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Biochemical reactions, Bacteriology

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<ul><li> Common Pathogenic Bacterial Isolates<br>Dr.T.V.Rao MD<br>BiochemicalReactions in<br>1<br>Dr.T.V.Rao MD<br></li> <li> Laboratory Investigation of Microbial infections<br>Examining specimens to detect isolate and identify pathogens:<br>1- Microscopy<br> 2- Culture techniques<br> 3- Biochemical reactions<br> 4- Serological identification:<br> 5- Molecular biology techniques <br> 6- Bacteriophage typing<br></li> <li> Identification of an Unknown Bacterium:<br>Dr.T.V.Rao MD<br>3<br>Microbiologists use biochemical tests, noting a particular microbe's ability to utilize or produce certain chemicals<br></li> <li> Biochemical tests help in Identification of several Bacterial isolates<br>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 &amp; IDENTIFY that organism<br>4<br>Dr.T.V.Rao MD<br></li> <li> Biochemical Reaction<br>Use of substrates and sugars to identify pathogens:<br> a- Sugar fermentation:<br> Organisms ferment sugar with production of acid only<br> Organisms ferment sugar with production of acid and gas<br> Organisms do not ferment sugar<br>b- Production of indole:<br> Depends on production of indole from amino acid tryptophan<br> Indole is detected by addition of Kovacs reagent<br> Appearance of red ring on the surface <br>e- H2S production:<br> Depends on production H2S from protein or polypeptides<br> Detection by using a strip of filter paper containing lead acetate<br></li> <li> Biochemical Reaction (cont.)<br>c- Methyl red reaction (MR): <br> Fermentation of glucose with production of huge amount of acid<br> Lowering pH is detected by methyl red indicator<br>d- Voges proskaurs reaction (VP):<br> Production of acetyl methyl carbinol from glucose fermentation<br> Acetyl methyl carbinol is detected by addition KOH<br> Color of medium turns pink (positive)<br>e-<br></li> <li> Biochemical Reaction (cont.)<br>f- Oxidase test:<br> Some bacteria produce Oxidase enzyme<br> Detection by adding few drops of colorless oxidase reagent<br> Colonies turn deep purple in color (positive)<br>g- Catalase test:<br> Some bacteria produce catalase enzyme<br> Addition of H2O2 lead to production of gas bubbles (O2 production)<br>h- Coagulase test:<br> Some bacteria produce coagulase enzyme<br> Coagulase enzyme converts fibrinogen to fibrin (plasma clot) <br> Detected by slide or test tube method<br>i- Urease test:<br> Some bacteria produce urease enzyme <br> Urease enzyme hydrolyze urea with production of NH3<br>Alklinity of mediaand change color of indicator from yellow to pink<br></li> <li> Common Tests To identify Bacterial isolates<br>Indole<br>Methyl Red/Voges Proskauer<br>Citrate<br>H2S production<br>Urea hydrolysis<br>Motility<br>Lactose fermentation<br>Sucrose fermentation<br>Glucose fermentation &amp; gas production<br>8<br>Dr.T.V.Rao MD<br></li> <li> Catalase test .<br>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.<br>The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result. <br>9<br>Dr.T.V.Rao MD<br></li> <li> Catalase test<br>'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<br>10<br>Dr.T.V.Rao MD<br></li> <li> OXIDASE TEST<br>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<br>11<br>Dr.T.V.Rao MD<br></li> <li> Oxidase test<br>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.<br>12<br>Dr.T.V.Rao MD<br></li> <li> Filter strip method<br>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.<br>13<br>Dr.T.V.Rao MD<br></li> <li> Oxidase testing needs controls <br>Positive control: Pseudomonas aeruginosa<br>Negative control<br>Enterobactericia<br> E.coli. Klebsiella spp<br>14<br>Dr.T.V.Rao MD<br></li> <li> HYDROGEN SULFIDE PRODUCTION<br>Dr.T.V.Rao MD<br>15<br>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).<br></li> <li> PROCEDURE and Reading result<br>Dr.T.V.Rao MD<br>16<br>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.<br>Bacteria growing in TSI degrade amino acids forming ferrous sulfide which blackens the medium<br></li> <li> Hydrogen sulphide production <br>Dr.T.V.Rao MD<br>17<br>e- H2S production:<br> Depends on production H2S from protein or polypeptides<br> Detection by using a strip of filter paper containing lead acetate<br>H2S production. H2S production, either via cysteine catabolism or thiosulfate reduction, produces a black precipitate in the media.<br></li> <li> Nitrate Medium Nitrate reduction Test<br>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. <br>18<br>Dr.T.V.Rao MD<br></li> <li> Nitrate reduction Test<br>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. <br>19<br>Dr.T.V.Rao MD<br></li> <li> Nitrate reduction Test<br>The reduction of nitrate to nitrite was detected with dimethyl-a-naphthylamin (Wallace &amp; 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<br>20<br>Dr.T.V.Rao MD<br></li> <li> I M Vi C Tests<br>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.<br>21<br>Dr.T.V.Rao MD<br></li> <li> Indole Test<br>How to Perform Test:Inoculate Tryptone broth with inoculating loop.<br>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. <br>Media and Reagents Used:Tryptone broth contains tryptophan. Kovacs reagentcontains hydrochloric acid, dimethylaminobenzaldehyde, and amyl alcoholyellow in color.<br>Reading Results:Kovacs reagent reacts with indole and creates a red color at the top part of the test tube.<br>22<br>Dr.T.V.Rao MD<br></li> <li> Principles of Indole Test<br>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, an enzyme that cleaves tryptophan, producing indole and other products. When Kovac's reagent (p-dimethylamino benzaldehyde) is added to a broth with indole in it, a dark pink colour develops. The indole test must be read by 48 hours of incubation because the indole can be further degraded if prolonged incubation occurs. The acidic pH produced by Escherichia coli limits its growth.<br>23<br>Dr.T.V.Rao MD<br></li> <li> Indole Test<br>Indole is a product of the breakdown of another amino acid, tryptophan by the enzyme TRYPTOPHANASE. To test for indole Kovacs reagent is added to the SIM medium following growth. If indole is present a red ring formsaround the surface of the medium. <br>24<br>Dr.T.V.Rao MD<br></li> <li> Tryptone Broth after addition of Kovacs(+) Indole test on left --- (-) Indole test on right<br>Dr.T.V.Rao MD<br>25<br></li> <li> Indole test reactions<br>26<br>Dr.T.V.Rao MD<br></li> <li> Methyl Red/Voges Proskauer (MR/VP)<br>How to Perform Tests: Inoculate 2 glucose broths with inoculating loop. After 48 hours of incubation, add a few drops of MR to one tube, and VP reagents to the other tube.<br>Properties they test for: Both tests are used to help differentiate species of the family Enterobacteriaceae. <br>MRtests for acid end products from glucose fermentation. <br>VPtests for acetoin production from glucose fermentation.<br>Media and Reagents Used: <br>Glucose Broth<br>Methyl Red indicator for acid<br>Voges Proskauer reagentsA: 5% Alpha-Naphthol, &amp; ethanol, B: Potassium Hydroxide, &amp; Deionized Water.<br>27<br>Dr.T.V.Rao MD<br></li> <li> Methyl red (MR) and Voges-Proskauer (VP) tests<br>The methyl red (MR) and Voges-Proskauer (VP) tests are read from a single inoculated tube of MR-VP broth. After 24-48 hours of incubation the MR-VP broth is split into two tubes. One tube is used for the MR test; the other is used for the VP test.<br>MR-VP media contains glucose and peptone. All enterics oxidize glucose for energy; however the end products vary depending on bacterial enzymes. Both the MR and VP tests are used to determine what end products result when the test organism degrades glucose.<br>28<br>Dr.T.V.Rao MD<br></li> <li> MR/VP continued<br>Reading Results: <br>MR a + result is red (indicating pH below 6) and a result is yellow (indicating no acid production)<br>VPA + result is red after VP reagents are added (indicating the presence of acetoin) and a result is no color change.<br><br>Methyl Red: left and right +<br>29<br>Dr.T.V.Rao MD<br></li> <li> Methyl Red<br>This test is used to determine which fermentation pathway is used to utilize glucose. In the mixed acid fermentation pathway, glucose is fermented and produces several organic acids (lactic, acetic, succinic, and formic acids). The stable production of enough acid to overcome the phosphate buffer will result in a pH of below 4.4. If the pH indicator (methyl red) is added to an aliquot of the culture broth and the pH is below 4.4, a red color will appear (first picture, tube on the left).<br>30<br>Dr.T.V.Rao MD<br></li> <li> Methyl Red Test<br>If the pH indicator (methyl red) is added to an aliquot of the culture broth and the pH is below 4.4, a red color will appear (first picture, tube on the left). If the MR turns yellow, the pH is above 6.0 and the mixed acid fermentation pathway has not been utilized (first picture, tube on the right).<br>31<br>Dr.T.V.Rao MD<br></li> <li> Methylene- blue reduction<br>Standardized methylene blue in concentrations of 0.1 and 0.01 yo are mixed,with suspension and sealed. Readings are made after 4 and 24 hr. at 37".<br>32<br>Dr.T.V.Rao MD<br></li> <li> Voges-Proskauer (VP) test<br>The reagents used for the VP test are Barritt's A (alpha-napthol) and Barritt's B (potassium hydroxide). When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a pink-burgundy colour (a positive VP test). This colour may take 20 to 30 minutes to develop. E. coli does not produce acetyl methyl carbinol, but Enterobacter and Klebsiella do.<br>33<br>Dr.T.V.Rao MD<br></li> <li> Citrate Test<br>How to Perform Test:Inoculate slant with inoculating loop.<br>Property it tests for:This test is used to help differentiate species of the family Enterobacteriaceae. It is selective for bacteria that has the ability to consume citrate as its sole source of carbon and ammonium as sole nitrogen source.<br>Media and Reagents Used:Simmons Citrate Agar contains sodium citrate (carbon source), ammonium ion (nitrogen source), &amp; pH indicatorbromthymol blue.<br>Reading Results:<br>A + result is blue (meaning the bacteria metabolised citrate and produced an acid end product) and a result remains green<br>34<br>Dr.T.V.Rao MD<br></li> <li> Simmon's citrate agar <br>Uninoculated Simmon's citrate agar has a pH of 6.9, so it is an intermediate green color. Growth of bacteria in the media leads to development of a Prussian blue color (positive citrate). Enterobacter and Klebsiella are citrate positive while E.coli is negative.<br>35<br>Dr.T.V.Rao MD<br></li> <li> Citrate Test<br>Left positive and right negative.<br>36<br>Dr.T.V.Rao MD<br></li> <li> Urea Hydrolysis<br>How to Perform Test: Inoculate Urea broth with inoculating loop.<br>Property it tests for: This test is done to determine a bacterias ability to hydrolyze urea to make ammonia using the enzyme urease.<br>Media and Reagents Used: Urea broth contains a yeast extract, monopotassium phosphate, disodium phosphate, urea, and phenol red indicator.<br>Reading Results: Urea broth is a yellow-orange color. The enzyme urease will be used to hydrolyze urea to make ammonia. If ammonia is made, the broth turns a b...</li></ul>