Lecture 3 Learning Objectives

Question 1

List basic steps for tissue fixing and embedding

Answer 1

Fixing
Dehydration
Removal of alcohol
Embedding

Question 2

What is the purpose of tissue fixing

Answer 2

Prevents further deterioration of the tissue specimen and helps harden the tissue before embedding and sectioning

Question 3

What is the purpose of embedding tissues and sectioning

Answer 3

Embedding in was preserves the cells and tissue structure, sectioning allows us to view various cross sections throughout the tissue

Question 4

What are the advantages and disadvantages of using formalin as a fixing agent

Answer 4

Formalin reacts with amino acids of the tissue proteins and stabilized tissue structures
Con - not good for fine cytological detail - distorts specimen

Question 5

What is the purpose for the dehydration and hydration cycle used for tissue processing?

Answer 5

Dehydration is used to clear the water after fixing in order to prepare the tissue for embedding. A series is used to gradually replace water with alcohol to avoid excessive distortions or plasmolysis. Hydration is done to prepare the specimen to be stained, most stains being water soluble. Another round of dehydration is done to preserve the specimen for long-term storage and viewing.

Question 6

Describe the methods used to prepare thin slices of tissue

Answer 6

MIcrotomes - hand and rotary

Question 7

What are the advantages of using a rotary microtome over hand-held microtomes?

Answer 7

Faster processing, weight allows user to be more precise -reduces error to vibration. Fixed thickness of each section

Question 8

How does tissue sectioning for TEM differ from that used in paraffin embedded specimens

Answer 8

Thinner cross sections, more fragile. Can’t be normally handles must be floated onto grid

Question 9

Why is it preferred that tissues be stained for observation

Answer 9

Tissues are typically colorless, color brings detail. Can selectively stain for functional groups, structures ect.

Question 10

What steps are necessary for staining of paraffin embedded structures?

Answer 10

Removal of paraffin wax switch xylene. Removal of xylene with rehydration using alcohol. Stains are applied here before dehydration series and replacement with xylene.

Question 11

What are the components of the most common staining techniques for tissues in general

Answer 11

Hemotoxylin and Eosin

Question 12

Hemotoxylin

Answer 12

Stains nuclear materials dark blue, light blue or purple and behaves like a basic dye due to mordant

Question 13

Eosin

Answer 13

Stains cytoplasmic components and extracellular material with yellow and pinkish color - acidic dye

Question 14

Compare basic dyes to acidic dyes and give examples

Answer 14

Acidic dyes bind tissue components by forming electrostatic linkages with cationic groups such as amino acids in proteins - cytoplasmic material will by dyed - Mallory’s stain, acid fuchsin, aniline blue, eosin
Basic dyes react with ionic groups of tissue components such as phosphate groups, sulfate groups, carbonyl groups - methyl green, methylene blue, pyronine G, Toluidine blue

Question 15

What does metachromasia refer to? Give an example

Answer 15

Dye changes color after reacting with tissue component

Toluidine blue stains cartilage ground substance or mast cell granules

Question 16

What does histochemistry refer to?

Answer 16

The study of chemistry of cells and tissues

Question 17

What does Immunochemistry refer to?

Answer 17

The study of presence of specific tissue constituents

Question 18

Describe the Schiff reaction

Answer 18

Depends on formation of aldehyde groups after exposure to HCL or acid (exposes aldehyde groups on deoxyribose)

Question 19

Compare PAS reaction with the Feulgen reaction

Answer 19

Feulgen and PAS use mild hydrolysis with HCL or periodic acid, respectively

Question 20

Acid Fixatives fix

Answer 20

Chromatin, Nucleoli, Spindle fibers

DO NOT STAIN mitochondria or nucleoplasm

Question 21

Basic Fixatives fix

Answer 21

Mitochondria. Chromatin is dissolved

Question 22

Basic dyes

Answer 22

React with anionic groups of tissues such as phosphate groups, sulfate groups, and carboxyl groups

Question 23

Acidic Dyes

Answer 23

Bind to tissue components by forming electrostatic linkages with cationic groups such as the AA of proteins

Question 24

Best Carmine

Answer 24

Stains glycogen deposits

Question 25

RNA organelles can be stained with

Answer 25

Basic dyes