The term "mordant" originally comes from cloth dying. It's a chemical that fixes a dye's color in a material. In microbiology the mordant in question is also the agent that gives the Gram-staining process its name. This mordant is called Gram's iodine solution and contains iodine mixed with potassium iodide.
Bacteria are extremely small, so small it can be tough to make out individual details even with a scanning electron microscope. Yet proper identification is essential for things like diagnosis and treatment of illness. Biologists use dyes to make individual bacteria and their identifying characteristics stand out more clearly. Bacteria are broadly grouped as either Gram-positive or Gram-negative based on their sensitivity to one of these dyeing processes, known as the Gram stain.
Bacterial cell membranes are composed of carbohydrates, proteins and fats. The proportions of each vary by species. A common bacterial membrane macromolecule is the peptidoglycan. Peptidoglycans are made up of proteins and sugars. Gram-positive bacteria have lots peptidoglycans, gram-negative ones have only a few. The number of peptidoglycans in the outer membrane determines a bacterium's sensitivity to the initial Gram-stain dye.
If you're doing a Gram stain, first you flood your bacterial culture with a dye called crystal violet, which gets through the cell membranes of all the bacteria, whether they're Gram-positive or negative. Now comes the mordant. When you add Gram's iodine solution to the violet-stained cells, the iodine binds to the crystal violet and the potassium iodide prevents the bound complexes from dissolving in water. This makes the Gram's solution a mordant, or dye-fixer, within the bacterial cells.
The next step is "bleaching" the dyed Gram-negative cells by adding acetone or ethyl alcohol. These chemicals dehydrate the bacteria's outer membranes. Bacterial membranes with lots of peptidoglycans shrink but don't break, which traps the violet-iodine crystals inside the cells. These types are Gram-positive and stay violet. Membranes with few peptidoglycans snap open, releasing the purple crystals. These are Gram-negative bacteria.
Of Two Colors
The last step is adding a red stain, usually safranin. This doesn't affect the dark, fixed color in the gram-positive bacteria, but stains the ruptured Gram-negative cells a pinkish red. The purpose of this last step is simply to make Gram-negative cells contrast with Gram-positive ones and the background so they're easier to see. This step does not use a mordant.
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