The famous “Gram Stain”

So far, somewhere between 30% and 50% of this blogs’ current readers have expressed an interest in this, so what the heck. Here, put simply, is why Gram stains work.

Actually, I’ll start with how it works, for anyone unfamiliar with the process, which was invented about 125 years ago. In short:

1. First, the bacterial sample is put on a slide, allowed to dry, then the slide’s passed over a flame to “heat fix” the bacteria (basically, slightly cooked onto the surface of the slide, like meat stuck to an ungreased hot frying pan)
2. The sample is soaked with a purple dye (“Crystal Violet”)
3. The sample is soaked with an iodine solution
4. The slide is “decolorized” with a quick rinse of alcohol, which should remove the dye from anything that’s “Gram negative”.
5. Finally the sample is soaked with a pink or red dye to color the “Gram negative” cells so they can be seen as well (unstained bacteria are often nearly colorless so they can be harder to see in a normal microscope.)

“How” is boring, though. What’s interesting is why it works.

Bacteria (with the exception of one group) have cell wall – basically a relatively rigid protein “shell”. The difference between “Gram positive” and “Gram negative” turns out to be that Gram positive bacteria have a thick cell wall, while “Gram negative” bacteria have a thin cell wall, that is also covered by an outer membrane (think of it as a bubble of grease…).

The iodine in the gram stain is a mordant – the combination with the Crystal Violet is “chunky”, and sticks into the cell walls. When the alcohol wash is done, it rinses away exposed Crystal-violet-and-iodine. It also dissolves the outer membrane of gram negative bacteria. Either because there’s not enough cell wall to hold it or because perhaps it sticks in the outer membrane instead, this step washes the dye out of gram negative bacteria, but the gram positive bacteria keep some dye in their cell walls.

The pink/red dye that’s then added actually stains both kinds of bacteria, but you can’t really see it on the gram positive cells because they’re already dyed dark purple.

So, in summary, the Gram stain turns out to be a test for bacterial cell wall types. The results aren’t always reliable (there are “gram variable” bacteria) and it takes some practice to get right (too much alcohol wash and you end up washing the dye out of the gram-positives, too – not enough and some of the gram negatives can look gram positive), but it’s a quick way to start categorizing bacteria.

There, how’s that for some boring science?