OR University of Pennsylvania's How to Gram Stain
Gram staining (or Gram's method) is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical, primarily the presence of high levels of peptidoglycan, and physical properties of their cell walls. The Gram stain is almost always the first step in the identification of a bacterial organism. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique, thus forming Gram-variable and Gram-indeterminate groups as well. A Gram positive results in a purple/blue color while a Gram negative results in a pink/red color.
The word Gram is always spelled with a capital, referring to Hans Christian Gram, the inventor of Gram staining.
Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain (safranin or fuchsine) and appearing red or pink. Gram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall. Gram-positive cell walls typically lack the outer membrane found in Gram-negative bacteria.
REMEMBER: GRAM-POSITIVE... P FOR POSITIVE, FOR PURPLE AND PETIDOGLYCAN
Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counterstain (commonly safranin) is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color. The test itself is useful in classifying two distinct types of bacteria based on the structural differences of their bacterial cell walls. Gram-positive bacteria will retain the crystal violet dye when washed in a decolorizing solution.
The pathogenic capability of Gram-negative bacteria is often associated with certain components of Gram-negative cell walls, in particular, the lipopolysaccharide layer (also known as LPS or endotoxin layer). In humans, LPS triggers an innate immune response characterized by cytokine production and immune system activation. Inflammation is a common result of cytokine (from the Greek cyto, cell and kinesis, movement) production, which can also produce host toxicity.
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- GRAM POSITIVE
- LESS LIPID MORE PEPTIDOGLYCAN
- OVER DECOLORIZATION AND OLD AGE: LOOSE ABILITY TO RETAIN PRIMARY STAIN: GRAM VARIABLE
- REMEMBER: GRAM-POSITIVE... P FOR POSITIVE, FOR PURPLE AND PETIDOGLYCAN
- GRAM NEGATIVE
- MORE LIPID LESS PEPTIDOGLYCAN
- DURING DECOLORIZATION: LIPID WASHED AWAY
- UNABLE TO RETAIN PRIMARY STAIN
- RETAIN: SAFRANIN: COUNTER STAIN