The Gram stain, developed in 1884 by the physician Hans Christian Gram, is a differential stain that separates bacteria into two groups, gram-positive and gram-negative, based on differences in the structure of the cell wall. This staining technique is differential because it allows the discrimination of one cell from another based on differential staining properties, thus providing not only morphological information (size, shape, and arrangement), but also a clue into the physiological differences between groups of bacteria.
Gram staining begins with staining cells with the primary stain crystal violet. Both gram-positive and gram-negative cells are colored a deep purple at this point. To enhance the stain of cells with crystal violet, iodine is added as a mordant, a chemical that serves to fix a dye in a staining process. In this case, the iodine binds with the crystal violet to create an insoluble complex within the thick peptidoglycan layer of gram-positive cells. Cells are then decolorized with ethyl alcohol. The alcohol dissolves the lipids that are found in the outer membrane of gram-negative cells, allowing the crystal violet-iodine complex to escape. At this point, gram-negative cells are colorless while gram-positive cells are still dark purple. To reveal the shape of gram-negative cells, the counterstain safranin is added to stain the gram-negative cells, rendering them red/pink. Gram-positive cells are dyed pink as well, but the darker color of the crystal violet masks the lighter color of the safranin. In the end, those cells staining purple are designated gram-positive while those staining pink are designated gram-negative.
There are three main factors that can affect the outcome of a Gram stain and should be remembered: (1) Young cultures should be used for Gram staining. Gram-positive cultures older than 24 h may sometimes stain gram-negative due to changes in the peptidoglycan layer of the cell wall that accompany aging. (2) Smears must not be too thick. Thick smears can entrap crystal violet so that it is not removed by the alcohol, leading to a false gram-positive result. (3) Decolorization must be done for an appropriate period of time. Alcohol, if left on too long, will remove the crystal violet from even gram-positive cells, leaving them red/pink. Conversely, if cells are not decolorized long enough, gram-negative cells will be seen as purple, leading to a false impression that the cells are gram-positive.
How do gram-positive and gram-negative bacteria differ in their cellular structure?
a. Number of ribosomes
b. Presence of a nucleus
c. Composition of cell membrane
d. Cell wall structure
