MICROBIOLOGY

10) Carbohydrate Fermentation Test                                    The term fermentation is applied to the anaerobic degradation of carbohydrates.Ultimately the purpose of fermentation is to make energy available to a living cell,since carbohydrate molecules are quite rich in stored energy. Whether or not a particular carbohydrate is fermented depends upon the enzymes produced by the organism. During fermentation,most bacteria produce different types of acids & gases. The end product of fermentation varies from one organism to also upon the enzymes present in them. Some of these ends can be detected in lab. Thus, the ability of organisms to break a single   sugar, combination of sugars or various other carbohydrates  in media provides information for the classification of a bacterial spp.           Acid production can be determined by the used of indicators of pH media containing carbohydrates.A commonly used pH indicator is phenol red, which is red at pH 8.5 & yellow at

9) Nitrate Reduction Test :                       Many microorganisms can reduce nitrate salts such as potassium nitrate to nitrite salt, KNO2, under anaerobic conduction. This is an example of anaerobic respiration in which the nitrate molecule acts as electron acceptor in place of oxygen. This reduction of nitrate can easily be demonstrated experimentally by formulating a medium containing broth to which a very small amount of potassium nitrate is added.               Some mos possess the enzymatic capacity to act further on nitrates to reduce them to ammonia or molecule Nitrogen. After growing the organism in nitrate medium, its ability to reduce nitrate to nitrite is determined by the addition of two reagents solution A containing sulfanilic acid & solution B containing alpha-naphthylamine. Nitrite gives cherry red colored complex with sulfanilic acid & alpha-naphthylamine. The culture may not produce red color due to : a) nitrates not reduced by mos or  b) the mos p

8.3) CITRATE UTILIZATION TEST :                      The basic principle of this test is to detect the ability of an organism which can utilize citrate as sole source of carbon for their metabolism with resulting alkalinity. All coliforms metabolize citrate when molecule is generated inside the bacterial cell. But not all coliforms produce transport enzymes that bring citrate from the environment across the cytoplasmic membrane & into the cell.Normally, the citrate metabolism involves the condensation of acetyl with Coenzyme A resulting in the formation of oxaloacetate which enters the TCA cycle.      In the bacteria that utilize citrate, the cleavage of citrate involves an enzyme system without the intervention  of  Coenzyme A. This enzyme system is known as citrase or citrate permease. This enzyme system requires a divalent cation for its activity which supplied by the Magnesium+ ion or Mn+ ion. The  product obtained  from the citrate metabolism depends on the pH of the med

8) IMViC Tests :( Contd...)  8.2)   Methyl Red/Voges Proskauer Test :(MR-VP)                   Microorganisms when grown in MR-VP or Clark and Lub's medium containing  a limited amount of carbohydrate ferment it to produce acids which lowers the pH of the medium. Certain genera go on producing more acid in a mixed acid fermentation making the pH too low. Basic principle of this test is to detect the ability of an organism to produce & maintain stable acid end products. If an organism produces large amount of organic acids like formic acid, acetic acid ,lactic acid, succinic acids as end products from glucose, the pH of the medium lowers down.       To test if sufficient amount of acid has been produced in the culture, a pH indicator methyl red is added to it. This indicator turns red at Ph 4.4 & below & becomes yellow at pH 6.2 & above. In a positive MR test, the reagent remains red indicating that large amounts of acids were produced by bacteria growing in the

8) IMViC Test:                                 IMViC are a group of tests naming Indole, Methyl red, Voges Proskauer, & Citrate tests. Microbiologists have developed groups of biochemical tests that they use to differentiate morphologically & physiologically similar organism. One such group is IMViC test. It is used, primarily, to separate coliforms. Coliforms are defined as Gram negative, aerobic or facultative anaerobic, non spore former, rod shaped bacteria that produces acid & gas from lactose within 48 hours at 37 degree celsius. The coliforms are placed in family Enterobacteriaceae, commonly called enterics. Among the enterics are many species that normally residue in human & in other animal intestines including the common sewage indicator organism E.coli .          Using IMViC test, E.coli   is easily differentiated from other morphologically & physiologically similar coliforms like Enterobacter aerogenes . This differentiation is important in testing th

6)   Coagulase Test :                            The coagulase test identifies whether an organism produces the exoenzyme coagulase, which causes the fibrin of blood plasma to clot. Organisms that produce coagulase can form protective barriers of fibrin around themselves, making themselves highly resistant to phagocytosis, other immune responses, & some other antimicrobial agents. Staphylococcus aureus is the only significant human pathogen that produces the enzyme coagulase. Thus, it is a confirmatory test for    Staphylococcus aureus  & also helps to differentiate from non pathogenic S. epidermidis . The coagulase enzymes exist in two forms- free & bound coagulase.                                       The extracellular free  coagulase is a heat labile enzyme secreted free into culture medium. It requires the cooperation of plasma factor called coagulase reacting factor(CRF) for its clotting action. CRF is present in rabbit & human plasma. Free coagulase has thromb

5) Urea Hydrolysis Test :                                     Urea is a common metabolic waste product of protein digestion in most vertebrates that is toxic to most living organisms. The enzyme Urease catalyzes the breakdown of urea into ammonia & carbon dioxide.  The basic principle of this test is to determine the ability of certain microorganisms to produce the enzyme Urease. It is one of the useful diagnostic tests for  identifying bacteria, particularly Proteus spp   from other Gram negative bacteria.  Urease test is performed by growing the test organisms on urea agar medium containing the pH indicator, phenol red. During incubation microorganisms capable of producing the enzyme urease will liberate ammonia which cause the rise in pH of the medium. As, the pH increases, the color of the phenol red changes from an orange to deep pink.   In the clinical laboratory, members of the genus Proteus can be distinguished from other enteric nonlactose-fermenting bacteria ( Salm

4) Oxidative/Fermentative Test:                       During the process of metabolism ie breakdown of complex organic molecules, certain amount of energy is released . Most mos have the capability of catabolizing carbohydrates . Both the extracellular & intracellular enzymes are necessary for the degradation of carbohydrates . Extracellular enzymes degrade complex carbohydrate into simpler carbohydrate which can pass to the cell. The intracellular enzymes catabolize these simpler molecules to generate energy. The end product of this degradation is used in glycolysis for the generation of energy.   Glycolysis is one of the metabolic pathway in which simple sugar is converted in a form of energy as ATP. If the glucose after entering a cell is catabolized aerobically the process is called oxidative metabolism where oxygen serves as final electron acceptor. If glucose is catabolized anaerobically, the fermentative metabolism takes place where organic molecules serve as final elect

3)   Triple Sugar Iron(TSI) Agar Test:                         The TSI agar test  is used to differentiate among the members of the family Enterobacteriaceae and between Enterobacteriaceae and other intestinal bacteria. The authors have chosen three common bacteria in the family Enterobacteriaceae: Escherichia coli, Proteus vulgaris, and Shigella flexneri .  All three are facultatively anaerobic gram-negative rods. In a TSI tube, E. coli produces an acid butt, an acid or alkaline slant, and no H2S, but does produce gas. P. vulgaris produces an acid butt, an acid or alkaline slant, H2S, and gas. S. flexneri produces an acid butt, an alkaline slant, no H2S, and no gas. For the other intestinal bacteria, the authors have chosen Alcaligenes faecalis and Pseudomonas aeruginosa. Both of these intestinal bacteria are gram-negative aerobic rods. In a TSI tube, A. faecalis produces an alkaline butt, alkaline slant, H2S, and gas; P. aeruginosa, an acid butt, alkaline slant, H2S,

BIOCHEMICAL TESTS( Cont..) 2)  Oxidase Test:  The oxidase test is a means of specifically detecting the presence of the terminal enzyme system in aerobic respiration called Cytochrome C oxidase.  The aerobic respiratory mechanism is composed of a number of enzymes which alternately oxidize & reduce each other by donating or accepting electrons derived from hydrogen. Hydrogen atoms are removed from various substrate compounds during oxidation reaction & ultimately the hydrogen atoms are combined with oxygen to form water. Thus, cytochrome C oxidase is the terminal or last hydrogen electron acceptor in this chain, its function being to transfer the hydrogen electrons to oxygen to form water molecules. This is the classic aerobic respiration scheme that most microorganisms utilize to derive energy from the food they take in as molecules of ATP.                The ability of an organism to produce cytochrome C oxidase can be determined by using the reagent tetramethyl-p-phenyle

BIOCHEMICAL TESTS TO IDENTIFY BACTERIA: While several commercial systems for identifying bacteria are available, these are often difficult to obtain or too expensive to use in developing countries. This subunit includes a range of conventional biochemical tests and tablet identification tests which most district laboratories will be able to perform. The following tests are described in this part:  Test                          Purpose Beta-glucuronidase  To identify E. coli Bile solubility:       To differentiate S. pneumoniae from other alpha-haemolytic streptococci Catalase: To differentiate Staphylococci from  Streptococci Citrate utilization :To differentiate Enterobacteria Coagulase: To identify S. aureus DNA-ase  To help identify S. aureus Indole  To differentiate Gram negative rods, particularly E. coli Litmus milk  To help identify decolorization Enterococcus and some clostridia Lysine decarboxylase  To assist in the identification of