8 cell homogenisation + microscopy

Question 1

define cell homogenisation

Answer 1

• the creation of a suspension of small broken fragments of cellular / tissue material
• the suspension is called a homogenate

Question 2

what two methods is homogenisation achieved through?

Answer 2

mechanical
non- mechanical

Question 3

name four non mechanical methods of homogenisation

Answer 3

1. enzymatic
2. osmotic shock
3. freeze thaw cycling
4. detergent

Question 4

what is the enzymatic method of homogenisation?

Answer 4

enzymes disrupt the cell wall / membrane

Question 5

what is the osmotic shock method of homogenisation?

Answer 5

rapid influx of water into the cells causes bursting of the cell

Question 6

what is the freeze thaw cycling method of homogenisation?

Answer 6

damages cell walls / membranes and forms internal ice crystals

Question 7

what is the detergent method of homogenisation?

Answer 7

permeabilising the membrane

Question 8

name four mechanical methods of homogenisation

Answer 8

1. blenders
2. french press
3. sonicators
4. cell fractionation

Question 9

what is the french press method of homogenisation?

Answer 9

cells forced via a liquid through a narrow opening

Question 10

what is the sonicators method of homogenisation?

Answer 10

ultrasonic waves disrupting cells in a homogenate

Question 11

define cell fractionation

Answer 11

separation of cell components based on size and density

Question 12

give 5 steps for cell fractionation

Answer 12

1. cells homogenised in a blender to break them up
2. homogenate is centrifuged
3. pellet forms at the bottom of the tube and the liquid above is called the supernatant
4. supernatant poured into separate tube and centrifuged for longer at a higher speed
5. process repeats - differential centrifugation

Question 13

what organelles might be visible in the first four tubes after differential centrifugation?

Answer 13

1. nuclei / cellular debris
2. mitochondria / chloroplasts
3. microsomes
4. ribosomes

Question 14

what is a centrifuge?

Answer 14

• a spinning vessel device
• which applies high speed centrifugal forces to separate fluids
• based on density

Question 15

give four types of a centrifuge

Answer 15

1. microcentrifuge
2. ultracentrifuge
3. bench top centrifuge
4. fixed arm centrifuge

Question 16

what speed can a microcentrifuge go at?

Answer 16

14k - 21k g

Question 17

what speed can an ultracentrifuge go at?

Answer 17

up to 1 mil g

Question 18

what are each of the centrifuges designed for?

Answer 18

micro - small volumes
ultra - tiny particles
bench top - routine tasks
fixed angle - rapid separation

Question 19

define the sedimentation rate

Answer 19

how fast it takes a particle to settle

Question 20

what is the unit for the sedimentation rate?

Answer 20

Svedberg unit (S)

Question 21

what does 1 S equal?

Answer 21

10^-13 seconds

Question 22

what does the sedimentation rate depend on?

Answer 22

• mass
• density
• shape

Question 23

what is an alternative method of centrifugation?

Answer 23

density gradient centrifugation

Question 24

describe how density gradient centrifugation works

Answer 24

1. spins particles through a liquid gradient
2. separates particles by their density

Question 25

what is a pro and a con for density gradient centrifugation?

Answer 25

pro - more precise
con - takes longer