Danish bacteriologist Hans Christian Gram developed this staining technique in 1884. Interestingly, his original purpose was not to differentiate bacteria, but to differentiate lung tissue from bacterial cells. However, his discovery turned out to be a game-changer in microbiology, as it allowed scientists to categorize bacteria into distinct groups.
Imagine there are two types of bacterial cells in front of you: Gram-positive and Gram-negative. We stain them with crystal violet, which colors all cells purple. Then, we add iodine, which forms a complex with crystal violet, locking the color inside the cells. Next comes the critical step: decolorization with alcohol. This step is like a wash that affects the cells' color retention based on their cell wall structure. Gram-positive cells, with their thick peptidoglycan layer, hold onto the purple stain, while Gram-negative cells, with their thinner peptidoglycan and outer membrane, lose the stain. Finally, we apply safranin, which stains the previously decolorized Gram-negative cells pink.
Gram-positive bacteria have sturdy cell walls with a thick peptidoglycan layer. This layer gives them structural strength and retains the crystal violet stain effectively. On the other hand, Gram-negative bacteria have a more complex structure. Their cell walls consist of a thinner peptidoglycan layer surrounded by an outer membrane containing lipopolysaccharides, which can make the stain removal process more likely during decolorization.
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