Staining techniques

Staining techniques

Bacteria are semi transparent difficult to visualized in unstained condition. Identification of bacteria is aided by their staining reaction.
Stain: are coloring agent used for biological purposes to identify microscopical agents. There are alkaline and acidic stains. There are three types of staining methods:
1. Simple stain: is composed of one type of stain, e.g. crystal violet (C.V.), safranin.
2. Differential stain: it gives different color to different bacteria e.g. Gram’s stain, Albert’s stain and Ziehle Neelsen (ZN) stain.
3. Special stain: e.g.:
• Negative staining (have background colored in which bacteria remain unstained).
• India ink stain for capsule staining.
• Special stains used for staining of some bacterial components like spore stain and flagella stain.
Gram stain technique:
The Gram stain procedure was originally developed by the Danish Physician Hans Christian Gram in 1884 to differentiate pneumococci fro Klebsiella pneumonia. In brief, the procedure involves the application of a solution of iodine (potassium iodide) to cells previously stained with crystal violet or gentian violet. This procedure produces purple colored iodine-dye complexes in the cytoplasm of bacteria. The cells that are previously stained with crystal violet and iodine are next treated with a decolorizing agent such as 95% ethanol or a mixture of acetone and alcohol. The difference between Gram-positive and Gram negative bacteria is in the permeability of the cell wall to these purple colored iodine-dye complexes when treated with the decolorizing solvent. While Gram-positive bacteria retain purple iodine dye complexes after the treatment with the decolorizing agent. Gram-negative bacteria do not retain complexes when decolorized. To visualize decolorized Gram-negative bacteria, a red counter stain such as safranin is used after decolorization treatment.
The first consideration is the correct preparation of the smear. Make a thin film of the material on a clean glass slide, using a sterile loop. Air dry, then heat fix the slide by passing it several times through a flame (the slide should not become too hot to tough). Failure to follow these directions may disrupt the normal morphology of bacteria and cells.