Lecture 1 (Revised)

Question 1

Steps required in preparation of living tissues for microscopic examination include?

Answer 1

-Fixation
-Dehydration
-Clearing
-Embedding
-Sectioning
-Mounting
-Staining

Question 2

Light Microscope is composed of?

Answer 2

A light source, condenser lens, objective lens, occular lens and a mechanical stage for supporting the specimen

Question 3

Transmission election microscope is composed of?

Answer 3

Uses an electron beam as an “illuminating” beam, magnets for lens and a fluorescent screen to visualize structures of interest

Question 4

Fixation?

Answer 4

To avoid autolysis or degradation by bacteria and to preserve the structure and molecular composition

Question 5

What chemicals do you use for Fixation?

Answer 5

-Formaldehyde (LM)
-Glutaraldehyde (EM)

Question 6

Formaldehyde?

Answer 6

(LM) Reacts with amine groups to stabilize same

Question 7

Glutaraldehyde?

Answer 7

(EM) Cross-links proteins, followed by a second fixation in buffered Osmium Tetroxide that preserves membranes

Question 8

Dehydration?

Answer 8

Water is extracted from fixed tissue fragments by bathing them successively in a graded series of ethanol/water mixtures

Question 9

Ethanol?

Answer 9

-Used for Dehydration
-Fixed tissue is bathed in serious of ethanol and water mixtures
-Last solution is 100% ethanol

Question 10

Clearing?

Answer 10

Making tissue clear or transparent so we can see through it

Question 11

Xylene?

Answer 11

-Solution used in Clearing
-Xylene replaces ethanol is tissue to allow paraffin to penetrate tissue
-Xylene is miscible with embedding medium which makes tissue clear

Question 12

Embedding?

Answer 12

Embedding tissue and making it a harder mold so we can cut it, if it is soft it’ll be hard to cut

Question 13

Paraffin Wax?

Answer 13

-Step 1: Paraffin is melted to 58-60 degrees Celsius
-Step 2: Tissue is placed in melted wax –> heat causes xylene to evaporate
-Step 3: Paraffin cools and hardens

Question 14

Epoxy Resin?

Answer 14

(EM) Used in cases where tissue has been cleared with plastic solvents

Question 15

Chemicals used in Embedding?

Answer 15

-Paraffin Wax
-Epoxy Resin (EM)

Question 16

Sectioning?

Answer 16

Cooled and hardened blocks of paraffin containing tissue are sectioned on a slicing machine called a microtome for LM or on an ultramicrotome for transmission EM

Question 17

Methods used in Sectioning?

Answer 17

-Microtome Machine
-Section Mounting
-Section Staining

Question 18

Microtome Machine?

Answer 18

Cuts 6u thick for LM and 0.06u thick for EM via ultramicrotome

Question 19

Section Mounting?

Answer 19

Glass slides for LM and copper wire grid for EM

Question 20

Section Staining?

Answer 20

-Step 1: Dissolve paraffin wax with xylene
-Step 2: Re-hydrate section through a series of alcohols stained with Hematoxylin (H-stain) in water
-Step 3: De-hydrate section through a series of alcohols stained with Eosin (E-stain)

Question 21

H & E Stain?

Answer 21

-Most Used
-Hematoxylin + Eosin

Question 22

H?

Answer 22

-Hematoxylin
-Stains the cell nuclei and RNA-rich organelles blue/purple
-Basic dye (+ charge) –> basophilia (reacts with negatively charged groups)

Question 23

E?

Answer 23

-Eosin
-Stains mitochondria, secretory granules and extracellular material such as collagen pink
-Acidic dye (- charge) –> acidophilic (reacts with positively charged groups)

Question 24

Artifacts of Preparation?

Answer 24

Artificial spaces, loss of molecules, shrinkage caused by tissue preparation

Question 25

Shrinkage causes?

Answer 25

-Fixation -Dehydration (in ethanol) -Heating (in melted paraffin during clearing)

Question 26

Artificial Spaces?

Answer 26

Holes between cells and extracellular components

Question 27

Artificial Spaces cause?

Answer 27

-Shrinkage -Loss of molecules (due to chemicals used in tissue preparation)

Question 28

Artifacts of Prep?

Answer 28

Collection of distortions caused by prep procedures (shrinkage, artificial spaces, etc.)

Question 29

Artifacts of Prep?

Answer 29

Collection of distortions caused by prep procedures (shrinkage, artificial spaces, etc.)

Question 30

Resolution?

Answer 30

-Resolving Power -Smallest distance seen between 2 particles -Depends on quality of objective lens -Quality of an image (clarity and detail) depends on resolving power

Question 31

Light Microscope?

Answer 31

(LM) -Max resolving power= 0.2u -Magnification= 1,000-1,500x

Question 32

Transmission Electron Microscope?

Answer 32

(EM) -Max resolving power= 3nm -Max magnification= 400,000x -Magnification= 120,000x

Question 33

H&E preparations do not reveal much about the?

Answer 33

Chemical composition