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 on biochemical testing. The types of biochemical reactions each organism undergoes act as a "thumbprint" for its identification. This is based on the following chain of logic:

Each different species of bacterium has a different molecule of DNA (i.e., DNA with a unique series of nucleotide bases). Since DNA codes for protein synthesis, then different species of bacteria must, by way of their unique DNA, be able to synthesize different protein enzymes. 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.

When 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.

1 、 Macromolecules hydrolysis Exoenzymes diastase （ amylase ）、 protease 、 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 diastase （ amylase ）.

Starch hydrolysis

protein 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.

protein hydrolysis

2 、 fermentation 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.

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.

3 IMViC and hydrogen sulfide production Indole test Methyl red test Voges-Proskauer test Citrate test Hydrogen sulfide (H 2 S) test

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.

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.

The Methyl Red Test: Left to Right: positive, positive, negative, control.

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.

Voges-Proskauer Test Left: uninoculated control Right: negative (copper color) Left: uninoculated control Right: positive (red color)

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.

Citrate Utilization Enterobacter cloacae: positive Eschericia coli: negative Klebsiella pneumoniae: positive

Hydrogen sulfide (H 2 S) 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 (H 2 S). 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.

三 材料与器材 菌种：枯草芽胞杆菌，大肠杆菌，金黄色葡萄 球菌，普通变形杆菌 (Proteus vularis). 培养基：固体油脂培养基，固体淀粉培养基， 明胶培养基试管，石蕊牛奶试管，尿素琼脂试 管. 试剂：革兰染色用卢戈氏碘液. 无菌平板，无菌试管，接种环、接种针等.

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 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. Let sit, undisturbed, for at least 15 minutes. 4 、 Citrate test 接种、培养、观察。

五 注意事项 在进行糖发酵培养基接种时，要注意防止倒置 的小管进入气泡。 配柠檬酸盐培养基时，要注意调节好 pH ；另外， 接种时要适当加大接种量。 六 思考题 试设计一种可用于蛋白酶产生菌株初筛的筛选 模型。 产气肠杆菌也可发酵葡萄糖产生有机酸，但其 甲基红反应却为阴性。试分析其原因。