Taxonomy relies on three key elements: characterization, classification and nomenclature. All three elements are dynamic fields, but each step depends on the one which precedes it. Thus, the nomenclature of a group of organisms depends on the way they are classified, and the classification (among other elements) depends on the information gathered as a result of characterization. While nomenclature is governed by the Bacteriological Code, the classification and characterization of prokaryotes is an area that is not formally regulated and one in which numerous changes have taken place in the last 50 years. The purpose of the present article is to outline the key elements in the way that prokaryotes are characterized, with a view to providing an overview of some of the pitfalls commonly encountered in taxonomic papers.

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Notes on the characterization of prokaryote strains for taxonomic purposes.

In performing phenotypic characterization tests, the organisms used for the inoculation of test media should be from fresh transfers and in good physiological condition. The methods described in this chapter are based primarily on methods developed for the characterization and identification of organisms which have usually been isolated on nutrient-rich media. While zoologists may be content to classify all insects or all vertebrates, prokaryote systematics generally deals with all prokaryotes and in an evolutionary context attempts to extend this study back to the origin of life itself. To test for acetamide hydrolysis, streak the surface of an acetamide agar slant with a sample from a dilute suspension. Incubate for up to 7 days. Care should be taken to ensure that the basal medium to which the carbohydrates are added does not contain fermentable or oxidizable sugars, and control media without any added carbohydrates should always be included during testing of cultures. Methanogenic members of the Archaea also fluoresce under UV light. Some facultative anaerobes, such as Escherichia coli and Staphylococcus aureus, grow aerobically by respiration with oxygen and grow anaerobically by fermentation.

Phenotypic Characterization and the Principles of Comparative Systematics

Surface layers of bacteria.

Intracellular lipid transport is poorly understood. Genetic studies to identify lipid-transport factors are complicated by the essentiality of many lipids, whereas biochemical and cell biology approaches aiming to determine localization and mechanisms of lipid transport are often challenged by the lack of adequate technology. Here, we review the epic history of how different approaches, technological advances and ingenuity contributed to the recent discovery of a multi-protein pathway that transports lipopolysaccharide across the envelope of Gram-negative bacteria.

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Transport of lipopolysaccharide across the cell envelope: the long road of discovery

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be "gram-positive," whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be "gram-negative." This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.

Use of the Gram stain in microbiology

Crystal violet (hexamethyl-para-rosaniline chloride) interacts with aqueous KI-I2 during the Gram stain via a simple metathetical anion exchange to produce a chemical precipitate. There is an apparent 1:1 stoichiometry between anion (I-) and cation (hexamethyl-para-rosaniline+) during the reaction and, since the small chloride anion is replaced by the bulkier iodide, the complex formed becomes insoluble in water. It is this same precipitate which forms in the cellular substance of bacteria (both gram-positive and gram-negative types) and which initiates the Gram reaction. Potassium trichloro(eta 2-ethylene)-platinum(II), as an electronopaque marker for electron microscopy, was chemically synthesized, and it produced an anion in aqueous solution which was compatible with crystal violet for the Gram stain. It interacted with crystal violet in a similar manner as iodide to produce an insoluble complex which was chemically and physically analogous to the dye-iodide precipitate. This platinum anion therefore allows the Gram staining mechanism to be followed by electron microscopy.

Chemical mechanism of the Gram stain and synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain.

J A Davies

Papers

Chemical mechanism of the Gram stain and synthesis of a new electron-opaque marker for electron microscopy which replaces the iodine mordant of the stain.