Lecture 2: Tools of Histology

Question 1

Histology:

Answer 1

Histology: microscopic study of cells, tissues, organs

Question 2

Cell:

Answer 2

Cell: smallest Living Unit

Question 3

Tissue:

Answer 3

Tissue: organized group of cells and their products that function in a collective manner

Question 4

Organ:

Answer 4

Organ: structure composed of 2 or more tissue types that performs and specific function

Question 5

Organ system:

Answer 5

Organ system: 2 or more organs that perform a common function

Question 6

• Decimeter
• Centimeter
• Millimeter
• Micrometer (microns)
• Nanometer

Answer 6

Question 7

Resolution (definition)

Answer 7

The smallest distance at which 2 points can be distinguished as seperate entities

• Smaller resolution = stronger

Question 8

Resolution of:

• human eye =
• light microscope =
• transmission electron microscope =
• scanning electron microscope =

Answer 8

• Resolution of human eye: 100 um
• Resolution of light microscope: 0.2 um (200 nm)
• Resolution of transmission electron microscope: 3 nm
• Resolution of scanning electron microscope = 1 nm

Question 9

Ultrastructure

Answer 9

Ultrastructure = cellular structures that can only been seen using an electron microscope

Question 10

Light microscope:

1. ____ travels through ____
2. The ____ lens in the ____ (_x) and the ____ lenses (_x, _x, _x, _x) magnify the image
   
   • Low = _x
   • Medium = _x
   • High = _x
   • Oil immersion = _x

Answer 10

• Light travels through a thin section of tissue.
• The ocular lens in the eyepiece (10x) and the objective lenses (4x, 10x, 40x, 100x) magnify the image.
  
  • Low = 4x
  • Medium = 10x
  • High = 40x
  • Oil immersion = 100x

Question 11

Virtual microscope (5 steps)

Answer 11

1) Slide collections
2) Slide Scanner
3) Servers
4) Virtual microscope software
5) Histology lab and mobile devices

Question 12

Routine slide preparation for light microscopy (7)

Answer 12

1) Fixation
2) Dehydration
3) Clearing
4) Infiltratoin
5) Embedding
6) Sectioning
7) Mounting on slide, removal of paraffin,

hydration, staining.

Question 13

Fixation

Answer 13

① Fixation = Preserve with formalin (so does not decompose)

• cross-linking of proteins and inactivation of enzymes (Formaldehyde polymerizes so we add something to it to to stop it - now called formalin)
• Macromolecules (like glycogen) get taken out of tissue

Question 14

Dehydration with alcohol

Answer 14

② Dehydration = use of alcohol to remove all water

• To later embed in block of wax (so we can cut it into thin slices) we need to remove water with alcohol because water does not mix well with wax

Question 15

Clearing

Answer 15

③ Clearing = use of organic solvent (e.g. xylol) to remove alcohol. The tissue is now saturated with organic solvent, which can dissolve paraffin used in the next step

• Called “clearing” because this step renders the tissue transparent
• Will also wash out lipids with clearing agent

Question 16

Infiltration

Answer 16

④ Infiltration = melted paraffin penetrates the tissue

Question 17

Embedding

Answer 17

⑤ Embedding = paraffin placed in a mold and hardens, Block is trimmed

Question 18

Sectioning

Answer 18

⑥ Sectioning = tissue is sectioned into thin slices (5-10um)

Question 19

Mounting on slide, removal of paraffin,

hydration, staining.

Answer 19

⑦ Tissue slice is mounted on a glass slide.

• Paraffin is removed and the tissue is rehydrated.
• Stain is applied to color the clear cellular components so that they may be seen.

Question 20

Artefact

Answer 20

Artefact = structural abnormality not present in the living tissue as a result of the preparation process

• Something you see under microscopy that is not there in real life
  
  • tear, fold, air bubble

Question 21

Planes of cut (3)

Answer 21

• Cross section = the specimen was sectioned along its short axis (cut in top/bottom halves)
• Longitudinal section = the specimen was sectioned along its long axis (cut in left/right halves)
• Oblique section = the specimen was sectioned on an angle

Question 22

4 chemical building blocks of tissues

Answer 22

1) nucleic acids
2) proteins
3) carbohydrates
4) lipids
* Tissue becomes colorless during “clearing”, so we need to color it. You can color (stain) any of the basic macromolecules*

Question 23

Hematoxylin:

• behaves like
  
  • has what charge
• stains what (generally)
  
  • most commonly stains what (and why)
• causes what color change

Answer 23

Hematoxylin:

• behaves like a base
  
  • ​has positive charge
• stains things that are basophilic
  
  • stains nucleic acids (most common example)
    
    • why: + charged in solution, attracted to - charge in cell (- phosphate in nucleic acids)
    • So, anything that turns blue = contains nucleic acids

(Base = Blue = Basophilic stains)

Question 24

Eosin:

• acts like:
  
  • what charge:
• what does it stain (generally):
  
  • most commonly stains what:
    
    • why?
• what color change:

Answer 24

Eosin:

• Acts like an acid
  
  • has a negative charge
• Stains things that are acidophilic
  
  • Stains proteins (most common example)
    
    • why: - charge = acidic in solution, attracted to the + structures in cells like proteins (with ionized amino groups +)
• Stains things pink

Question 25

Basophilic vs. Acidophilic

Answer 25

* _Basophilic:_ a structure that attracts basic dyes and is therefore stained by basic dyes (stained by hematoxylin) * _Acidophilic:_ a structure that attracts acidic dyes and is therefore stained by acidic dyes (stained by eosin)

Question 26

Using H&E together: * which is used first? * what colors?

Answer 26

* Often the two stains are used one after the other (hematoxylin always first) on the same tissue. * hematoxylin can look purple in the final specimen (eosin is pink)

Question 27

Metachromatic

Answer 27

_Metachromatic:_ the property of staining a different color than the dye. * Occurs when a basic dye reacts with a tissue component. * *Example: Purple cell shown is metachromatic because it shifted the dye's color from blue to purple*

Question 28

Periodic acid-Schiff (PAS) stain * what does it stain? * why is it used? * color changes?

Answer 28

_Periodic acid-Schiff (PAS):_ stain is used for **carbohydrates** * in routine slide preparation with H&E: you lose glycogen during fixation, so will look like white holes where glycogen used to be * *if on a low carb diet, you want to PAS on the sugars* * can use other slide prep techniques to keep the sugars in the cell and then use PAS to stain * PAS stains sugars **magenta**

Question 29

_Basement membrane:_ * function * what stain is best for basement membranes and why * what does it look like when stained in PAS with hematoxylin vs. H&E

Answer 29

The _basement membrane_ provides an attachment site for certain cell types. * contains sugars so it's best stained with PAS * when stained in PAS with hematoxylin = pink * when stain in H&E = white (hard to see)

Question 30

_Osmium tetroxide:_ * what is it used to stain * common example * why is it used to stain this * what color does it stain

Answer 30

_Osmium tetroxide:_ * used to stain **lipids** * *example:* used to stain **myelin sheath** surrounding axons (because myelin sheath contains lipids) * in routine slide preparation with H&E, lipids get washed out during clearing, so can prep cells in other ways that leave lipids in and then use Osmium tetroxide to stain * will stain lipids **black**

Question 31

* Glycogen lost during: * Lipids lost during:

Answer 31

* Glycogen lost during **fixation** * Lipids lost during **clearing**

Question 32

What stains each of the following macromolecules? * lipids * proteins * nucleic acids * carbohydrates

Answer 32

What stains each of the following macromolecules? * lipids = **Osmium Tetroxide (black)** * proteins = **Eosin (pink)** * nucleic acids = **Hematoxylin (blue)** * carbohydrates = **PAS (magenta)**

Question 33

which macromolecule is lost during routine slide preparation? A) Lipids B) Nucleic Acids C) Proteins D) Carbohydrates

Answer 33

which macromolecule is lost during routine slide preparation? **A) Lipids** * *The organic solvent used during clearing will dissolve and wash out the lipids*

Question 34

Answer 34

* **B = Oblique angle (correct answer)** * *This plane lies neither along the short nor the long axis of the tube. It is at an oblique angle.* * A = a cross-section because the plane is along the short axis of the tube * C = longitudinal cut because the plane is along the long axis of the tube.