The Gram Stain

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Individual bacterial cells are hard to see, partly because they are small, but also because they are almost transparent. In addition to magnification under a microscope, optical tricks must also be used to be able to see them, such as phase contrast microscopy ans staining. In 1884, Hans Christian Gram, a Danish doctor working in Berlin, accidentally stumbled on a method which still forms the basis for the identification of bacteria. While examining lung tissue from patients who had died of pneumonia, he discovered that certain stains were taken up and retained by bacterial cells. Over the course of the next few years, Gram developed a procedure which divided almost all bacteria into two large groups - the Gram stain. Because it’s a proper noun, Gram stain is spelt with a capital G.
Gram staining is one of the most useful procedures in bacteriology. Why? It’s cheap and quick, giving results in minutes compared to hours or days for bacterial culture, but also, if you’re attempting to classify or divide up objects, the most useful tests are those which split the things you’re looking at into two roughly equal groups. And in general terms, that’s exactly what the Gram stain does.

So how does it work? To perform a Gram stain, you place a small amount of bacteria on a glass microscope slide. Because the slide will be washed repeatedly, the next step is to stick the cells to the slide by heating them. After that, the cells are stained with a dye. The fixed cells are first stained with crystal violet, or sometimes methyl violet, dye. The dye soaks into the surface of the cells.
After 1-2 minutes, the stain is poured off and the surface of the slide flooded with iodine solution for 1-2 minutes. This solution acts as a mordant, a substance which fixes a dye, in this case the violet stain. The next step is to decolourize the slide with ethanol or acetone, which washes the stain off, but some bacteria have a thick mesh-like cell wall made of of a polymer called peptidoglycan. If it is present, the cell wall traps and retains the violet dye-iodine complex. If no cell wall is present, the primary violet stain is washed away.
This is where the Gram stain procedure gets clever. After decolourizing, the primary stain is followed by a pink secondary counterstain, a dye called safranin. After 1-2 minutes staining, the counterstain is washed off with water and the slide is dried and the cells are examined under a microscope. The presence of a cell wall creates purple Gram-positive cells, but if no cell wall is present, pink Gram-negative cells are seen.
So the Gram stain procedure is not only quick and cheap, it also tells you something about the structure of bacterial cells, although Dr Gram didn’t know that when he developed the technique. Some bacterial cells have different structures which defeat the Gram stain. A good example of this are Mycobacteria such as Mycobacterium tuberculosis, which causes TB. Although they have a cell wall, this is heavily impregnated with lipids and other compounds so the violet stain does not penetrate it and the cells appear Gram negative, even though a cell wall is present. For these cells, alternative procedures such as the Ziehl-Neelsen acid-fast staining procedure are commonly used to diagnose Mycobacteria.
Some cells are Gram-variable, and will stain differently depending on their age. Bacillus cereus is a Gram-positive rod. However, like many bacteria, this species can give conflicting results, particularly from old cultures. An alternative explanation for this type of appearance is a Gram-positive organism which has been over-decolourized with acetone, a common mistake with beginners!
Nevertheless, Gram was onto something when he developed this method, and you’re not a real microbiologist until you’ve done your first Gram stain!

Links:

• LabWork: Virtual Gram Stain Experiment
• Microbiology Video Library

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