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本课件为《现代生物学实验》的实验三－七大分子物质的水解实验和IMVIC实验部分，依次展开实验三－七大分子物质的水解实验和IMVI、Starchhydrolysis等内容，并说明目的要求、原理与操作步骤，便于课前预习和实验
实验三－七大分子物质的水解实验和IMVIC实验
一、教学要求了解微生物一些生化反应的原理及其在微生物鉴定中的应用。二、简介To identify bacteria, we must rely heavily onbiochemical testing. The types of biochemicalreactions each organism undergoes act as a"thumbprint" for its identification. This is based onthe following chain of logic:
When identifying a suspected organism, youinoculate a series of differential media Afterincubation, you then observe each medium to seeifspecific end productsof metabolism arepresent. This can be done by addingindicatorstothe medium that react specifically with the endproduct being tested, givingsome form of visiblereaction such as a color change. The results ofthese tests on the suspected microorganism arethen compared to known results for that organismto confirm its identification.
Starch hydrolysis
protein hydrolysisProteins are made up of various amino acidslinked together in long chains by means ofpeptide bonds. Many bacteria can hydrolyze avariety of proteins into peptides (short chains ofamino acids) and eventually into individualamino acids. They can then use these aminoacids to synthesize their own proteins and othercellular molecules or to obtain energy. Thehydrolysis of protein is termedproteolysisandthe enzyme involved is called aprotease.
protein hydrolysis
In order to test for these fermentation products, youinoculate and incubate tubes of media containing asinglecarbohydrate(such as lactose or maltose), apHindicator(such as phenol red) and adurham tube(asmall inverted tube to detect gas production). If theparticular carbohydrate is fermented by the bacterium,acid end products will be produced which lowers the pH,causing the pH indicator to change color (phenol redturns yellow) .If gas is produced along with the acid, itcollects in the durham tube as a gas bubble.
Indole testIndole is a component of the amino acidtryptophan. Some bacteria have the abilityto break down tryptophan for nutritionalneeds using the enzyme tryptophanase.When tryptophan is broken down, thepresence of indole can be detected throughthe use of Kovacs' reagent. Kovac'sreagent, which is yellow, reacts with indoleand produces a red color on the surface ofthe test tube.
Methyl Red (MR) TestThe combination medium used for this test,MR/VP broth, includes peptone, glucose, and aphosphate buffer. Bacteria that are able to performa mixed-acid fermentation of glucose and producelarge amounts of stable acids. The pH indicator,methyl red, is added to a 48 hour culture. If the pHis less than 4.4, the indicator will turn red. A redcolor is read as positive, a yellow color (pH greaterthan 6.0) is negative, and an orange color,indicating a pH between the two, will usuallyrequire further incubation.
The Methyl Red Test: Left to Right:positive, positive, negative, control.
Voges-Proskauer (VP) TestThe Voges-Proskauer test uses the same MR/VPbroth. Some fermentative organisms do notproduce enough stable acids to lower the pH of themedium. For these organisms, the chief endproducts of glucose metabolism are acetoin and2,3-butanediol. After 48 hours of incubation,Barritt's Reagent A (alpha-napthol) and Barritt'sReagent B (potassium hydroxide) are added to thesample. After gently shaking the tube for aeration,formation of a red color will indicate a positivereaction. No color change or a copper color arenegative results.
Voges-Proskauer TestLeft: uninoculated controlRight: negative (copper color)Left: uninoculated controlRight: positive (red color)
Citrate Utilization Testcitrate agar is a defined medium containing sodiumcitrate as the sole carbon source and the ammoniumion 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, theresulting gowth will produce alkaline products (pH>7.6), changing the color of the medium from green toblue.
Citrate UtilizationEnterobactercloacae: positiveEschericiacoli: negativeKlebsiellapneumoniae: positive
Hydrogen sulfide (H2S) testSome bacteria are capable of breaking down sulfur containingamino acids (cystine, methionine) or reducing inorganicsulfur-containing compounds (such as sulfite, sulfate, orthiosulfate) to producehydrogen sulfide(H2S). This reducedsulfur may then be incorporated into other cellular amino acids,or perhaps into coenzymes. The ability of an organism toreduce sulfur-containing compounds to hydrogen sulfide canbe another test for identifying unknown organisms such ascertainProteusandSalmonella. To test for hydrogen sulfideproduction, a medium with a sulfur-containing compound andiron salts is inoculated and incubated. If the sulfur is reducedand hydrogen sulfide is produced, it will combine with theiron salt to form a visibleblackferric sulfide (FeS) in the tube.
四操作步骤1、Starch hydrolysis接种、培养、碘液染色、观察2、Lactose Fermentation接种、培养、观察。
3、Methyl red and Voges-Proskauer testsInoculate into the MRVP broth.AFTER INCUBATION（48 hours）:Pour 1/3 of thesuspension into a clean nonsterile tube:run the MRtest in the tube with 2/3, and the VP test in the opentube with 1/3.for methyl red:Add 6-8 drops of methyl red reagent.for Voges-Proskauer:Add 12 drops of Barritt'sA(alpha-napthol) , mix, 4 drops of Barritt's B (KOH--potassium hydroxide) , mix.Let sit,undisturbed, for atleast 15 minutes.4、Citrate test接种、培养、观察。
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