In the second half of nineteenth century, scientists proved that specific bacterial organisms caused specific diseases, and the field of microbiology was on its way to becoming a distinct science. The microscope was also further developed during that time, and scientists were concerned with identifying and classifying bacteria . Most bacteria are difficult to see with a compound microscope, but can be seen when there is obvious contrast between the bacteria cells and their surrounding medium. Various dyes are used to stain cells so that they are more easily seen. As early as the late eighteenth century, scientists had developed some basic methods of staining cells to aid in their study and used natural substances such as saffron, which stained some parts of a cell. The discovery of synthetic dyes in the mid-1800s enabled scientists to utilize more colors to stain cells.
In 1884, the Danish physician Hans Christian Joachim Gram further developed a method of staining bacteria originally developed by the German biologist Paul Ehrlich . Ehrlich used aniline water and gentian violet (a cationic dye) to stain cells, and the cell walls would appear purple after staining. Gram added a potassium triiodide solution, which acted as a mordant for the gentian violet dye, and then poured ethanol over the cells to wash away the unfixed dye. Gram found that some of the cells remained purple, while others did not. Bacteria that remained purple were termed positive and those that did not remain purple were called negative. A few years later, Carl Weigert, director of the Senckberg Foundation in Frankfurt, Germany, added another step to the staining method. Weigert followed Gram's procedure with a final staining using saffranine (an anionic dye), which subsequently stained the negative bacteria red. The Gram stain is still considered the definitive, differential test to determine the chemical make-up of a bacterium cell wall. On the basis of a cell's reaction to the Gram stain, bacteria are divided into two groups, Gram positive and Gram negative.
The distinguishing feature between Gram positive and negative bacteria is the difference in the structure of the cell walls. The cell wall of a Gram positive is a thick, single layer of a cross-linked polysaccharide that is easily stained by gentian violet, while the cell wall of a Gram negative bacterium consists of a thin layer of polysaccharide and is covered by a lipid layer that resists the gentian violet, but can be stained by saffranine. Many dyes, which are organic compounds, are positively charged and easily combine with the negatively charged, acidic polysaccharide wall. Other dyes are negatively charged and combine with protein-based cell constituents.
The chemical make-up of the cell wall also determines the penetrability of the wall by various drugs. Knowing if a bacterium is Gram positive or negative determines what type of antibiotic is suitable for treatment, as some antibiotics act against Gram positive bacteria (i.e., penicillin ), while others act against Gram negative bacteria (i.e., tetracycline or streptomycin). Another important consideration is the fact that some Gram negative bacteria release endotoxins, which can be fatal. When pharmaceutical companies develop new antibacterial drugs, the Gram stain is the method by which scientists determine the effectiveness of the drug.
See also Laboratory techniques in microbiology