells
• Yeast cell
• Mould cells
• Cultured mammalian cells
• Cultured plant cells
• Ground tissue
                                 Bacteria
                                                      Periplasm

                                                        Cell membrane

                     Cell membrane

                                                      Peptidoglycan
                     Cell wall

                                                     Lipopolysaccharides +
                                                     proteins


•Sub-micron to 2 microns in size     •Sub-micron to 1 micron in size
•Have thick cell walls, 0.02-0.04    •Cell capsule present
microns, peptidoglycan +
                                     •Peptidoglycan layer is thin
polysaccharide+ teichoic acid
                                     •Periplasmic space present
•Phospholipid cell membrane
present                              •Mechanically less robust than gm+
                                     bacteria
                                     •Chemically more resistant than
                                     gm+ bacteria
               Yeast and mould

•Yeast: 2-20 microns in size, spherical or ellipsoid
•Moulds: Bigger and filamentous
•Yeasts are unicellular while moulds are multicellular
•Very thick cell walls are present in both
•Cell wall is mainly composed of polysaccharides such as
glucans, mannans and chitins
•Plasma membranes are mainly made up of phospholipids
        Animal and plant cells
•Animal cells do not have cell walls
•Animal cells are very fragile
•Cultured animal cells are several microns in size
•Spherical or ellipsoid


•Plant cells can be bigger
•Plant cells have thick and robust cell walls mainly composed
of cellulose
•Plant cells are difficult to disrupt
•Cultured plant cells are less robust than real plant cells
         Cell disruption methods

Physical methods
•   Disruption in bead mill
•   Disruption using a colloid mill
•   Disruption using French press
•   Disruption using ultrasonic vibrations

Chemical and physicochemical methods
•   Disruption using detergents
•   Disruption using enzymes e.g. lysozyme
•   Disruption using solvents
•   Disruption using osmotic shock
                         Bead mill

                                                  Cascading
                                                  beads
              Rolling
              beads                            Cells being
                                               disrupted



•Disruption takes place due to the grinding action of the rolling
beads and the impact resulting from the cascading ones
•Bead milling can generate enormous amounts of heat
•Cryogenic bead milling : Liquid nitrogen or glycol cooled unit
•Application: Yeast, animal and plant tissue
•Small scale: Few kilograms of yeast cells per hour
•Large scale: Hundreds of kilograms per hour.
          Theory of grinding
Kick’s law
                                        r1 
 dE K K f c
                       E  K K f c ln  
                                       r 
 dr   r                                 2

Rittinger’s law

 dE K R f c                        1 1
     2                 E  KR fc   
                                  r r 
 dr   r                            2 1
Product release from disrupted cells

          C




                    Time
       C                t
              1  exp          Single pass
      C m ax            

                               N
       C             t 
            1  exp   
      C max             
                                   Multi pass
                         
                     Colloid mill
                                           Rotor
                 Disrupted
                      cells




                              Cell                 Stator
                              suspension


•Typical rotation speeds: 10,000 to 50,000 rpm
•Mechanism of cell disruption: High shear and turbulence
•Application: Tissue based material
•Single or multi-pass operation
                      French press


                                               Plunger


               Cell                            Cylinder
         suspension

                                     Jet
                      Orifice
                                      Impact
                                      plate


•Application: Small-scale recovery of intracellular proteins and
DNA from bacterial and plant cells
•Primary mechanism: High shear rates within the orifice
•Secondary mechanism: Impingement
•Operating pressure: 10,000 to 50,000 psig
              Ultrasonic vibrations
                                    Ultrasound
                                    generator


                                    Ultrasound tip


                                   Cell suspension




•Application: Bacterial and fungal cells
•Mechanism: Cavitation followed by shock waves
•Frequency: 25 kHz
•Time: Bacterial cells 30 to 60 seconds, yeast cells 2 to 10 minutes
•Used in conjunction with chemical methods
Chemical and physicochemical
          methods

•Detergents
•Enzymes
•Organic solvents
•Osmotic shock

				
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posted:5/15/2011
language:English
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