Principles of Histology Staining

Question 1

What other chemical bonds are involved in selectivity and exceptional circumstances, in relation to staining?

Answer 1

• Hydrogen bonds: important in the staining of amyloid by Congo red
• Van der Waals: important in keeping tissue fixed to the slides
• Covalent bonding: important in attaching dyes to antibodies in immunofluorescence
• Hydrophobic bonds: important in selectivity and play a major role in the staining of lipids

Question 2

What are mordants?

Answer 2

Metal salts that help bind dyes to tissues.

Question 3

What is a dye lake?

Answer 3

The dye and Mordant complex = dye lake

Question 4

What is the effect of a mordant?

Answer 4

Gives greater stability to the stain so that it is less easily removed by water, alcohol or weak acid

Question 5

Give some examples of mordants.

Answer 5

Haemotoxylin and aluminium potassium/ammonium sulphate

Question 6

What are the 2 types of staining?

Answer 6

1) progressive staining

2) Regressive staining

Question 7

What is progressive staining?

Answer 7

The dye is applied gradually to the section until the desired density of colour is reached

Question 8

What is regressive staining?

Answer 8

More common to use mordanted stains regressively. Acids or excess mordant can be used to differentiate the tissue.

Question 9

What is metachromasia?

Answer 9

Some dyes exhibit this behaviour which means they can stain different tissue components a different colour.

Question 10

What is haemotoxylin?

Answer 10

The most commonly used stain in any routine histopathology department. It is used as a nuclear stain which depicts the chromatin pattern in a dark blue/black colour.

Question 11

What cellular structures does the Periodic Acid-Schiff reaction show?

Answer 11

Demonstrates various cellular structures including carbohydrates, mucins, basement membranes and viable fungi. Histochemical method.

Question 12

What are 2-step chemical reactions involved in the PAS Reaction?

Answer 12

1) Periodic Acid
-oxidises sugars to form aldehyde groups
periodic acid is chosen as it stops the oxidation process once these aldehyde groups are formed

2) Schiff’s reagent
- contains fuchsin that has been treated to displace the chromophore group resulting in loss of colour
- reaction with aldehyde groups restores the chromophore to give a strong red/purple colour

Question 13

What causes false positive PAS reactions?

Answer 13

• improper schiff reagent preparation. dye may not have fully decolourised, may have deteriorated, out of date etc.
• certain fixatives can cause artificial production of aldehyde groups within the tissue
• preformed aldehydes can also occur naturally in small amounts in tissues

Question 14

What causes false negative PAS reactions?

Answer 14

-usually associated with poor technique, timing or reagent preparation

Question 15

What does the Perls Prussian Blue stain demonstrate?

Answer 15

• demonstrates the presence of iron within the tissue

- hemosiderosis: excess iron i the liver

Question 16

What does the Oil Red O stain demonstrate?

Answer 16

• demonstrates the presence of lipid within the tissue

- this technique can only be performed on frozen sections

Question 17

What does the Orcein stain demonstrate?

Answer 17

• presence of hepatitis B surface antigen.

- positive cells have “ground glass” like appearance.

Question 18

What is Silver Staining used for?

Answer 18

AKA silver impregnation

• involves the reduction of silver ions in solution to leave a very fine deposition of silver and silver oxide
• structures such as reticulin actually become plated with silver rather than being reversibly bound like other tinctorial dyes.

Question 19

What is Reticulin Silver staining?

Answer 19

• Oxidisation: first hexose groups are oxidised to form aldehydes
• sensitisation: aldehydes reduce silver nitrate solution in solution to leave a deposition of silver ions
• developing: transferring the section into formaldehyde further reduces the diamine silver
• toning: silver is replaced with metallic gold and the colour of the reticulin fibres change from brown to black

Question 20

What does the reticulin stain demonstrate?

Answer 20

-the structure or pattern of reticulin fibres based upon their argyrophilic nature

Question 21

What does Van Gieson’s stain demonstrate?

Answer 21

(mixture of picric acid and acid fuchsin)

-differentiates collagen from other connective tissues

Question 22

When cellular components are stained with Van Gieson’s stain, what results are observed?

Answer 22

Nuclei - Blue
Collagen - Bright Red
Cytoplasm, Fibrin, Muscle, RBCs - Yellow

Question 23

What are the functions of the epithelium?

Answer 23

• protection
• absorption
• filtration
• excretion
• secretion

Question 24

What features are characteristic of the epithelium?

Answer 24

• cells are in close proximity to each other and form sheets
• epithelial cells have an apical and basal surface
• basal surface rests of the membrane
• avascular
• regenerative capacity (undergo mitosis)