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Biochemical reactions, Bacteriology

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  • Common Pathogenic Bacterial Isolates
    Dr.T.V.Rao MD
    BiochemicalReactions in
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    Dr.T.V.Rao MD
  • Laboratory Investigation of Microbial infections
    Examining specimens to detect isolate and identify pathogens:
    1- Microscopy
    2- Culture techniques
    3- Biochemical reactions
    4- Serological identification:
    5- Molecular biology techniques
    6- Bacteriophage typing
  • Identification of an Unknown Bacterium:
    Dr.T.V.Rao MD
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    Microbiologists use biochemical tests, noting a particular microbe's ability to utilize or produce certain chemicals
  • Biochemical tests help in Identification of several Bacterial isolates
    EVERYTHING that a living organism does is the result of the activity of an ENZYME, the SUMMATION of the activities of all an organism's enzymes equals its BIOCHEMICALFINGERPRINT. That is, an organism is the totality of its enzymes, so by determining which enzymes are present in an unknown organism one can DESCRIBE & IDENTIFY that organism
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    Dr.T.V.Rao MD
  • Biochemical Reaction
    Use of substrates and sugars to identify pathogens:
    a- Sugar fermentation:
    Organisms ferment sugar with production of acid only
    Organisms ferment sugar with production of acid and gas
    Organisms do not ferment sugar
    b- Production of indole:
    Depends on production of indole from amino acid tryptophan
    Indole is detected by addition of Kovacs reagent
    Appearance of red ring on the surface
    e- H2S production:
    Depends on production H2S from protein or polypeptides
    Detection by using a strip of filter paper containing lead acetate
  • Biochemical Reaction (cont.)
    c- Methyl red reaction (MR):
    Fermentation of glucose with production of huge amount of acid
    Lowering pH is detected by methyl red indicator
    d- Voges proskaurs reaction (VP):
    Production of acetyl methyl carbinol from glucose fermentation
    Acetyl methyl carbinol is detected by addition KOH
    Color of medium turns pink (positive)
    e-
  • Biochemical Reaction (cont.)
    f- Oxidase test:
    Some bacteria produce Oxidase enzyme
    Detection by adding few drops of colorless oxidase reagent
    Colonies turn deep purple in color (positive)
    g- Catalase test:
    Some bacteria produce catalase enzyme
    Addition of H2O2 lead to production of gas bubbles (O2 production)
    h- Coagulase test:
    Some bacteria produce coagulase enzyme
    Coagulase enzyme converts fibrinogen to fibrin (plasma clot)
    Detected by slide or test tube method
    i- Urease test:
    Some bacteria produce urease enzyme
    Urease enzyme hydrolyze urea with production of NH3
    Alklinity of mediaand change color of indicator from yellow to pink
  • Common Tests To identify Bacterial isolates
    Indole
    Methyl Red/Voges Proskauer
    Citrate
    H2S production
    Urea hydrolysis
    Motility
    Lactose fermentation
    Sucrose fermentation
    Glucose fermentation & gas production
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    Dr.T.V.Rao MD
  • Catalase test .
    This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas.
    The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result.
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    Dr.T.V.Rao MD
  • Catalase test
    'Ten vol.' H2O2, is run into a capillary tube, followed by suspension. Gas is usually evolved immediately and only tubes not showing gas within 10 sec. Are sealed for longer observation
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    Dr.T.V.Rao MD
  • OXIDASE TEST
    The Oxidase test (also known as the Cytochrome Oxidase test) is used to look for oxidase enzymes produced by certain bacteria. Oxidases catalyse electron transport between substrates acting as electron donors in the bacterium and tetramethyl-p-phenylenediamine OR dimethyl-p-phenylenediamine - a redox dye present as the hydrochloride or oxalate salt The dye is reduced to a deep violet-blue colour in the presence of oxidase enzymes
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    Dr.T.V.Rao MD
  • Oxidase test
    The oxidase test is a test used in microbiology to determine if a bacterium produces certain cytochrome c oxidases. It uses disks impregnated with a reagent such as N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) or N,N-Dimethyl-p-phenylenediamine (DMPD), which is also a redox indicator.
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    Dr.T.V.Rao MD
  • Filter strip method
    Soak strips of filter paper in a fresh dye solution, drain and freeze dry. Strips should be stored in an air-tight bottle and kept in a cool dark environment. Strips prepared in this manner will keep for several months, and have a faint pastel-violet color. To use, take a strip and soak in distilled water. Pick the colony to be tested with a loop and rub onto moistened strip. A color change within 10 seconds indicates a positive reaction.
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    Dr.T.V.Rao MD
  • Oxidase testing needs controls
    Positive control: Pseudomonas aeruginosa
    Negative control
    Enterobactericia
    E.coli. Klebsiella spp
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    Dr.T.V.Rao MD
  • HYDROGEN SULFIDE PRODUCTION
    Dr.T.V.Rao MD
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    Some bacteria have the enzymatic capability to degrade amino acids (cysteine, cystine etc.) that contain sulfhydryl group (-SH) producing hydrogen sulfide. Hydrogen sulfide reacts with heavy metals such as lead or iron forming a black precipitate. You can use TSI medium (contains iron) or prepare a nutritive agar with lead acetate (1g Pb acetate to 100 ml nutritive agar).
  • PROCEDURE and Reading result
    Dr.T.V.Rao MD
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    Harvest a well isolated colony and inoculate a TSI tube by stabbing the medium. Incubate at 37 C, 24 hours. Reaction is positive if a black color appears.
    Bacteria growing in TSI degrade amino acids forming ferrous sulfide which blackens the medium
  • Hydrogen sulphide production
    Dr.T.V.Rao MD
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    e- H2S production:
    Depends on production H2S from protein or polypeptides
    Detection by using a strip of filter paper containing lead acetate
    H2S production. H2S production, either via cysteine catabolism or thiosulfate reduction, produces a black precipitate in the media.
  • Nitrate Medium Nitrate reduction Test
    This is a differential medium. It is used to determine if an organism is capable of reducing nitrate (NO3-) to nitrite (NO2-) or other nitrogenous compounds via the action of the enzyme nitratase (also called nitrate reductase). This test is important in the identification of both Gram-positive and Gram-negative species.
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    Dr.T.V.Rao MD
  • Nitrate reduction Test
    After incubation, these tubes are first inspected for the presence of gas in the Durham tube. In the case of non fermenters, this is indicative of reduction of nitrate to nitrogen gas. However, in many cases gas is produced by fermentation and further testing is necessary to determine if reduction of nitrate has occurred.
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    Dr.T.V.Rao MD
  • Nitrate reduction Test
    The reduction of nitrate to nitrite was detected with dimethyl-a-naphthylamin (Wallace & Neave, 1927) and sulphanilic acid. The reaction was rapid with all the species tested; at 30 min. the results were consistent with the usual cultural method
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    Dr.T.V.Rao MD
  • I M Vi C Tests
    I M Vi C is an acronym that stands for indole , methyl red, Voges-Proskauer , and citrate . To obtain the results of these four tests, three test tubes are inoculated: tryptone broth (indole test), methyl red - Voges Proskauer broth (MR-VP broth), and citrate test.
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    Dr.T.V.Rao MD
  • Indole Test
    How to Perform Test:Inoculate Tryptone broth with inoculating loop.
    Property it tests for: This test is performed to help differentiate species of the family Enterobacteriaceae. It tests for the bacteria species ability to produce indole. Bacteria use an enzyme, tryptophanase to break down the amino acid, tryptophan, which makes by-products, of which, indole is one.
    Media and Reagents Used:Tryptone broth contains tryptophan. Kovacs reagentcontains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcoholyellow in color.
    Reading Results:Kovacs reagent reacts with indole and creates a red color at the top part of the test tube.
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    Dr.T.V.Rao MD
  • Principles of Indole Test
    The test organism is inoculated into tryptone broth, a rich source of the amino acid tryptophan. Indole positive bacteria such as Escherichia coli produce tryptophanase,