performed by the rapid passage of air-dried smear two or three times over the flame of the bunsen burner
the bacterial smear is stained w/a single reagent, which produces a distinctive contrast between the organisms and its background. it is used to show the morphology and arrangement of bacterial cells, most commonly used:
requires the use of an acidic stain such as India ink or nigrosin, it stains around the bacteria because it cannot penetrate the cell wall, it colors the slides background leaving the organisms transparent. "negative copy" of a picture
requires the use of at least three chemical reagents that are applied sequentially to a heat-fixed smear. the first reagent is called the primary stain. the second one is a decolorizing agent, final the counterstain
it may or may not remove the primary stain from the entire cell or only from certain cell structures (due to the cellular composition)
cannot be absorbed and the cell or its components will retain the color of the primary stain. if the primary stain is removed, the decolorized cellular components will accept and assume the contrasting color of the counterstain.
it divides bacterial cells into two major groups, gram-positive and gram-negative, which makes it an essential tool for classification and differentiation of microorganisms.
Gram's Iodine (Mordant)
this reagent serves not only as a killing agent but also as a mordant, a substance that increases the cells' affinity for a stain. it does this by binding to the primary stain, thus forming an insoluble complex. the result: crystal-violet-iodine (CV-I) complex serves to intensify the color of the stain. cells appear purple-black
Ethyl Alcohol, 95% (Decolorizing agent)
serves a dual function as a protein-dehydrating agent and as a lipid solvent, its action is determined by two factors, the concentration of lipids and the thickness of the peptidoglycan layer in bacterial cell walls. in gram-negative cells it increases the porosity of the cell wall by dissolving the lipids in the outer layers. thus CV-I can be easily removed from the thinner and less highly cross-linked peptido layer, the washing-out effect facilitates the release of the unbound CV-I, leaving the cell colorless. the much thicker peptido layer in gram-positive cells is responsible for the more stringent retention of the CV-I complex, as the pores are smaller, due to the dehydrating affect of the alcohol. it is dificult to remove, cells remain purple.
the final reagent, used to stain pink those cells that have been previously decolorized. since only gram-negative cells undergo decolorization, they may now absorb the counterstain. gram-positive cells retain the purple color of primary stain.