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homogenate preparation and lyophilization (freeze drying) (lyophilization…
homogenate preparation and lyophilization (freeze drying)
homogenization
prepare uniform dilution of a known quantity of tissue suspended within a known quantity of a suitable diluents and uniformly crush the tissue in such manner as to disperse minute fragments of tissue evenly throughout the mixture
methods
osmotic lysis.
suitable for cell breakage. suitable for the cell that have only a cell membrane and exist as single cells such as bacteria and yeast. useless for cells that have strong carbohydrate containing cell wall such as bacteria, plant and fungi
lysozyme
works effectively for E. coli
gram +ve bscteria
chemical method
toluene (methyl benzene)
yeast
grinding
mortar and pestle
gentle grinding
animal and plant tissues
bacteria and yeast, add abrasive to the mixture. fine alumina often used to break bacteria
add fine glass beads also work well
add liquid nitrogen function to lower the temperature
avoid denaturation of compound protein
limited to small sample size
tissue homogenizer
glass or electric
for soft tissue - animal tissue (liver)
blender
relatively harsh method of breakage
handles large and small samples
plant and animal tissues
ineffective for yeast and bacteria
add fine glass beads
ultrasonic homogenizers
bacteria and yeast - can be used for long of period of time
can save time by adding glass beads in the suspension
use ultrasonic signal of high intensity
the shock and vibration due to high energy - tissue destruction
lyophilization (freeze-drying)
for concentration of samples
can be kept in an active state
involve the removal of solvent from a frozen sample
most effective fro drying or concentrating heat-sensitive materials
ice formed from the aqueous solution sublimes and is pumped from the sample vial
most freeze-dried biological materials are stable for log periods of time and remain viable for many years
precaution and limitaion
limited to aqueous solution
organic solvent lower the melting point of aqueous solution and increase the chances that the sample will melt and become denatured during freeze-drying
use solvent trap can caused damage