Article: Gram staining

Gram-positive anthrax bacteria (purple rods) in cerebrospinal fluid sample. If present, a gram-negative bacterial species would appear pink. (The other cells are white blood cells)

Gram staining (or the Gram's method) is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls.

The method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938), who developed the technique in 1884 to discriminate between pneumococci and Klebsiella pneumoniae bacteria.

Uses

Research

Gram staining is one of the most useful staining procedures in bacteriological laboratory. The technique is widely used as a tool for differentiating Gram-negative and Gram-positive bacteria, as a first step to determine the identity of a particular bacterial sample.

Medical

Gram stains are performed on body fluid or biopsy when infection is suspected. It yields results much quicker than culture, and is especially important when infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis.

Procedure

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Gram staining

The procedure consists of retrieving a sample from the culture using an inoculation loop, adding a dye, an iodine solution, and ethanol to the sample, followed by basic fuchsin as a counter stain, and observing color change. Gram-positive samples will appear black-blue or purple, and Gram-negative samples red or pink. Some samples may not fit into these groups. These are known as gram-variable.

Mechanism

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan which is capable of retaining the violet dye/iodine complex. Gram-negative bacteria have a thin cell wall made of a layer of peptidoglycan. In addition to an inner membrane, they also have an outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space.

The decolorising mixture causes dehydration of the multilayered peptidoglycan in the Gram-positive cell wall, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell. But in Gram-negative bacteria, the decolorising mixture acts as a lipid solvent and dissolves the outer membrane of the Gram-negative cell wall. The thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the Gram-negative cell is decolorized. The decolorisation step is the crucial one, and requires some degree of skill, as being Gram-positive is not an all-or-none phenomenon.

As a general rule of thumb (which has exceptions), Gram-negative bacteria are more dangerous as disease organisms, because their outer membrane is often hidden by a capsule or slime layer which hides the antigens of the cell and so acts as "camouflage" - the human body recognises a foreign body by its antigens; if they are hidden, it becomes harder for the body to detect the invader. Often the presence of a capsule will increase the virulence of a pathogen. Additionally, Gram-negative bacteria have lipopolysaccharide in their outer membrane, an endotoxin which increases the severity of inflammation. This inflammation may be so severe that septic shock may occur. Gram-positive infections are generally less severe because the human body does not contain peptidoglycan; in fact, the human body produces an enzyme called lysozyme which attacks the open peptidoglycan layer of Gram-positive bacteria. Gram-positive bacteria are also frequently much more susceptible to beta-lactam antibiotics, such as penicillin.

See also

• Staining (biology)