Staining Techniques: Gram stain, Acid Fast Stain, Endospore Stain | Techniques in Microbiology
The many types of dyes used to stain microorganisms have two features in common: they have chromophore groups, groups with conjugated double bonds that give the dye its color, and they can bind with cells by ionic, covalent, or hydrophobic bonding. Most dyes are used to directly stain the cell or object of interest, but some dyes (e.g., India ink and nigrosin) are used in negative staining, where the background but not the cell is stained; the unstained cells appear as bright objects against a dark background.
Dyes that bind cells by ionic interactions are probably the most commonly used dyes. These ionizable dyes may be divided into two general classes based on the nature of their charged group.
1. Basic dyes:
Basic dyes bind to negatively charged molecules like nucleic acids, many proteins, and the surfaces of procaryotic cells.
have positively charged groups (usually some form of pentavalent nitrogen) and are generally sold as chloride salts.
Examples of Basic Dyes: Methylene Blue, Basic fuchsin, Crystal Violet, Safranin, Malachite green
2. Acidic dyes:
Acidic dyes, because of their negative charge, bind to positively charged cell structures.
possess negatively charged groups such as carboxyls (—COOH) and phenolic hydroxyls (—OH).
Example of Acidic Dyes: Eosin, Rose bengal, and Acid fuchsin
The staining effectiveness of ionizable dyes may be altered by pH, since the nature and degree of the charge on cell components change with pH. Thus acidic dyes stain best under acidic conditions when proteins and many other molecules carry a positive charge; basic dyes are most effective at higher pHs.
Dyes that bind through covalent bonds or because of their solubility characteristics are also useful. For instance,
DNA can be stained by the Feulgen procedure in which the staining compound (Schiff’s reagent) is covalently attached to its deoxyribose sugars.
Sudan III (Sudan Black) selectively stains lipids because it is lipid soluble but will not dissolve in aqueous portions of the cell.
Differential Staining : Gram Staining
The Gram stain, developed in 1884 by the Danish physician Christian Gram, is the most widely employed staining method in bacteriology. It is an example of differential staining procedures
that are used to distinguish organisms based on their staining properties. Use of the Gram stain divides Bacteria into two classes: Gram negative and Gram positive.
Acid-fast staining is another important differential staining procedure. It is most commonly used to identify Mycobacterium tuberculosis and M. leprae, the pathogens responsible for tuberculosis and leprosy, respectively. These bacteria have cell walls with high lipid content; in particular, mycolic acids—a group of branched-chain hydroxy lipids, which prevent dyes from readily binding to the cells. However, M. tuberculosis and M. leprae can be stained by harsh procedures such as the Ziehl-Neelsen method, which uses heat and phenol to drive basic fuchsin into the cells. Once basic fuchsin has penetrated, M. tuberculosis and M. leprae are not easily decolorized by acidified alcohol (acid-alcohol), and thus are said to be acid-fast. Nonacid- fast bacteria are decolorized by acid-alcohol and thus are stained blue by methylene blue counterstain.
Endospore staining, like acid-fast staining, also requires harsh treatment to drive dye into a target, in this case an endospore. An endospore is an exceptionally resistant structure produced by some bacterial genera (e.g., Bacillus and Clostridium). It is capable of surviving for long periods in an unfavorable
environment and is called an endospore because it develops within the parent bacterial cell. Endospore morphology and location vary with species and often are valuable in identification; endospores may be spherical to elliptical and either smaller or larger than the diameter of the parent bacterium. Endospores are not stained well by most dyes, but once stained, they strongly resist decolorization. This property is the basis of most endospore staining methods .
In the Schaeffer-Fulton procedure, endospores are first stained by heating bacteria with malachite green, which is a very strong stain that can penetrate endospores. After malachite green treatment, the rest of the cell is washed free of dye with water and is counterstained with safranin. This technique yields a green endospore resting in a pink to red cell.
