Introductory Microbiology

Gram staining

• One of the most important and widely used differential staining technique in microbiology is gram staining. This technique was introduced by Christian gram in 1884. In this process the fixed bacterial smear is subjected to the following staining reagents in the order listed; crystal violet, iodine solution, alcohol (decolorizing agent) and safranin or some other suitable counter stain. Bacteria stained by the gram method fall into two groups; gram positive bacteria, which retain the crystal violet and hence appear deed violet in color and gram negative bacteria which lose the crystal violet are counter stained by the safranin, and hence appear red in color.
• The most possible explanations for this phenomenon are associated with the structure and composition of cell wall. Differences in the thickness of cell walls between these two groups may be important; the cell wall of gram negative bacteria are generally thinner than those of gram positive bacteria. Gram negative bacteria contain a higher percentage of lipid than gram positive bacteria. Experimental evidence suggests that during staining of gram negative bacteria the alcohol treatment extracts the lipid, which results in increased porosity or permeability of cell wall. Thus the crystal violet iodine (CV-I) complex can be extracted and the gram negative organism is decolorized. These cells subsequently take on the color of the safranin counterstain. The cell walls of gram-positive bacteria because of their different composition (lower lipid content) become dehydrated during treatment with alcohol. The pore size decreases, permeability is reduced and the CV-I complex cannot be extracted. Therefore these cells remain purple violet.
• The gram stain has its greatest use in characterizing bacteria. This staining technique is not generally applicable for other groups of microorganisms such as protozoa and fungi, however, yeasts consistently stain gram-positive.

Other differential stains
There are numerous other staining techniques designed to identify some particular feature of cell structure or composition. These techniques are summarized below.

Sl. No	Name of the staining technique	Application
1	Acid fast stain	Distinguishes acid fast bacteria such as Mycobacterium Spp. from non-acid fast bacteria.
2	Endospore stain	Demonstrates spore structure in bacteria as well as free spores.
3	Capsule stain	Demonstrates presence of capsules surrounding cells.
4	Flagella stain	Demonstrates presence and arrangement of flagella.
5	Cytoplasmic inclusion stains.	Identifies intracellular deposits of starch, glycogen, poly phosphates, hydroxyl butyrate and other substances.
6	Giemsa stain	Particularly applicable for staining ricketsia and some protozoa.

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