After more than 130 years, scientists may finally have figured out how a common laboratory method for identifying bacteria works. Invented by Hans Christian Gram in 1884 and thus known as the gram stain, the test differentiates bacteria based on the properties of their membranes. Researchers have long thought that the dye used in the gram stain, crystal violet, infiltrates the innermost confines of bacterial cells. But a new study shows that this is not the case.
Dai’s team measured the light-scattering behavior of crystal violet in Escherichia coli, which is gram-negative. After adding crystal violet to the bacteria, the researchers observed a sharp spike in signal as the dye aligned with the outer membrane, and then a dip as the molecules passed into the space between the membranes, where they have greater freedom to move. They saw a second rise in signal that corresponded to crystal violet’s alignment along the inner membrane. But instead of declining a second time as expected, the signal leveled off, suggesting that the dye did not cross the inner membrane. When the researchers repeated the experiment with a similar dye, malachite green, that is known to cross the bacteria’s inner membrane, they observed a second decline in the light-scattering signal, demarcating its entry into the cytoplasm. Overall, the results suggest that crystal violet doesn’t infiltrate the deepest recesses of bacterial cells. Instead it distinguishes gram-negative bacteria from gram-positive ones based on how well the dye sticks to the peptidoglycans.
A New Spin On The Old Gram Stain