Lecture 1

Question 1

Preparation For Light Microscopy

Answer 1

1. Fixation- Formalin (formaldehyde) which reacts with tissues to cross link proteins and other substances
2. Dehydration- Alcohol or acetone
3. Embedding- paraffin or plastic
4. Sectioning- generally 2-10 um thick
5. Mounting- glass slide

Question 2

Hematoxylin and eosin (H and E)

Answer 2

H has positive charge and stains negatively charged structures (nucleus). E is negatively charged and stains positively charged substances (cytoplasm).

Question 3

Periodic Acid Schiff Stain

Answer 3

For carbohydrates like sugars (glycogen and glycoproteins)

Question 4

Aldehyde Fuchsin

Answer 4

Stains Elastic Fibers and b cells of the pancreatic islets. Connective tissue

Question 5

Orcein

Answer 5

Stains collagen fibers

Question 6

Silver Stain

Answer 6

Stains golgi apparatus and reticular fibers

Question 7

Resolution of Light Microscopy

Answer 7

• about 0.2 microns.

- useful Magnification of about 1,500 times

Question 8

Resolution of Transmission Electron Microscope

Answer 8

• about 1-1.45 nanometer

- useful magnification greater than 500,000 times

Question 9

Resolution of Scanning Electron Microscope

Answer 9

• about 2 nm

- useful magnification is greater than x100,000

Question 10

Preparation for TEM

Answer 10

1. Fixation - with glutaraldehyde that fixes proteins and nucleic acids followed by osmium tetroxide that fixes lipids.
2. Dehydration
3. Embeddign: plastic such as Epon
4. Sectioning: 20-100 nm thick using ultramicrotome using glass or diamond knife
5. Mounting: wire mesh
6. Staining: heavy metals are used to enhance electron density

Question 11

Freeze Fracture

Answer 11

• method of specimen preparation
• follows the hydrophobic region of the membrane exposing 2 fracture faces
• the E face- inwardly facing outer half
• P face- the outwardly facing inner half

Question 12

Preparation of Scanning Electron Microscopy

Answer 12

1. Fixation and dehydration - same as TEM
2. Dried using the critical point method to avoid drying artifacts
3. mounting - glued to a metal stub
4. coated with metal (gold/palladium) so it will enhance emission of electrons