In microbiology and bacteriology, Gram stain, is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique in 1884.
In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall.
Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane.
Mycobacterium is a genus of over 190 species in the phylum Actinomycetota, assigned its own family, Mycobacteriaceae. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis and leprosy in humans. The Greek prefix myco- means 'fungus', alluding to this genus' mold-like colony surfaces. Since this genus has cell walls with a waxy lipid-rich outer layer that contains high concentrations of mycolic acid, acid-fast staining is used to emphasize their resistance to acids, compared to other cell types.
Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology, in cytology, and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of diseases at the microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.
The cell envelope comprises the inner cell membrane and the cell wall of a bacterium. In gram-negative bacteria an outer membrane is also included. This envelope is not present in the Mollicutes where the cell wall is absent.
The Ziehl-Neelsen stain, also known as the acid-fast stain, is a bacteriological staining technique used in cytopathology and microbiology to identify acid-fast bacteria under microscopy, particularly members of the Mycobacterium genus. This staining method was initially introduced by Paul Ehrlich (1854–1915) and subsequently modified by the German bacteriologists Franz Ziehl (1859–1926) and Friedrich Neelsen (1854–1898) during the late 19th century.
The auramine–rhodamine stain (AR), also known as the Truant auramine–rhodamine stain, is a histological technique used to visualize acid-fast bacilli using fluorescence microscopy, notably species in the Mycobacterium genus. Acid-fast organisms display a reddish-yellow fluorescence. Although the auramine–rhodamine stain is not as specific for acid-fast organisms as the Ziehl–Neelsen stain, it is more affordable and more sensitive, therefore it is often utilized as a screening tool.
Acid-fastness is a physical property of certain bacterial and eukaryotic cells, as well as some sub-cellular structures, specifically their resistance to decolorization by acids during laboratory staining procedures. Once stained as part of a sample, these organisms can resist the acid and/or ethanol-based decolorization procedures common in many staining protocols, hence the name acid-fast.
The bacteria capsule is a large structure common to many bacteria. It is a polysaccharide layer that lies outside the cell envelope, and is thus deemed part of the outer envelope of a bacterial cell. It is a well-organized layer, not easily washed off, and it can be the cause of various diseases.
Mycobacterium smegmatis is an acid-fast bacterial species in the phylum Actinomycetota and the genus Mycobacterium. It is 3.0 to 5.0 µm long with a bacillus shape and can be stained by Ziehl–Neelsen method and the auramine-rhodamine fluorescent method. It was first reported in November 1884 by Lustgarten, who found a bacillus with the staining appearance of tubercle bacilli in syphilitic chancres. Subsequent to this, Alvarez and Tavel found organisms similar to that described by Lustgarten also in normal genital secretions (smegma). This organism was later named M. smegmatis.
Franz Ziehl was a German bacteriologist. He was a professor in Lübeck.
Bartonellosis is an infectious disease produced by bacteria of the genus Bartonella. Bartonella species cause diseases such as Carrión's disease, trench fever, cat-scratch disease, bacillary angiomatosis, peliosis hepatis, chronic bacteremia, endocarditis, chronic lymphadenopathy, and neurological disorders.
Auramine O is a diarylmethane dye used as a fluorescent stain. In its pure form, Auramine O appears as yellow needle crystals. It is insoluble in water and soluble in ethanol and DMSO.
Burkholderia pseudomallei is a Gram-negative, bipolar, aerobic, motile rod-shaped bacterium. It is a soil-dwelling bacterium endemic in tropical and subtropical regions worldwide, particularly in Thailand and northern Australia. It was reported in 2008 that there had been an expansion of the affected regions due to significant natural disasters, and it could be found in Southern China, Hong Kong, and countries in America. B. pseudomallei, amongst other pathogens, has been found in monkeys imported into the United States from Asia for laboratory use, posing a risk that the pathogen could be introduced into the country.
Mycobacterium bohemicum is a species of the phylum Actinomycetota, belonging to the genus Mycobacterium.
The Wayson stain is a basic fuchsin-methylene blue, ethyl alcohol-phenol microscopic staining procedure. It was originally a modified methylene blue stain used for diagnosing bubonic plague. With this stain, Yersinia pestis appears purple with a characteristic safety-pin appearance, which is due to the presence of a central vacuole.
Proteus penneri is a Gram-negative, facultatively anaerobic, rod-shaped bacterium. It is an invasive pathogen and a cause of nosocomial infections of the urinary tract or open wounds. Pathogens have been isolated mainly from the urine of patients with abnormalities in the urinary tract, and from stool. P. penneri strains are naturally resistant to numerous antibiotics, including penicillin G, amoxicillin, cephalosporins, oxacillin, and most macrolides, but are naturally sensitive to aminoglycosides, carbapenems, aztreonam, quinolones, sulphamethoxazole, and co-trimoxazole. Isolates of P. penneri have been found to be multiple drug-resistant (MDR) with resistance to six to eight drugs. β-lactamase production has also been identified in some isolates.
Pseudescherichia vulneris is a Gram-negative bacterial species. P. vulneris is a fermentative, oxidase-negative, motile rod, which holds characteristics of the family Enterobacteraceae. This bacterium can colonize in the respiratory tract, genital tract, stool, and urinary tract. However, P. vulneris is most often associated with wounds and has been known to colonize open wounds of both humans and animals. This association gave the bacterium its species name, vulneris, which is Latin for wound. It has also been infrequently reported in cases of meningitis. It was identified as Escherichia vulneris in 1982 with a 2017 genomic analysis of its original genus resulting in the creation of its new genus Pseudescherichia.
The Kinyoun method or Kinyoun stain, developed by Joseph J. Kinyoun, is a procedure used to stain acid-fast species of the bacterial genus Mycobacterium. It is a variation of a method developed by Robert Koch in 1882. Certain species of bacteria have a waxy lipid called mycolic acid, in their cell walls which allow them to be stained with Acid-Fast better than a Gram-Stain. The unique ability of mycobacteria to resist decolorization by acid-alcohol is why they are termed acid-fast. It involves the application of a primary stain, a decolorizer (acid-alcohol), and a counterstain. Unlike the Ziehl–Neelsen stain, the Kinyoun method of staining does not require heating. In the Ziehl–Neelsen stain, heat acts as a physical mordant while phenol acts as the chemical mordant. Since the Kinyoun stain is a cold method, the concentration of carbol fuschin used is increased.
