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Enzymes catalyze all the various chemical reactions of which the organism is capable. This in turn means that different species of bacteria must carry out different and unique sets of biochemical reactions. {  (" " " " "  2 H  0޽h ? ̙33 yqP (    6  kWhen identifying a suspected organism, you inoculate a series of differential media After incubation, you then observe each medium to see if specific end products of metabolism are present. This can be done by adding indicators to the medium that react specifically with the end product being tested, giving some form of visible reaction such as a color change. The results of these tests on the suspected microorganism are then compared to known results for that organism to confirm its identification. V  6  P 4  H  0޽h ? ̙33R `(  Z  6  |10Macromolecules hydrolysis Exoenzymes diastaseamylase 0protease0 lipase endoenzymes Starch hydrolysis Some bacteria are capable of using starch as a source of carbohydrate but in order to do this, they must first hydrolyze or break down the starch so it may enter the cell. The bacterium secretes an exoenzyme which hydrolyzes the starch by breaking the bonds between the glucose molecules. This enzyme is called a diastaseamylase .  &!O ?     rH  0޽h ? ̙33 p;(  H  C A9-2P`   B) mG  AStarch hydrolysisH  0޽h ? ̙33 e](    6h2+ Wprotein hydrolysis Proteins are made up of various amino acids linked together in long chains by means of peptide bonds. Many bacteria can hydrolyze a variety of proteins into peptides (short chains of amino acids) and eventually into individual amino acids. They can then use these amino acids to synthesize their own proteins and other cellular molecules or to obtain energy. The hydrolysis of protein is termed proteolysis and the enzyme involved is called a protease. b   $   H  0޽h ? ̙33  @(   J  C "A pv4lP`@   BD   Dprotein hydrolysis H  0޽h ? ̙33 rj$(  $ $ 6L d 20fermentation of carbohydrates Facultative anaerobic and anaerobic bacteria are capable of fermentation, an anaerobic process during which carbohydrates are broken down for energy production. A wide variety of carbohydrates may be fermented by various bacteria in order to obtain energy and the types of carbohydrates which are fermented by a specific organism can serve as a diagnostic tool for the identification of that organism. We can detect whether a specific carbohydrate is fermented by looking for common end products of fermentation. When carbohydrates are fermented as a result of bacterial enzymes, the following fermentation end products may be produced: acid end products. acid and gas end products. 2         $$H $ 0޽h ? ̙33 (X(  (  ( 6d`<  In order to test for these fermentation products, you inoculate and incubate tubes of media containing a single carbohydrate (such as lactose or maltose), a pH indicator (such as phenol red) and a durham tube (a small inverted tube to detect gas production). If the particular carbohydrate is fermented by the bacterium, acid end products will be produced which lowers the pH, causing the pH indicator to change color (phenol red turns yellow) .If gas is produced along with the acid, it collects in the durham tube as a gas bubble.Vi!  E,-H ( 0޽h ? ̙33 , (  ,H , C A9-3` H , C A9-4`p H , C A9-5` H , 0޽h ? ̙33 ~v0(  0 0 6}  p3 IMViC and hydrogen sulfide production Indole test Methyl red test Voges-Proskauer test Citrate test Hydrogen sulfide (H2S) test z-_$ %$$U   F#1H 0 0޽h ? ̙33 HC(  H  H 68  Indole test Indole is a component of the amino acid tryptophan. Some bacteria have the ability to break down tryptophan for nutritional needs using the enzyme tryptophanase. When tryptophan is broken down, the presence of indole can be detected through the use of Kovacs' reagent. Kovac's reagent, which is yellow, reacts with indole and produces a red color on the surface of the test tube. L}  "$"$"$}$" / (  !5'<H H 0޽h ? ̙33@ (  H  C A9-6pP @H  0޽h ? f3f3 {L(  L L 6dn0  u Methyl Red (MR) Test The combination medium used for this test, MR/VP broth, includes peptone, glucose, and a phosphate buffer. Bacteria that are able to perform a mixed-acid fermentation of glucose and produce large amounts of stable acids. The pH indicator, methyl red, is added to a 48 hour culture. If the pH is less than 4.4, the indicator will turn red. A red color is read as positive, a yellow color (pH greater than 6.0) is negative, and an orange color, indicating a pH between the two, will usually require further incubation. >" "  H L 0޽h ? ̙33=  X}(  XP X C (A MRtest1@h  X 60p 0 KThe Methyl Red Test: Left to Right: positive, positive, negative, control.  L 9 H X 0޽h ? ̙33^ P(  Pf P 6, : Voges-Proskauer (VP) Test The Voges-Proskauer test uses the same MR/VP broth. Some fermentative organisms do not produce enough stable acids to lower the pH of the medium. For these organisms, the chief end products of glucose metabolism are acetoin and 2,3-butanediol. After 48 hours of incubation, Barritt's Reagent A (alpha-napthol) and Barritt's Reagent B (potassium hydroxide) are added to the sample. After gently shaking the tube for aeration, formation of a red color will indicate a positive reaction. No color change or a copper color are negative results. ,  v3  H P 0޽h ? ̙33 |0\(  \N \ C &A vp_pos  N \ C &A vp_neg U \ 6В P  OVoges-Proskauer Test Left: uninoculated control Right: negative (copper color) P;   9  , ( \ 6ڒ P  m7Left: uninoculated control Right: positive (red color)  &H \ 0޽h ? ̙33 F>T(  T T 6  8 Citrate Utilization Test citrate agar is a defined medium containing sodium citrate as the sole carbon source and the ammonium ion as the sole nitrogen source. The pH indicator, bromthymol blue, will turn from green at neutral pH (6.9) to blue when a pH higher than 7.6 is reached (basic or alkaline). If the citrate is utilized, the resulting gowth will produce alkaline products (pH >7.6), changing the color of the medium from green to blue.   4 aH T 0޽h ? ̙33 4,@`(  `R ` s *&p& ` 6  |nCitrate Utilization Enterobacter cloacae: positive Eschericia coli: negative Klebsiella pneumoniae: positive o               P     H ` C Agjx H ` 0޽h ? ̙33 kc(    6 ]1 Hydrogen sulfide (H2S) test Some bacteria are capable of breaking down sulfur containing amino acids (cystine, methionine) or reducing inorganic sulfur-containing compounds (such as sulfite, sulfate, or thiosulfate) to produce hydrogen sulfide (H2S). This reduced sulfur may then be incorporated into other cellular amino acids, or perhaps into coenzymes. The ability of an organism to reduce sulfur-containing compounds to hydrogen sulfide can be another test for identifying unknown organisms such as certain Proteus and Salmonella. To test for hydrogen sulfide production, a medium with a sulfur-containing compound and iron salts is inoculated and incubated. If the sulfur is reduced and hydrogen sulfide is produced, it will combine with the iron salt to form a visible black ferric sulfide (FeS) in the tube.      #Zg R GH  0޽h ? f3f3B P4(  4J 4 C "A NH2S H 4 0޽h ? ̙33$   $(  r  S `hP   r  S 4 h  H  0޽h ? f3f380___PPT10.0y&[ ld`8(  8* 8 0$Pp  RV d\Oek 10Starch hydrolysis cy0W{Q0xmgr0‰[  * 2$#4H 8 C A 9-1`h B 8 B&  B20Lactose Fermentation cy0W{Q0‰[0 >"$ $$, H 8 0޽h ? ̙33 jb (    c $5h0   V30 Methyl red and Voges-Proskauer tests Inoculate into the MRVP broth. AFTER INCUBATION 48 hours : Pour 1/3 of the suspension into a clean nonsterile tube: run the MR test in the tube with 2/3, and the VP test in the open tube with 1/3. for methyl red: Add 6-8 drops of methyl red reagent. for Voges-Proskauer: Add 12 drops of Barritt's A(alpha-napthol) , mix, 4 drops of Barritt's B (KOH--potassium hydroxide) , mix. 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AFTER INCUBATION (48 hours ):  Pour 1/3 of the suspension into a clean nonsterile tube:  run the MR test in the tube with 2/3, and the VP test in the open tube with 1/3. for methyl red:  Add 6-8 drops of methyl red reagent. for Voges-Proskauer:  Add 12 drops of Barritt's A(alpha-napthol) , mix, 4 drops of Barritt's B (KOH--potassium hydroxide) , mix. Let sit, undisturbed, for at least 15 minutes. 4、 Citrate test 接种、培养、观察。 幻灯片 25 已用的字体演示文稿设计模板幻灯片标题_`arenaren  !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~Root EntrydO)PicturesFCurrent UserSummaryInformation(cPowerPoint Document(FDocumentSummaryInformation8