Vital, supravital and intravital staining are three different types of staining techniques. However, vital and supravital terms are often used interchangeably.
Let’s look at some of the major differences in staining between the three and why these terms shouldn’t be used interchangeably.
1. VITAL STAINING
1.Contrary to the name, vital stains are taken up by dead cells and not by living cells.
2. Demonstration of nuclear staining using a vital stain signifies cells death, because living cells are impermeable to the stain.
3. Example: Tryptan blue and propiodine iodine
VITAL STAINS ATE TOO BULKY OR TOO CHARGED THEY CANNOT ENTER LIVE CELLS- Hence only stain dead cells.
2. SUPRAVITAL STAINS
1. Supravital staining is a method of staining used in microscopy to examine living cells that have been removed from an organism.
2. Those that enter and stain living cells are called supravital stains
3. Examples New Methylene Blue and Brilliant Cresyl Blue for reticulocyte staining.
3. INTRAVITAL STAIN
1. Intravital staining of living cells is done by injecting the dye into any part of the animal body (either intravenous, intraperitoneal or subcutaneous), producing specific coloration of certain cells, particularly those of the reticulo-endothelial system.
2. Common dyes used are lithium, carmine and India ink.
MORDANT and ACCENUATOR both are terms used in staining, for substances which enhance staining. However, both have different mechanisms of action. We will be looking at the difference between mordant and accenuator in staining.
Differences between mordant and accenuator
MORDANT serves as a link or bridge between the tissue and the dye, to make the staining reaction possible.
The mordant combines with a dye to form a colored “lake”, which in turn combines with the tissue to form a “tissuemordant-dye-complex”.
Examples of mordants are potassium alum with hematoxylin in Ehrlich’s hematoxylin, and iron in Weigert’s hematoxylin.
Diseases in which PAS staining can be used for diagnosis
Glycogen storage disease
Paget’s disease of the breast
Staining macrophages in Whipple’s disease
Alveolar soft part sarcoma
Pulmonary alveolar proteinosis
3. LET’S MOVE ON TO ALCIAN BLUE STAINING
Group 1 is always Alcian blue negative
Group 2 is always Alcian blue positive. Hyaluronic acid is positive at a pH of 2.5 and Chondroitin sulfate is positive at a pH of 0.5.
Group 3 is the confusing part, we need to divide it into three parts for better understanding
1.Neutral mucins– Always alcian blue negative.
2. Sialomucins or carboxylated mucins– Alcian blue positive at a pH of 2.5
3. Sulfated mucins– Alcian blue positive at a pH of 1.
Group 4 is Alcian blue negative.
Alcian blue stain is frequently combined with PAS and High iron diamine for diagnostic purposes (discussed below).
4. COMBINED PAS AND ALCIAN BLUE STAINING
Primary use of PAS+Alcian blue combined staining is to distinguish neutral and acidic especially sialomucns.
Diagnostic utility: 1. To distinguish eccentric gastro-esophageal junction from Barret’s mucosa
2. Gastric intestinal metaplasia
Remember: Key finding an diagnostic feature intestinal metaplasia is the presence of goblet cells. Goblet cells and surface epithelial cells of stomach may be difficult to distinguish in routine Hematoxylin and Eosin stained sections.
Goblet cells are positive with both PAS and Alcian blue but gastric epithelial cells are positive with PAS alone. When combined PAS+Alcian blue staining is performed, Goblet cells stain PURPLE and Gastric foveolar cells stain MAGENTA.
5. COMBINED ALCIAN BLUE AND HIGH IRON DIAMINE
Combined PAS + High iron diamine is used to distinguish between sulfomucins and sialomucins.
Diagnostic utility: Large bowel metaplasia can be identified, since it contains both sulfomucins and sialomucins, in contrast to small bowel metaplasia (sialomucins only)
With Alcian blue + High iron diamine staining- Sulfomucins: BROWN COLOR and Sialomucins: BLUE COLOR
6. SUMMARY OF CARBOHYDRATE STAINING
PAS is always negative in mesenchymal and connective tissue mucopolysaccharides
Alcian blue is always negative in neutral mucins
PAS+Alcian blue is key to distinguish ectopic gastric mucosa or eccentric gastroesophageal junction from Barret’s mucosa in the esophagus.
Hematoxylin is extracted from the heartwood of the tree Hematoxylin campechianum.
Hematoxylin is not a stain itself. Hematin, a major oxidation product of hematoxylin is responsible for the color.
Hematin is produced in two ways (a) Natural oxidation OR Ripening (b) Chemical oxidation.
Ripening is done by exposing the solution to light and air. This is a slow process and takes up to 3-4 months.
Ehrlich’s hematoxylin and Delafield’s hematoxylin are obtained by natural oxidation.
A mordant is a substance, usually a metal which helps bind the dye with the tissue strongly.
Sodium Iodate is the mordant used in Meyer’s hematoxylin and Mercuric Oxide used in Harris hematoxylin.
Aluminum is the most common mordant.
Carrazi’s hematoxylin has a staining time of 1 minute- shortest time for any hematoxylin stain. Hence it is used to stain frozen section slides.
Iron hematoxylins are preferred connective tissue stains. Due to the acidity of dye solutions in connective tissue staining ( picric acid in Van Gieson staining), standard alum hematoxylins are decolorized. Iron hematoxylins such as Wiegert’s hematoxylin are resistant to the acidic environment and should be used in these techniques