3.2.1.3 methods of studying cells

What is centrifugation?

A technique used to isolate different organelles in a cell → separates structures of different density

What is cell fractionation?

The process used to separate cellular components while preserving individual functions of each component

What is step 1 of centrifugation?

Homogenisation → cells are first broken open by grinding a tissue in an ice, cold isotonic buffer solution using a blender (homogeniser) → releases the organelles

Explain why an ice cold, isotonic, buffer solution is used

Ice cold → low kinetic energy minimises enzyme reactions within the cells that might cause self digestion (autolysis) of the organelles

Isotonic → prevents the osmotic movement of water in and out of organelles that might cause them to burst / shrivel

Buffer → maintains pH so proteins, especially enzymes, aren’t denatured

What is step 2?

Filtration → resulting suspension is filtered to remove cell debris e.g cell wall that haven’t burst in homogenisation → passed through a gauze

Explain the ultracentrifugation procedure that happens next

• Homogenate is poured into tube + placed into centrifuge

• Centrifuged at low speed → densest organelles e.g nuclei form a pellet / sediment at the bottom of tube

• Supernatant liquid above sediment contains rest of organelles + can be spun at a higher speed for a longer period of time → second most densest organelle forms pellet

• Procedure is repeated, increasing the speed + duration of centrifugation to obtain a series of pellets in order of decreasing density

What is the order of the organelles in decreasing density → order they’re isolated

nuclei, chloroplasts, mitochondria, endoplasmic reticulum, ribosomes, soluble proteins remain in supernatant

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Define resolution

Ability to distinguish between 2 separate points

How does a light microscope work

Light rays by lenses pass through specimen usually mounted on a glass slide

How must the specimen be prepared for light / optical microscopes

• Thin → light can pass through it + a single layer of cells is visible

• Stained → structures are visible

Which type of specimens can be looked at with a light microscope

Dead + living samples

What magnification + resolution do light microscopes have?

Magnification - 1500

Resolution - 200nm → relatively poor so small structures aren’t visible

How does an electron microscope work?

Uses beam of electrons focused by electromagnets → electrons have a shorter wavelength than light → greater resolution of 0.05nm

Why is a vacuum required for electron microscopes

Electrons are absorbed by air molecules → only dead specimens can be looked at

Why are electron microscopes important?

They can investigate the fine structure (ultrastructure) of a cell

What happens if you have a low resolution but a high magnification

Images become blurred

How to calculate magnification?

image / actual

How to convert between units?

What are the 2 main types of electron microscopes?

• Transmission electron microscope

• Scanning electron microscope

How does a TEM work?

• Beam of electrons is transmitted through specimen

• Specimen must be thin + stained using electron dense substances e.g heavy metal salts

• These substances deflect the electrons in beam + pattern that the remains electrons produce as they pass through the specimen is converted to an image

How does a SEM work?

• Specimen is coated with a thin film of heavy metal e.g gold

• Electron beam is scanned to and fro across specimen

• Electrons are reflected from surface are collected + produce an image on a viewing screen

Compare SEM + TEM