Gram stain: the iodine mordant and decolourisation
The four-step Gram stain uses iodine, a mordant (trapping agent), to lock the purple dye crystal violet into thick walls; decolourisation then makes cells with thin walls lose it.
Remember
Iodine locks crystal violet inside thick walls, so rapid decolourisation washes the dye out of cells with thin walls (Gram-negative) but not out of cells with thick walls (Gram-positive).
Memory aid
stain everything purple, lock the purple in, wash it out, then colour what is left pink
Each phrase matches one reagent in turn: crystal violet, Gram's iodine, the decolouriser and safranin.
The Gram stain separates bacteria into Gram-positive bacteria, which have thick peptidoglycan walls, and Gram-negative bacteria, which have thin walls. Peptidoglycan is a mesh made of sugars and short protein chains. The bacteria are spread as a smear (a thin film of the sample on a slide), and heat fixation attaches the smear to the slide.
The reagents are added in order. Crystal violet, a purple dye, goes on first and stains every cell. Gram's iodine follows. It acts as a mordant, meaning a trapping agent that sets a dye in place, and it joins the dye into a larger complex that clumps inside the wall.
Next comes decolourisation, using the decolouriser, which is ethanol or acetone applied rapidly. It washes the dye out of cells that cannot hold it. This is the step that separates the two groups: Gram-positive cells keep the purple complex in their thick walls, whereas Gram-negative cells lose it and become colourless.
Finally, safranin, a pink counterstain (the dye that colours cells that have already lost the purple), makes the colourless Gram-negative cells visible.
Iodine does not separate the groups by itself: it holds the dye in. If the iodine step is omitted, the decolouriser can strip crystal violet from Gram-positive cells as well, so they come out pink and look Gram-negative, which could lead to the wrong antibiotic choice.
Key facts
- Mordant
- Gram's iodine, a trapping agent that sets a dye in place, joins crystal violet into a larger complex.
- Order of reagents
- Crystal violet goes on first, then Gram's iodine, then the decolouriser, and the pink safranin counterstain comes last.
- Step that separates the groups
- Decolourisation, with ethanol or acetone, is the step that treats the two groups differently.
- Gram-positive result
- The thick peptidoglycan wall keeps the purple complex, so the cells stay purple.
- Gram-negative result
- The thin wall loses the complex, so the cells become colourless and then take up pink safranin.
- Heat fixation
- It attaches the bacteria to the slide and acts on all the cells equally, so it does not separate the groups.
Exam traps
- Iodine helps Gram-positive walls keep the dye, yet the separation of the two groups comes from the washing step, not from the iodine.
- Leaving out heat fixation means many bacteria would wash off the slide, leaving few or no cells to see, so it does not turn Gram-positive bacteria pink.
- Iodine does not decolourise Gram-negative bacteria: washing out the dye is the job of the alcohol or acetone decolouriser, and iodine is added earlier, to help the dye stay in.
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