Saeb Aliwaini Saeb Aliwaini 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 ID: 268978
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Slide1
Cell disruption
Saeb Aliwaini
Saeb AliwainiSlide2
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 AliwainiSlide3
Type of cells
Gram positive bacterial cells Gram negative bacterial cells
Yeast cell
fungi
Cultured mammalian cells
Cultured plant cells Ground tissue
Saeb AliwainiSlide4
Saeb Aliwaini
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.Slide5
But in
These don't have distinct cell walls but instead have multilayered envelops.
Saeb Aliwaini
Gram negative bacteria
*
The peptidoglycan layer is significantly thinner than in gram positive bacteria.
*
An external layer composed of lipopolysaccharides and proteins is usually present
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The presence of the
p
eriplasm
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 lipopolysaccharidesSlide6
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
Mould
s
are similar to yeast but multicellularMammalian cells are easy to disrubt
Saeb AliwainiSlide7
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.
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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
Saeb Aliwaini
This can be achieved simply by transferring the cell fro m Isotonic medium to distilled waterSlide9
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 AliwainiSlide10
Cell disruption using bead mill
Saeb Aliwaini
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 tissueSlide11
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 AliwainiSlide12
Saeb Aliwaini
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 disruptionSlide13
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
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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 AliwainiSlide15
Cell disruption using ultrasonic vibrations
Ultrasound emitting tips of various sizes are available
Saeb Aliwaini
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