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Comment :
Protoplasmic constituents of both gram positive and gram negative cells combine with crystal violet by an ionic bond between theiracidic groups and the basic groups of the dye .Iodine, in aqueous solution, enters the cells and reacts with the dye , either removing it from the cell protein or adding the dye-protein complex .
The gram stains differentiates bacteria into two different groups based on a differences in the nature of their cell wall. While the gram-positive wall in most bacteria is primarily a Sick layer of peptidoglycan, the gram-negative wall is a multilayered st ed structure composed of a thin a layer of peptidoglycan surrounded by an
outer layer of protein and lipopolysaccharide.

It is believed that 95 per cent alcohol dehydratesYhe mordanted walls of gram positive cells and forms a a barrier, which traps the crystal violet-iodine complex.
Such a barrier iS not formed in gram im negative e cells. It is known that the cell walls of these organisms contain a higher percentage of lipid (10-22%) than those of most gram positive organisms 0-2 %), and hence the solubility of the surface lipids in alcohol may be a factor in the greater case of decolorization of gram negative cells
The cytoplasmic membrane of gram positive bacteria contain Mg-ribonucleate (Mg", protein, and nucleic acid which containsribose,i.e. Ribonucleic acid). Such component are more sensitive to crystal violet iodine complex and fix it.

Observation

Gram positive bacteria retain the purple primary stain (crystal Violet) while gram negative bacteria stain with the relatively weak secondary pink dye ,safranin


b- Differential stains

Differential stains distinguish structures within a cell or distinguish one type of cell from another. Two dyes are usually employed, and certain structures or cell types appear in one color in the final preparation with the remaining structures or cell types in the second color. The first dye applied is id primary stai) while the second dye is the counter stain. Differential stains are employed(n identifying bacteria.

procedure:

1- Prepare a fixed smear.

2- Flood the fixed smear with the primary stain, crystal violet for 1-2 minutes.

3- Rinse with water.

4- Cover the film with gram’s iodine (behave as a(mordant) and allow it to act for
about 60 seconds.
5- Rinse with water.
6- Decolorize with 95 % alcohol.
7- Rinse with water
8 Countetstain with safranin for 20-30 seconds.
9- Rinse with water and dry.
10-Examine under the oil-immersion objective and make drawings.