The Gram stain test identifies bacterial species by detecting lipopolysaccharides and peptidoglycans in cell walls. Gram-positive bacteria have high peptidoglycan levels, while Gram-negative bacteria have lower levels with lipopolysaccharides. The test involves treating a bacterial sample with gentian violet iodine solution, rinsing with water, applying Gram’s solution, rinsing with ethyl alcohol, and applying a counterstain. Gram-negative bacteria can cause serious illnesses and are often antibiotic-resistant. The test was developed by Hans Christian Gram in the late 1800s to detect bacteria in sputum samples.
A Gram stain refers to a positive or negative test result produced when a wash of iodine is introduced into a culture of bacteria to identify its species. This test, known as a Gram stain, works by detecting the presence of lipopolysaccharides (lipoglycans) and peptidoglycans (mureins) contained within the cell walls of the bacterial sample. Bacteria that have a high level of peptidoglycans are called Gram-positive. Conversely, 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 where it is harmless in terms of infection for the conductor. Subsequently, the bacterial sample is treated with a gentian violet iodine solution for up to sixty seconds. The slide is then rinsed gently under clean water and Gram’s 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 bleeds the color in some bacteria samples, but not in others. A final coloring solution using a contrasting color, usually a variation of red, is applied. 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 illnesses, such as cholera and typhoid. Many Gram-negative bacteria are also resistant to antibiotics and vaccines cannot be made from them. Also, not all bacteria produce a positive or negative result. Some species are in fact considered Gram indeterminate or Gram variable. Other species are not completely affected by the test simply because they have 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 noted Danish bacteriologist Hans Christian Gram. However, the original purpose of the Gram stain test was not at all to distinguish between different bacterial species. In fact, Dr. Gram just came up with a better way to detect the presence of bacteria in sputum samples provided by patients with pneumonia. It is also interesting to note that Dr. Gram’s discovery, while unintentional, would have a major impact on the study of antibiotic-resistant bacteria half a century later.
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