Coloração de Gram

Gram staining (or Gram's method) is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative). The name comes from its inventor, Hans Christian Gram. 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. This gives rise to Gram-variable and Gram-indeterminate groups as well. History[edit]The method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938), who developed the technique while working with Carl Friedländer in the morgue of the city hospital in Berlin in 1884. Gram devised his technique not for the purpose of distinguishing one type of bacterium from another but make bacteria more visible in stained sections of lung tissue.[2] He published his method in 1884, and included in his short report the observation that the Typhus bacillus did not retain the stain.[3] Staining mechanism[edit]Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell envelope), and as a result are stained purple by crystal violet, whereas Gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by the Safranin. There are four basic steps of the Gram stain: Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixing kills some bacteria but is mostly used to affix the bacteria to the slide so that they don't rinse out during the staining procedure. The addition of iodine, which binds to crystal violet and traps it in the cell, Rapid decolorization with alcohol or acetone, and Counterstaining with safranin.[9] Carbol fuchsin is sometimes substituted for safranin since it more intensely stains anaerobic bacteria, but it is less commonly used as a counterstain.[10] Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl− ) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple. Iodine (I− or I− 3) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV–I complex and, therefore, color the cell.[11] When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A Gram-negative cell loses its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the Gram-negative cell along with the outer membrane. In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the Gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet stain is removed from both gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds). After decolorization, the Gram-positive cell remains purple and the Gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color.[12][13]