STAINING PRINCIPLES
Dr. Manjunatha, K.P.1and Dr. Mohan, H.V.2
Assistant Professor, Department of Veterinary Pathology, Veterinary College, KVAFSU, Hebbal, Bengaluru-560024
Assistant Professor, Department of Veterinary Public Health and Epidemiology, Veterinary College, KVAFSU, Hebbal, Bengaluru-560024
Staining is a technique used in microscopy to enhance the contrast of the microscopic image.
Contrast is defined as the difference in light intensity between the image and the adjacent background relative to the overall background intensity. In general, a minimum contrast value of 0.02 (2 percent) is needed by the human eye to distinguish differences between the image and its background.
There are hundreds of stains available.
Classification of Stains/Dyes:
Based on nature of stain:
- Acid stains: Stains which have negative charge. So they will bind to positively charged cell structures.
Ex: Eosin, Nigrosin, Picric acid, Acid fuschin, India ink.
- Basic stains: Stains which have positive charge. So they will bind to negatively charged cell structures.
Ex: Haematoxylin, Methylene blue, Crystal violet, Safronin, Basic fuschin.
- Neutral stains: Combination of acidic and basic dyes.
Ex: Giemsa stain (Methylene blue & Eosin)
Based on the source:
- Natural dyes: Obtained from natural sources such as plants or animal.
Ex: Heamotoxylin:Obtained from heart wood of log wood tree Haemotoxylumcampechianum
- Synthetic dyes: They are chemically synthesized and majority of them are synthetic dyes.
Ex: Fuschins, methyl violet, methyl blue, aniline dyes etc.
H&E staining:
For routinehistopathologicalexamination Haemotoxylin& Eosin method is the stain of choice. The two stains were independently introduced in 1865 & 1875 by Bhomer& Fischer respectively. In 1876 Wissowzky used combination method. Till today over a century later H&E remains the most frequently used tissue stain worldwide.
Principle:
Staining technique involves electrostatic attraction between ions of opposite charge. Haemotoxylin is a complex with aluminium salt is cationic and acts as a basic dye. It is positively charged and reacts with negatively charged nucleic acids in the nucleus. Eosin is anionic and acts as an acidic dye. It is negatively charged and can react with positively charged acidophiliccomponents such as amino group of proteins in cytoplasm.
Romanowsky stains
They are more commonly used for examination of blood or bone marrow samples. They are preferred over H&E for inspection of blood cells because different types of leukocytes (white blood cells) can be readily distinguished. They are also suited to examination of blood to detect blood-borne parasites such as Babesia and Theileriaand also for cytological studies.
Principle:
The Romanowsky stains are all based on a combination of eosinate (chemically reduced eosin) and methylene blue (sometimes with its oxidation products azure A and azure B). They are neutral stains containing both acidic and basic stains.
Ex: Giemsa stain, Wright’s stain, Jenner’s stain, May-Grunwald stain and Leishman stain.
Gram staining
Gram staining is used to differentiate bacteria according to their Gram character as Gram positive or Gram negative. It was developed by Dr. Christian Gram in 1884, and named after him.
Principle:
- Crystal violet.
- Gram’s Iodine solution.
- 70% alcohol.
- Carbolfuschin orsafranin.
It is based on the composition of their cell wall.
Gram-positive bacteria are typically rich in peptidoglycan (90%) with less lipid content (10%). In contrast Gram-negative bacteria has Lipopolysaccharide layer with high lipid content and less peptidoglycan (10%).
So, high peptidoglycal layer in Gram-positive bacteriaresistdeclorization with primary stain after alcohol or acetone treatment, whereas Gram negativebacteria gets decolorized and then get counterstained with secondary stain.
Gram staining uses crystal violet as primary stain, iodine as a mordant, and a carbolfuchsin or safranin as counterstain.
Crystal violet (CV) dissociates into CV+ and Cl– ions in aqueous solutions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple.
Iodine (I), used as mordant interacts with CV+ and forms large complex of crystal violet and iodine (CV–I) within the inner and outer layers of the cell.
When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. Since Gram negative organism have thin peptidoglycan layer(1-2 layers) and have additional lipopolysaccharide layer which gets dissolved due to the addition of alcohol, so gram negative organism fails to retain the complex and gets decolorized as the complex is washed away.
In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. This closes the pores in the cell wall and prevents the stain from exiting the cell. The large CV–I complexes become trapped within the Gram-positive cell also due to the thick and multilayered (40 layers) nature of its peptidoglycan.
Ziehl-Neelsen Staining (ZN-Stain) or Acid Fast staining
The Ziehl-Neelsen stain is used to identify acid-fast organisms. Acid fast organisms are those which resist the decolorization with primary stain when treated with an acidor acid alcohol.The ability of the bacteria to resist decolorization with acid confers acid -fastness to the bacterium (fast=holding capacity).
Members of genus Mycobacteria are acid-fast due to presence of the high mycolic acid content of their cell walls. Some bacteria may also be partially acid-fast, such as Nocardia.Oocysts of coccidian parasites, such as Cryptosporidium and Isospora, are also acid-fast.
Principle:
- Acid alcohol.
- Methylene blue or malachite green.
Organisms such as Mycobacteria are extremely difficult to stain by ordinary methods like Gram stain because of the high lipid content in their cell wall.
The phenolic compound carbolfuchsin is used as the primary stain because it is lipid soluble and penetrates the waxy cell wall. Staining by carbolfuchsin is further enhanced by steam heating the preparation to melt the wax and allow the stain to move into the cell.
Acid or Acid alcohol is used to decolorize the primary stain. The acid-fast cells resist this decolorization,where as nonacid-fast bacteria decolorize and then get counterstained with secondary stains malachite green or methylene blue.
Special Stains for Spirochetes
Spirochetes are Gram-negative, spiral or corkscrew-shaped bacteria.Due to their small size, they are not demonstrated satisfactorily by the Gram stain.
They are usually identified with silver impregnation techniques because spirochetes are argyrophilic, which means they will absorb silver from silver solutions.
Spirochetes are diderms, contain double membrane, where peptidoglycan layer will be sandwitched between inner and outer lipid membranes.
Many silver impregnation staining methods make use of silver nitrate. In all of them, the desired end product is a deposit of black metalic silver, formed by reduction of silver ions.
Fontana’s Staining Technique
- Fontana’s fixative: Formalin and glacial acetic acid.
- Fontana’s mordant: Tannic acid.
- 95% Ethanol.
- Fontana’s stain: Ammonical silver nitrate.
Principle:
- The spirochete is very delicate organism they easily get destroyed by heat fixation so here heat fixation step is avoided.
- Smear is treated by Fontana’s fixative. This fixative contain chemicals like formalin and glacial acetic acid.
- After fixation, the smear is treated to Fontana’s mordant. This mordant contains tannic acid which increases the affinity of the stain.
- Further, the stain is treated with alcohol and Fontana’s stain.
- Fontana’s stain contains ammonical silver nitrate and when this stain is heated it forms silver oxide (metallic silver)
- This silver oxide precipitates on the organism and increases the diameter of the cell and stains the cell.
- The stain demonstrates brownish-black spirochetes against brown background.
Warthin Starry Staining Technique
Principle:
This technique involves the argyrophilic reaction. The spirochetes are argyrophilic, which means they will absorb silver from a silver solution, but need a separate reducing agent in a solution to reduce the adsorbed silver to visible metallic silver.
When an aqueous silver nitrate solution is combined with the reducing agent hydroquinone, visible metallic silver (silver diamine complex) is generated.
Staining for Fungi
Lactophenol cotton blue for wet mount
Principle:
It contains three components
- Phenol which kills the live organisms.
- Lactic acid preserves the fungal structures.
- Cotton blue stains the chitin and cellulose in the fungal cell wall.
Histological stains for fungi
- Grocottmethanmine silver (GMS) stain.
- Periodic acid-Schiff (PAS).
- Gridley’s staining.
They are used to detect rich amount carbohydrates or polysaccharides present in the cell wall.
Grocott’sMethenamine Silver Stain
- Chromic acid
- Grocottsmethenamine silver
- Sodium thiosulfate
Principle:
Chromic acid oxidizes the 1, 2 glycol groups of carbohydrates in the cell wall of the fungus to form aldehyde groups. These aldehyde groups reduce the silver ions fromthe methenaminesilver solution to visible metallic silver. Sodium thiosulfate is used toremove any unreduced silver.
Periodic Acid-Schiff-Green Stain
- Periodic acid.
- Schiffs reagent.
The periodic acid-Schiff-Green (PAS-green) stain which has a wide range ofapplications, can be used to aid in diagnosis of fungal infections.
Principle:
Periodic acid oxidizes the 1, 2 glycol groupsof polysaccharides in the fungal cell wall to aldehydes, which in turnreact with Schiff reagent to produce insoluble magenta red color.
Gridley’s staining:
- Chromic acid
- Coleman’s fuelgen
- Metanil yellow
Principle: Chromic acid oxidizes the 1, 2 glycol groups of carbohydrates in the cell wall of the fungus to form aldehyde groups. Aldehyde reacts with coleman’sfuelgen to give deep purple color. Metanilyellowis used as secondary stain foe counterstaining.
Reference:
Luna, L.G. (1968). Manual of histologic staining methods of the Armed Forces Institute of Pathology. 3rd Edition, McGraw-Hill, New York.
