The most widely used identification scheme involves determining the morphologic and metabolic properties of the unknown bacterium and comparing these with properties of known microorganisms. It is essential to start identification tests with pure bacterial isolates grown from a single colony.

 A. Single-enzyme tests

 Different bacteria produce varying spectra of enzymes. For example, some enzymes are necessary for the bacterium’s individual metabolism, and some facilitate the bacterium’s ability to compete with other bacteria or establish an infection. Tests that measure single bacterial enzymes are simple, rapid, and generally easy to interpret. They can be performed on organisms already grown in culture and often provide presumptive identification.

 1. Catalase test: The enzyme catalase catalyzes the degradation of hydrogen peroxide to water and molecular oxygen (H₂O₂ → H₂O + O₂). Catalase-positive organisms rapidly produce bubbles when exposed to a solution containing hydrogen peroxide (Figure 1). The catalase test is key in differentiating between many gram-positive organisms. For example, staphylococci are catalase positive, whereas streptococci and enterococci are catalase negative. The production of catalase is an important virulence factor because H₂O₂ is antimicrobial, and its degradation decreases the ability of neutrophils to kill invading bacteria.

Fig1. Tests commonly used in identifying bacteria.

2. Oxidase test: The enzyme cytochrome c oxidase is part of electron transport and nitrate metabolism in some bacteria. The enzyme can accept electrons from artificial substrates (such as a phenylenediamine derivative), producing a dark, oxidized product (see Figure 1). This test assists in differentiating between groups of gram-negative bacteria. Pseudomonas aeruginosa, for example, is oxidase positive.

 3. Urease: The enzyme urease hydrolyzes urea to ammonia and carbon dioxide (NH₂CONH₂ + H₂O → 2NH₃ + CO₂). The ammonia produced can be detected with pH indicators that change color in response to the increased alkalinity (see Figure 1). The test helps to identify certain species of Enterobacteriaceae, Corynebacterium urealyticum, and Helicobacter pylori.

 4. Coagulase test: Coagulase is an enzyme that causes a clot to form when bacteria are incubated with plasma (see Figure 1). The test is used to differentiate Staphylococcus aureus (coagulase positive) from coagulase-negative staphylococci.

B. Automated systems

 Microbiology laboratories are increasingly using automated methods to identify bacterial pathogens. For example, in the Vitek System, small plastic reagent cards containing microliter quantities of various biochemical test media in 30 wells provide a biochemical profile that allows for organism identification (Figure 2). An inoculum derived from cultured samples is automatically transferred into the card, and a photometer intermittently measures color changes in the card that result from the metabolic activity of the organism. The data are analyzed, stored, and printed in a computerized database. There are many commercial variants of these automated systems and several can be used for simultaneous identification and antimicrobial susceptibility determination.

Fig2. A. Vitek test card containing test wells. B. Color of well changes with time.

C. Tests based on the presence of metabolic pathways

These tests measure the presence of metabolic pathways in a bacterial isolate, rather than a single enzyme. Commonly used assays include those for oxidation and fermentation of different carbohydrates, the ability to degrade amino acids, and use of specific substrates. A widely used manual system for rapid identification of members of the family Enterobacteriaceae and other gram-negative bacteria makes use of twenty microtubes containing substrates for various biochemical pathways. The test substrates in the microtubes are inoculated with the bacterial isolate to be identified, and, after 5 hours incubation, the metabolic profile of the organism is con structed from color changes in the microtubes. These color changes indicate the presence or absence of the bacteria’s ability to metabolize a particular substrate. The results are compared with a data bank containing test results from known bacteria (Figure 3). The probability of a match between the test organism and known pathogens is then calculated.

Fig3. Rapid manual biochemical system for bacterial identification. Different appearances of the upper and lower pairs of wells indicate the positive or negative ability of a bacterium to utilize each substrate.

مواضيع ذات صلة

Immunologic Detection of Microorganisms

Growing Bacteria in Culture

Cultivation of Listeria, Corynebacterium, and Similar Organisms

Pathogenesis and Spectrum of Disease for Listeria, Corynebacterium, and Similar Organisms

The Growth Curve in Batch Culture

The Measurement of Microbial Concentrations

Bacterial Pathogenesis

Distribution of Normal Flora in The Body

Cultivation of Bacillus and Similar Organisms

Diagnostic Laboratory Tests for Borrelia Burgdorferi

Bacterial Cell wall

Bacillus cereus