8Thurs_Minor unknown

8Thurs_Minor unknown - Lab Exercise A Physiological...

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Lab Exercise: A Physiological & Cultural Examination of the Minor Unknown OBJECTIVES 1. Become familiar with media used in identification of microbes, and how these media work to test for different physiological characteristics. 2. Perform physiological tests and be able to determine a negative from a positive result. BACKGROUND In order to identify a bacterial species, microbiologists employ many techniques that will allow them to gather both physical and physiological data about a particular isolate. Initially, an isolate is microscopically examined for Gram stain reaction and the presence of sub-cellular/cellular characteristics ( e.g ., capsules, spores, acid-fastness and flagella). In addition, the approximate size, morphology and arrangement of the cells are also determined. Once these morphological characteristics have been determined, the cultural characteristics of the isolate are determined: the oxygen requirement; its growth patterns on various solid and liquid media are observed and the optimal temperature of the organism is determined. Finally, the isolate’s physiological characteristics are examined. Bacteria use catabolic chemical reactions in order to harness the energy released from the decomposition of organic molecules as ATP . These reactions, like all metabolic reactions, are governed by enzymes catalysts. Enzymes can be divided into two groups, endoenzymes which function inside the bacterial cell and exoenzymes which are secreted by the cell in order to accomplish digestion in the exterior environment, (Figure 1). Because many bacteria share the morphological and cultural characteristics mentioned above, the identification of the physiological capabilities of an unknown bacterium is paramount to its identification. The collection of these physiological data is the most time-consuming and complicated aspect, and employs the use of multiple selective and differential media . Each of these media infers the presence of a specific enzyme and/or enzymes by measuring the absence of a substrate or the presence of a product from a given enzyme-regulated reaction. Because each enzyme, as a protein, is coded in the genes of an organism, the results of these physiological tests indicates something about the genome of the organism you are studying. Figure 1: Action of enzymes inside (endoenzymes) and outside (exoenzymes) the bacterial cell on a particular substrate. These enzymes catalyze the reactions and results in ATP generation. Physiological characteristics can be subdivided into categories: carbohydrate metabolism; fermentation; protein catabolism; and respiration. Carbohydrate metabolism includes those reactions which break down molecules containing the subunit ratio of (CH 2 O) n : polysaccharides, oligosaccharides, disaccharides and monosaccharides. Fermentation reactions , you will remember from lecture, are catabolic redox reactions whose terminal electron acceptor is an organic molecule, typically an acidic byproduct of pyruvate and/or carbon dioxide (CO 2 ) gas or acetoin. Protein catabolism
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