The gram stain was developed in 188

The gram stain was developed in 1884 by the danish bacteriologist hans christian gram.It is one of the most useful staining procedures because it classifies bacteria into two large groups: gram psitive and gram negative.
in this procedure;
1. A heat-fixed smear is covered with a basic purple dye, usually crystal violet. Because the purple stain imparts its color to all cells, it is reffered to as a primary stain.
2. After a short time, the purple dye is washed off, and the smear is covered with iodine, a mordant. When the iodine is washed off, both gram postive and gram negative bacteria appear dark violet or purple.
3. Next, the slide is washed with alcohol or an alcohol-acetone solution.This solution is a decolorizing agent, which removes the purple from the cells of some species but not from others.
4. The alcohol is rinsed off, and the slide is then stained with safranin, a basic red dye. The smear is washed again, blotted dry, and examined microscopically.
The purple dye and the iodine combine in the cytoplasm of each bacterium and color it dark violet or purple.Bacteria that retain this color after the alcohol has attempted to decolorize them are classified as gram positive;bacteria that lose the dark violet or purple color after decolorization are classified as gram negative.Because gram negative bacteria are colorless after the alcohol wash,the are no longer visible. This is why the basic dye safranin is applied;it turns the gram negative bacteria pink.Stains such as safranin that have a contrasting color to the primary stain are called counterstains.Because gram positive bacteria retain the original purple stain,they are not affected by the safranin counterstain.
As you will see in chapter 4,different kinds of bacteria react differantly to the gram stain because structural differences in their cell walls affect the retention or escape of a combination of crystal violet and iodine,called the crystal violet-iodine (CV-I)complex.Among other differences,gram positive bacteria have a thicker peptidoglycan(disaccharides and amino acids)cell wall than gram negative bacteria.In addition,gram negative bacteria contain a layer of lipopolysaccharide(lipids and pilysaccharides)as part of their cell wall.When applied to both gram positive and gram negative cells,crystal violet and then iodine readily enter the cells.Inside the cells,the crystal violet and iodine combine to form CV-I.This complex is larger than the crystal violet molecule that entered the cells,and,because of its size,it cannot be washed out of the intact peptidoglycan layer of gram positive cells by alcohol.Consequently,gram positive cells retain the color of the crystal violet dye.In gram negative cells,however,the alcohol wash disrupts the outer lipopolysaccharide layer,and the CV-I complex is washed out through the thin layer of peptidoglycan.As a result,gram negtive cells are colorless until counterstained with safranin,after which they are pink.