Methods

Question 1

Light microscope

Answer 1

routine laboratory scope used for studying tissue sections

Question 2

Transmission electron microscope - TEM

Answer 2

study cytology/internal structures of cells; study of electron micrographs

Question 3

Scanning electron microscope - SEM

Answer 3

study surface features of cells/tissues; 3-D picture of tissue

Question 4

Polarizing microscope

Answer 4

facilitates determination of whether or not biological materials have different refractive indicies along different optical axes

Question 5

Phase microscope

Answer 5

study living tissue; works on principle of different refractive indicies of cellular & sub-cellular components

Question 6

interference microscope

Answer 6

modification of phase microscope used for study of living tissue

Question 7

fluorescence microscope

Answer 7

uses UV light as light source; used to examine the presence of fluorescent material in tissue sections

Question 8

confocal scanning microscope

Answer 8

uses laser energy beam; used to opticallhy section a cell and with the appropriate computer equipment can reconstruct a 3-D image of the cell

Question 9

Fixation techniques - preparation steps

Answer 9

1. Fixation w/ 10% buffered formalin, glutaraldehyde, alcohol or osmic acid
2. Dehydration w/ alcohol
3. Clearing of alcohol w/ agent miscible in paraffin (toluene, xylene, benzene)
4. Infiltration & Embedding - replace clearing agent w/ paraffin, methacrylate, celloidin or gelatin
5. Sectioning 5-7micrograms thick w/ microtome
6. Staining w/ Hematoxylin and eosin (H&E)

Question 10

How are frozen histological sections useful?

Answer 10

Used in surgical biopsies and in research studies for the localization of enzymes

Question 11

Name some artifacts

Answer 11

post-mortem degeneration, shrinkage, precipitates, wrinkles & folds, nick due to knife, mishandling/pinching of tissue
ALL of these can lead to misinterpretation

Question 12

What is the difference b/w acidic and basic staining?

Answer 12

Acidic dyes are negatively-charged and form salts w/ positively charged tissues. Basic dyes are positively-charged and form salts w/ negatively-charged tissues

Question 13

What is an H&E stain and how is it used?

Answer 13

Hematoxylin - blue to purple. +, basic stain that stains basophilic substances. E.g. nuclear components such as DNA due to large PO4 groups.
Eosin - red to pink. -, acidic stain that stains acidophilic (eosinophilic) substances. E.g. proteins w/ large numbers of basic groups associated w/ side chains.

Question 14

Match the following special staining techniques w/ appropriate tissues:
Stains: Trichrome, Elastic & Silver
Reticular fibers, silver impregnation, elastic

Answer 14

Trichrome - collagen
Elastic - elastic fibers/tissue
Silver - Reticular

Question 15

Match the following special staining techniques w/ appropriate tissues:
Stains/techniques: Feulgen, Periodic acid-Schiff (PAS), Oil red O & Sudan Black, Immunochemistry

Answer 15

Feulgen - DNA (leukofuchin –> aldehyde Schiff)
Periodic acid-Schiff (PAS) - carbohydrate
Oil Red O and Sudan Black - Fats
Immunochemistry - proteins

Question 16

Three common methods for labeling & identifying specific proteins by immunochemistry

Answer 16

1) antibody is coupled with a fluorescent compound & studied w/ a fluorescent microscope
2) antibody coupled to peroxidase & studied w/ light electron microscope
3) antibody is coupled to ferritin or gold and studied w/ an electron microscope

Question 17

Why are RBCs useful in determining approximate magnifcation?

Answer 17

RBCs ~ 7micrograms in diameter and paraffin sections are usually 6-7micrograms thick. plastic sections (methacrylate) are usually 1-3 micrograms thick.