Histology

Question 1

For Skin (structure), give the Type of Structure and Type of Basic Tissue.

Answer 1

Type of structure = Organ
Type of basic tissue = Epithelial and Connective Tissues

Question 2

For Fascia, Tendons, Aponeurosis, Muscle, Bone (structure), give the Type of Structure and Type of Basic Tissue.

Answer 2

Fascia = Organ/Tissue - Connective Tissue
Tendon = Organ/Tissue - CT
Aponeurosis = Organ/Tissue CT
Muscle = Organ/Tissue - Muscular Tissue
Bone = Organ/Tissue - Variety of CT

Question 3

For Nerves (structure), give the Type of Structure and Type of Basic Tissue.

Answer 3

Type of Structrue = Tissue/Organ/System
Type of basic Tissue = Nervous Tissue

Question 3

For Blood Vessels (structure), give the Type of Structure and Type of Basic Tissue.

Answer 3

Type of Structure = Organ/Tissues
Type of Basic Tissue = Epithelial, CT, Muscular

Question 4

What is the equivalence between nm and Angstrom units?

Answer 4

1nm = 10 A

Question 5

What is the average thickness of a section?
What is the average diameter of a cell?
What is the average diameter of a lysosome?
What is the average thickness of plasma membrane?

Answer 5

Section = 5um
Diameter of cell = 10um
Diameter of a lysosome = 200 nm
Thickness of PM = 75A (7.5 nm)

Question 6

For a light microscope, what are the steps required for preparation of a tissue for microscopic examination?

Answer 6

1. Chemical fixation (formalin)
2. Dehydration (ethanol because Xylene and water don’t mix and Xylene is needed to allow proper infiltration of paraffin)
3. Clearing (Xylene)
4. Infiltration (Xylene/paraffin)
5. Embedding (paraffin)
6. Sectioning (microtome)
7. Mounting (glass slide)
8. Removal of paraffin (Xylene)
9. Rehydration (100% ethanol → 100% H2O by steps)
10. Staining (H&E) and/or histochemical reaction)
11. Light microscopy

*Paraffin = Wax

Question 7

For electron microscopy, what are the steps required for preparation of a tissue for microscopic examination?

Answer 7

1. Chemical fixation (gluteraldehyde and osmium tetroxide)
2. Dehydration (ethanol)
3. Clearing (propylene glycol because it dissolves plastic)
4. Infiltration (propylene glycol/plastic)
5. Embedding (plastic)
6. Sectioning (ultramicrotome)
7. Mounting (copper grid)
8. Staining (lead citrate and/or histochemical reaction)
9. Transmission with EM

Question 8

For a light microscope, what are the steps required for preparation of a tissue for microscopic examination when freezing the tissue instead of doing chemical fixation?

Answer 8

1. Freezing
2. Sectioning (cryostat)
   From there, same steps:
3. Mounting (glass slide)
4. Removal of paraffin
5. Rehydration
6. Staining (H&E)
7. Light microscope

Question 9

What does the dehydration step of tissue preparation involve?

Answer 9

Start with tissue in 100% H2O solution and step-by-step go to a 100% ethanol solution
1. 50% EtOH/50% H2O
2. 60% EtOH/40% H2O
3. 70% EtOH/30% H2O
…

*Do opposit for rehydration

Question 10

What are the 2 main methods of chemical fixation?

Answer 10

*Fixation stops any biological process, freezes the organ in time
1. Perfusion = injecting a fix in the artery/circulation of the specimen freezing it inside-out
- Best to preserve resolution (faster)
- Used for EM or LM

2. Immersion = anesthetize the specimen, remove the organ and immerse it into fix solution, fixing it outside-in
   - for light microscope only

Question 11

What solutions where/are used for Light miscroscopy fixation?

Answer 11

Before:
- Formalin (modifies nucleic acids)
- Mercuric Chloride (toxic)
- Pitric Acid (explosive)

Now:
Boin’s Fluid = Acetic Acid, Pitric Acid, Formalin (formaldehyde), Water

*For perfusion or immersion

Question 12

What solutions are used for electron miscroscopy fixation?

Answer 12

Glutaaldehyde (2.5% - 5%)

Question 13

What substances are used as paraffin solvent and epoxy solvents in tissue preparation for microscopy?

Answer 13

Paraffin solvent = Xylene (LM)
Epoxy (plastic) Solvent = propylene glycol (EM)

Question 14

What are the most comon dyes in LM speciment preparation?

Answer 14

H&E:
1. Hematoxylin = basic dye
2. Eosin = acidic dye

Question 15

What structures do Hematoxylin and Eosin stain?

Answer 15

Hematoxylin = basic dye → stains basophilic structures with blue-purple hue
- stains cell nucleus and other acidic structure

Eosin = acidic dye → stains eosinophilic structure bright pink
- Stains cytoplasm and collagen

Question 16

What are basophilic and eosinophilic structures?

Answer 16

Basophilic structures → usually the ones containing nucleic acids, ex: ribosomes, chromatin-rich cell nucleus, RNA-rich cytoplasmic regions

Eosinophilic structures → generally composed of intracellular or extracellular proteins
- Most of cytoplasm is eosinophilic

*Structures do not have to be acidic or basic, terminology is based on affinity to the dye

Question 17

What is the end-point of a LM vs an EM?

Answer 17

LM = Eye
EM = Photographic plate/phosphorescent screen

Question 18

In the EM, what is responsible for generating electrons?

Answer 18

Cathode generate e- which then travel in a high vacuum

Question 19

What are electron dense and electron lucent structures?

Answer 19

Electron lucent structure = no e- because they are reflected when hitting lead citrate (more tissue)

Electron dense structure = more electrons on the screen as then pass direclty through the plastic

*Citrate binds to tissue

Question 20

What are the wavelengths of light and of electrons? What does it mean for microscopy definition?

Answer 20

wv length of Light = 550nm, therefore d=200nm
wv length of e- = 0.02nm, therefore d=1nm

Light microscopy = 1000X magnitude
EM = 1600,000X magnitude (1.6 million with camera zoom)