8 cell homogenisation + microscopy Flashcards
define cell homogenisation
- the creation of a suspension of small broken fragments of cellular / tissue material
- the suspension is called a homogenate
what two methods is homogenisation achieved through?
mechanical
non- mechanical
name four non mechanical methods of homogenisation
- enzymatic
- osmotic shock
- freeze thaw cycling
- detergent
what is the enzymatic method of homogenisation?
enzymes disrupt the cell wall / membrane
what is the osmotic shock method of homogenisation?
rapid influx of water into the cells causes bursting of the cell
what is the freeze thaw cycling method of homogenisation?
damages cell walls / membranes and forms internal ice crystals
what is the detergent method of homogenisation?
permeabilising the membrane
name four mechanical methods of homogenisation
- blenders
- french press
- sonicators
- cell fractionation
what is the french press method of homogenisation?
cells forced via a liquid through a narrow opening
what is the sonicators method of homogenisation?
ultrasonic waves disrupting cells in a homogenate
define cell fractionation
separation of cell components based on size and density
give 5 steps for cell fractionation
- cells homogenised in a blender to break them up
- homogenate is centrifuged
- pellet forms at the bottom of the tube and the liquid above is called the supernatant
- supernatant poured into separate tube and centrifuged for longer at a higher speed
- process repeats - differential centrifugation
what organelles might be visible in the first four tubes after differential centrifugation?
- nuclei / cellular debris
- mitochondria / chloroplasts
- microsomes
- ribosomes
what is a centrifuge?
- a spinning vessel device
- which applies high speed centrifugal forces to separate fluids
- based on density
give four types of a centrifuge
- microcentrifuge
- ultracentrifuge
- bench top centrifuge
- fixed arm centrifuge
what speed can a microcentrifuge go at?
14k - 21k g
what speed can an ultracentrifuge go at?
up to 1 mil g
what are each of the centrifuges designed for?
micro - small volumes
ultra - tiny particles
bench top - routine tasks
fixed angle - rapid separation
define the sedimentation rate
how fast it takes a particle to settle
what is the unit for the sedimentation rate?
Svedberg unit (S)
what does 1 S equal?
10^-13 seconds
what does the sedimentation rate depend on?
- mass
- density
- shape
what is an alternative method of centrifugation?
density gradient centrifugation
describe how density gradient centrifugation works
- spins particles through a liquid gradient
- separates particles by their density
what is a pro and a con for density gradient centrifugation?
pro - more precise
con - takes longer
