Besides Gram's stain, there is a wide range of staining methods available. The procedures for these other methods follow quite closely those of Gram's stain. By using appropriate dyes, different parts of the cell structure such as capsules, flagella, granules, or spores can be stained. Staining techniques are widely used to visualize those components that are otherwise too difficult to see under an ordinary light microscope either because of the lack of color contrast between the object under examination and the background or because of the limited resolving power of the light microscope. In addition, staining techniques are useful in detecting the presence or absence of certain cell components, thus allowing a relatively simple scheme of differentiation or identification of microorganisms. In this respect, Gram's stain ranks among the most important diagnostic tools in biological science.
In Gram's method, which is based on the ability of a cell in retaining the crystal violet dye during solvent treatment, it is the difference in the microbial cell wall that is amplified. The cell walls for gram-negative microorganisms have a higher lipid content than gram-positive cells. Originally, both kinds of cells are penetrated by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed too easily. This step is commonly referred to as fixing the dye. However, the subsequent treatment with the decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. In contrast, the solvent dehydrates the thicker gram-positive cell wall, closing the pores as the cell wall shrinks during dehydration. As a result, the the diffusion of the stain-iodine complex out from the cell is obstructed, and the cells remain stained. The actual mechanism of decolorization is currently not well understood and remains controversial. At any rate, if the decolorizer treatment is properly timed, there exists a period during which the crystal violet-iodine complex is effectively removed from the gram-negative cells but still retained in the gram-positive ones. Thus, the length of the decolorizer treatment is critical in clearly differentiating the gram-positive cells from the gram-negative cells. A prolonged exposure to the decolorizing agent will remove all the stain from both types of cells. The student is cautioned that some gram-positive cells lose the stain easily and therefore may appear gram negative.
Finally, although not essential, a counterstain of safranin is applied to the smear to dye the decolorized gram-negative cells with a pink color. Thus, the size and shape of both types of cells can be more easily observed under a microscope. At the same time, they can be differentiated by the imparted color. If desired, the slides can be permanently mounted and preserved for record keeping.
A mixture of S. cerevisiae and E. coli will be examined in this experiment, although Gram's stain is usually applied to differentiate bacteria, which are usually too small to be seen clearly under a light microscope. S. cerevisiae cells will be stained by the crystal violet-iodine complex and should appear purple-brown in color. In contrast, the much smaller E. coli, cells should appear pink, the color of safranin. Repeat the entire process until a satisfactory slide is prepared and properly focused in a microscope for the approval of the instructor. Note the improvement in cell visualization as compared to a plain unstained slide.