A Gram stain refers to a positive or negative test result produced when an iodine wash is introduced to a culture of bacteria in order to identify its species. This test, known as Gram staining, works by detecting the presence of lipopolysaccharides (lipoglycans) and peptidoglycans (mureins) contained within the cell walls of the bacteria sample. Bacteria that have a high level of peptidoglycans are said to be Gram-positive. In contrast, lower levels of peptidoglycans with lipopolysaccharides indicate that the sample is Gram-negative.
First, the bacteria sample is placed on a glass slide and heated only to the point of rendering it innocuous in terms of being infectious to the handler. Next, the bacteria sample is treated with a gentian violet-iodine solution for up to sixty seconds. The slide is then gently rinsed under clean water and the Gram solution is applied, which is a mixture of iodine and potassium iodide diluted in water. This step triggers a reaction to the gentian violet compound.
Initially, the reaction produces a dark blue color. However, a subsequent rinse with ethyl alcohol leads to the color in some bacteria samples to bleed out, but not in others. A final dye solution is applied that uses a contrasting color, usually a variation of red. A sample that accepts this counterstain will appear pink, and is designated as Gram-negative. However, a sample that retains the dark blue color is Gram-positive.
Aside from identification purposes, the significance of the Gram stain test lies in the fact that Gram-negative bacteria produce potent endotoxins that can cause serious diseases, such as cholera and typhoid. Many Gram-negative bacteria are also resistant to antibiotics and it is not possible to manufacture vaccines from them. In addition, not all bacteria produce a positive or negative result. In fact, some species are considered Gram indeterminate or Gram variable. Other species are entirely unaffected by the test simply due to having a wax-like protective layer in their cell walls that the stains cannot permeate.
The Gram stain test was developed in the late 1800s by the noted Danish bacteriologist, Hans Christian Gram. However, the original purpose of the Gram stain test was not to distinguish between different bacterial species at all. In fact, Dr. Gram merely set out to devise a better way to detect the presence of bacteria in sputum samples provided by pneumonia patients. It is also interesting to note that Dr. Gram’s discovery, while unintentional, would have a big impact on the study of antibiotic-resistant bacteria half a century later.