Gram Staining

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Gram Staining

Compared with Gram-positive bacteria, Gram-negative bacteria are more resistant against antibodies because of their impenetrable cell wall. These bacteria have a wide variety of applications ranging from medical treatment to industrial use and Swiss cheese production.

Danish scientist Hans Christian Gram devised a method to differentiate two types of bacteria based on the structural differences in their cell walls. In his test, bacteria that retain the crystal violet dye do so because of a thick layer of peptidoglycan and are called Gram-positive bacteria. In contrast, Gram-negative bacteria do not retain the violet dye and are colored red or pink.

PROCEDURE1) A heat-fixed smear is flooded with a basic purple dye, usually crystal violet. Because the purple stain imparts its color to all cells, it is referred to as a primary stain.2) After 1 minute, the crystal violet is drained off and washed with distilled water. The smear is then covered with Gram's iodine, a mordant or helper. When the iodine is washed off, both gram-positive and gram-negative bacteria appear dark violet or purple.3) Next, the slide is washed with alcohol (95% ethanol) 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. When the procedure is carried out, the slide is held at an angle and 95% ethanol is poured until the draining solution no longer has a purple tint.4) The alcohol is now rinsed off with distilled water and the slide is then stained with safranin, a basic red dye known as the counter stain. The staining process is allowed to be carried out for 2-3 minutes. The smear is washed again, heat dried and examined microscopically.


Gram Positive versus Gram Negative

Classifying Bacteria and gram-staining

90-95% of Gram negative bacteria are pathogenic. On the other hand, many Gram-positive bacteria are non-pathogenic.Not only do gram-negative bacteria tend to be harmful to humans, they are also more resistant to antibiotics. They also develop resistance sooner


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