Gram stain (Gram staining or Gram's method) is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. It may also be used to diagnose a fungal infection. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884. Gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Gram-positive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal violet. Gram-negative cells have a thinner peptidoglycan layer that allows the crystal violet to wash out on addition of ethanol. They are stained pink or red by the counterstain, commonly safranin or fuchsine. Lugol's iodine solution is always added after addition of crystal violet to form a stable complex with crystal violet that strengthens the bonds of the stain with the cell wall. Gram staining is almost always the first step in the identification of a bacterial group. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique. This gives rise to gram-variable and gram-indeterminate groups.
History The method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938), who developed the technique while working with Carl Friedländer in the morgue of the city hospital in Berlin in 1884. Gram devised his technique not for the purpose of distinguishing one type of bacterium from another but to make bacteria more visible in stained sections of lung tissue. Gram noticed that some bacterial cells possessed noticeable resistance to decolorization. Based on these observations, Gram developed the initial gram staining procedure, initially making use of Ehrlich's aniline-gentian violet, Lugol's iodine, absolute alcohol for decolorization, and Bismarck brown for counterstain. He published his method in 1884, and included in his short report the observation that the typhus bacillus did not retain the stain. Gram did not initially make the distinction between Gram-negative and Gram-positive bacteria using his procedure.
Uses
Gram staining is a bacteriological laboratory technique used to differentiate bacterial species into two large groups (gram-positive and gram-negative) based on the physical properties of their cell walls. Gram staining can also be used to diagnose a fungal infection. Gram staining is not used to classify archaea, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups. Gram stains are performed on body fluid or biopsy when infection is suspected. Gram stains yield results much more quickly than culturing, and are 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.
Staining mechanism
Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin. There are four basic steps of the Gram stain:
Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they do not rinse out during the staining procedure. The addition of iodine, which binds to crystal violet and traps it in the cell Rapid decolorization with ethanol or acetone Counterstaining with safranin. Carbol fuchsin is sometimes substituted for safranin since it more intensely stains anaerobic bacteria, but it is less commonly used as a counterstain.
Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl−) ions. These ions penetrate the cell wall of both gram-positive and gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple. Iodide (I− or I−3) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV–I complex and, therefore, colors the cell. When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A gram-negative cell loses its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the gram-negative cell along with the outer membrane. In contrast, a gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet stain is removed from both gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds). After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsine, is applied last to give decolorized gram-negative bacteria a pink or red color. Both gram-positive bacteria and gram-negative bacteria pick up the counterstain. The counterstain, however, is unseen on gram-positive bacteria because of the darker crystal violet stain.
Examples
Gram-positive bacteria
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