A New Spin On The Old Gram Stain

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 (ACS Chem. Biol. 2015, DOI: 10.1021/acschembio.5b00042).

Although the new mechanism contradicts the established theory of how the gram stain works, it does not affect the interpretation of results from the test, says Hai-Lung Dai of Temple University, coauthor of the study. But understanding why molecules such as crystal violet do or do not cross bacterial membranes could help scientists develop new dyes with different functions, he says.

Gram staining is typically the first step in any attempt to identify bacteria. Researchers had thought that staining with crystal violet worked because the dye passed freely through all the bacterial membranes in the cells. Generally, bacteria that are colored by crystal violet (gram-positive) have a single inner cytoplasmic membrane coated in a thick peptidoglycan layer, which seemed to trap the dye inside the cell. Bacteria with outer and inner membranes separated by a thin peptidoglycan layer reject the dye (gram-negative), and scientists suspected that the relatively thin peptidoglycan layer in gram-negative bacteria allows the dye to escape.

But no one had ever watched the journey of these dye molecules as they move inside bacterial cells. Dai wasn’t trying to overturn the gram-stain theory, but he realized that his pet spectroscopic method, second harmonic light scattering, would be great at studying the mechanism. The method is “quite unique in that we now have the ability to look at the transport of molecules into a living biological cell,” he says. In this method, molecules spinning randomly in solution don’t generate a signal, whereas those aligned along a membrane emit a strong signal.

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.

“This is an excellent piece of work,” says chemist Zhan Chen of the University of Michigan, Ann Arbor. “I’m happy to see people using nonlinear optical spectroscopy to solve real problems.” Chen would be interested in seeing data from additional bacterial species to confirm the results.