Gram stain

This procedure describes how to perform a Gram stain.

The Gram staining is used:

• to classify bacteria based on their shape, size, morphology and gram properties to assist in the identification
• to make a quick, presumptive diagnosis of infection
• to assess the quality of clinical samples

The Gram stain is a differential stain that enables classification of the bacteria into two large groups, the Gram-positive and the Gram-negative group. This classification is based on their reaction to the stain caused by differences in the structure of their cell walls. The Gram-positive bacteria have a thick peptidoglycan cell envelope and no membrane at the outside of their cell envelope. Crystal violet and lugol (iodine solution) form an alcohol-dissolvable complex that binds to this peptidoglycan layer. Alcohol-acetone has no decolorizing effect on the stain complex, so Gram-positive bacteria retain the violet stain. Gram-negative bacteria have a lipopolysaccharide on the membrane that covers the cell envelope and only a thin peptidoglycan layer that cannot retain crystal violet stain. Gram-negative bacteria will be decolorized by alcohol-acetone and take up the counterstain safranin or fuchsine (red color). Decolorizingagents and counterstains may differ. In this procedure we use the Gram stain set of bioMérieux, containing crystal violet, stabilized lugol-polyvinyl-pyrrolidone, alcohol-acetone and safranin.

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