CELL DISRUPTION

METHODS
Introduction to cell disruption
• Cell disruption is the process of obtaining intracellular
fluid via methods that open the cell wall.
• The overall goal in cell disruption is to obtain the
intracellular fluid without disrupting any of its components
• The method used may vary depending on the type of cell
and its cell wall composition. Irrespective of the method
used, the main aim is that the disruption must be effective
and the method should not be too harsh so that the product
recovered remains in its active form.
• Many techniques available in the lab- limited no. have
been proved to be suitable for large scale.
Elements of cell structure
Elements of cell structure
• Very thick cell wall
Gram • Stained purple
positive with crystal violet

• Thin cell wall

• Gram stain: red
Gram • Space between
negative plasma membrane
& peptidoglycan-
periplasm
Elements of cell structure
• Biological membranes- phospholipid bilayers
• Membrane stiffness is conferred by cholesterol & steroids
that partition into nonpolar layer.
• While flexibility is conferred by amphiphilic proteins
(consists of both hydrophilic & hydrophobic regions)
• The bacterial cell wall protects the plasma membrane &
cytoplasm from osmotic stress.
• The isoosmotic external conc. For most cells is 0.3
osmolar.
• Higher conc outside the cell draw out water & cause
cytoplasm to shrink- plasmolysis
Elements of cell structure
• The cell wall is rigid- the cytoplasm collapse within the
plasma membrane while cell wall does not.
• Conc of salts & neutral solutes lower than 0.3 osmolar
outside the cell- force water into the cell – turgor.
• Too much turgor can break the cell wall (lysis)- situation
that can aid cell disruption.
Elements of cell structure
Cell lysis
Cell lysis methods
Types of cell disruption
• Liquid shear
• Solid shear
• Agitation with
Mechanical
abrasives
• Freeze-thawing
• Ultrasonication

• Detergents
• Osmotic shock
Non-
mechanical • Alkali treatment
• Enzyme
treatment
Types of cell disruption
Mechanical method:
• Mechanical methods are those methods which required
some sort of force to separate out intracellular protein
without adding chemical or enzyme
Liquid shear
• Have been used in the large scale enzyme purification
procedures.
• High-pressure homogenizers- used for microbial cells
disruption.
• Microbial slurry passes through a non-return valve &
impinges against the operative valve set at the selected
operating pressure.
• The cells then pass through a narrow channel between the
valve & an impact ring followed by a sudden pressure drop at
the exit near the narrow orifice.
• The large pressure drop across the valve is believed to
cause cavitation in the slurry and the shock waves produced
disrupt the cells.
Liquid shear- homogenizer
Liquid shear- homogenizer
• With smaller industrial scale machine- a throughput of 6.4
kg soluble protein h-1, pressure at 550 kg cm-2- 90%
disruption could be achieved.
• Need to cool the slurry between 0-4oC to minimize loss in
enzyme activity because of heat generation during the
process.
Solid shear
• Pressure extrusion of frozen microorganisms at around
-25oC through a small orifice using Hughes press or X-
press
• Disruption caused by liquid shear through a narrow orifice
& the presence of ice crystals.
• This technique may be ideal for microbial products which
are temperature labile.
Agitation with abrasives
• Mechanical cell disruption can also be achieved in a
disintegrator containing series of rotating discs and a
charge of small beads.
• Beads- made of mechanically resistant materials- glass,
alumina ceramics, zirconia, or some titanium compounds
• but this kind of mechanical shear is gentle enough to keep
organelles intact.
• It can be used with all kinds of cells, just add beads to an
equal amount of cell suspension and vortex
Agitation with abrasives
Freezing and thawing
• Freezing & thawing of a microbial cells will inevitably
cause ice crystals to form & their expansion followed by
thawing will lead to subsequent disruption of cells.
• β- glucosidase has been obtained from S. cerevisiae by
this method.
Ultrasonication
• Ultrasonic homogenizers work by inducing vibration in a
titanium probe that is immersed in the cell solution.
• A process called cavitation occurs, in which tiny bubbles are
formed and explode, producing a local shockwave and
disrupting cell walls by pressure change.
• This method is very popular for plant and fungal cells but
comes at a disadvantage: It’s very loud and has to be
performed in an extra room.
• Very effective for small scale but a no. of drawbacks for
large-scale:
• Power requirements are high- large heating effect thus need cooling
• Probes have a short working life and only effective over a short range.
Ultrasonication
Other physico-mechanical methods
• Grinding/pestle & mortar
Just give the cells a good old grinding.
• This does not have to be in suspension and is often done
with plant samples frozen in liquid nitrogen.
• When the material has been disrupted, metabolites can
be extracted by adding solvents.
Other physico-mechanical methods
• The use of blenders (high speed can be used to disrupt
cell walls.
• This is the same process used by centrifugation, which
separates or concentrates materials suspended in a liquid
medium
CHEMICAL METHOD
Detergents
• Detergents will damage the lipoproteins of the microbial
cell membrane & lead to the release of intracellular
components.
• Compounds that can be used:
• Quartenary ammonium compounds
• Sodium lauryl sulphate
• Sodium dodecyl sulphate (SDS)
• Triton X-100
• Detergents may cause some protein denaturation and
may need to be removed before further purification stages
can be undertaken.
Osmotic shock
• Osmotic shock caused by a sudden change in salt conc
(0.15 M to 0.001 M) will cause disruption of a no. of cells
types.
• It has proved to be a successful technique for the
extraction of luciferase from Photobacterium fischeri
• Normally use for animal cells.
Alkali treatment
• Can be used for hydrolysis of microbial cell wall material
provided that the desired enzyme will tolerate a pH of 11.5
to 12.5 for 20 to 30 mins.
Chemical treatment
• Often used with plant cells (and sometimes in combination
with shearing), organic solvents such as toluene, ether,
benzene, methanol, surfactants, and phenyl ethyl alcohol
DMSO can be used to permeate cell walls.
• EDTA can be used specifically to disrupt the cell walls of
gram negative bacteria, whose cell walls contain
lipopolysaccharides that are stabilized by cations like
Mg2+ and Ca2+. EDTA will chelate the cations leaving
holes in the cell walls.
Chemical treatment
Enzymatic treatment
THANK YOU!