Lab 3 Microbial Metabolism Reading PDF

Part 5 Microbial Metabolism Exercises 13 Carbohydrate Catabolism 14 Fermentation 15 Protein Catabolism, Part 1 16 Protein Catabolism, Part 2 17 Respiration 18 Unknown Identification and Bergey's Manual ASM: Demonstrate ability in using appropriate microbiological media and test systems, incl...

Part 5 Microbial Metabolism Exercises 13 Carbohydrate Catabolism 14 Fermentation 15 Protein Catabolism, Part 1 16 Protein Catabolism, Part 2 17 Respiration 18 Unknown Identification and Bergey's Manual ASM: Demonstrate ability in using appropriate microbiological media and test systems, including using biochemical test media and recording accurately macroscopic observations. This statement from the first edition of Bergey's Manual summarizes the need for studying microbial metabolism: The earliest writers classified the bacteria solely on their morphological characters. A more detailed classification was not possible because the biologic characters of so few of the bacteria had been determined. With the accumulation of knowledge of the biologic characters of many bacteria it was realized that it is just as incorrect to group all rods under a single genus as to group all quadruped animals under one genus.\* \*Society of American Bacteriologists (D. H. Bergey, Chairperson). Bergey's Manual of Determinative Bacteriology. Baltimore: Williams & Wilkins, 1923, p. 1. "Biologic characters" refers to information about the metabolism of bacteria. The chemical reactions that occur within all living organisms are referred to as metabolism. Metabolic processes involve enzymes, which are proteins that catalyze biological reactions. The majority of enzymes function inside a cell---that is, they are endoenzymes. Many bacteria make some enzymes, called exoenzymes, that are released from the cell to catalyze reactions outside the cell (see the illustration on the next page). Because many bacteria share the same colony and cell morphology, additional factors, such as metabolism, are used to identify them. Moreover, studying the metabolic activities of microbes helps us understand their role in ecology. Exercises 13 through 17 in Part 5 introduce concepts in microbial metabolism and differential media used to detect various metabolic activities. In Exercise 18, you will identify unknown bacteria on the basis of metabolic characteristics. Exoenzymes breaking down large molecules outside a cell. Smaller molecules released by this reaction are taken into the cell and further degraded by endoenzymes. Case Study: Intestinal Gas The human large intestine (colon) contains up to 1012 bacteria/g (dry weight) of contents, representing 500 species in the intestinal microbiome. The metabolism of these bacteria affects host intestinal function and health. Your diet provides nutrients not only for you but also for these bacteria. An important metabolic function of intestinal bacteria is metabolism of nondigestible carbohydrates such as starch and cellulose. Bacteria produce short-chain fatty acids including butyric acid and propionic acid, which provide energy for human colonic cells. The odors of feces can be attributed to indole and H2S produced by bacteria from dietary proteins. Intestinal bacteria may produce highly carcinogenic nitrosamines from amino acid residues and nitrite. Use the following choices to answer the questions: Anaerobic respiration Deamination of amino acids Decarboxylation of amino acids Desulfurization of amino acids Fermentation Questions Butyric acid and propionic acid are produced from starch by what process? Nitrite is produced from nitrate by what process? Amino acid residues are produced by removing CO2 by what process? How do bacteria produce indole from tryptophan? How do bacteria produce H2S from cysteine and methionine? Background Chemical reactions that release energy from the decomposition of complex organic molecules are referred to as catabolism. Most bacteria catabolize carbohydrates for carbon and energy. Carbohydrates are organic molecules that contain carbon, hydrogen, and oxygen in the ratio (CH2O)n. Carbohydrates can be classified based on size: monosaccharides, oligosaccharides, and polysaccharides. Monosaccharides are simple sugars containing from three to seven carbon atoms. Oligosaccharides are composed of two to about 20 monosaccharide molecules; disaccharides are the most common oligosaccharides. Polysaccharides consist of 20 or more monosaccharide molecules. Play Lab Technique Video with Pre-Lab Quiz \@MasteringMicrobiology Carbohydrate Catabolism: Amylase Production Starch Hydrolysis Exoenzymes are mainly hydrolytic enzymes that leave the cell and break down, by the addition of water, large substrates into smaller components, which can then be transported into the cell. Amylase hydrolyzes the polysaccharide starch into smaller carbohydrates. Glucose, a monosaccharide, can be released by hydrolysis (Figure 13.1). In the laboratory, the presence of an exoenzyme is determined by looking for a change in the substrate outside a bacterial colony. Figure 13.1 Starch hydrolysis. A molecule of water is used when starch is hydrolyzed. OF Medium Glucose can enter a cell and be catabolized; some bacteria, using endoenzymes, catabolize glucose oxidatively, producing carbon dioxide and water. Oxidative catabolism requires the presence of molecular oxygen (O2). Most bacteria, however, can ferment glucose without using oxygen. Fermentative catabolism does not require oxygen but may occur in its presence. The metabolic end-products of fermentation are small organic molecules, usually organic acids. Some bacteria produce gases from the fermentation of carbohydrates. Whether an organism is oxidative or fermentative can be determined by using Rudolph Hugh and Einar Leifson's OF basal media with the desired carbohydrate added. OF medium is a nutrient semisolid agar deep containing a high concentration of carbohydrate and a low concentration of peptone (Figure 13.2). The peptone will support the growth of bacteria that don't use the carbohydrate. Two tubes are used: one open to the air and one sealed with mineral oil to keep air out. OF medium contains the indicator bromthymol blue, which turns yellow in the presence of acids, indicating catabolism of the carbohydrate. Alkaline conditions, caused by the use of peptone and not of carbohydrate, are indicated by a dark blue color due to ammonia production. If the carbohydrate is metabolized in both tubes, fermentation has occurred. An organism that can use the carbohydrate only under aerobic conditions will produce acid in the open tube only. Acids are produced as intermediates in respiration, and the indicator will turn yellow in the top of the open tube. This organism is called "oxidative" in an OF test. Figure 13.2 Reactions in OF-glucose medium. The species in tubes (a) and (b) is an oxidizer. The organism in tubes (c) and (d) is a fermenter. The culture in tubes (e) and (f) does not use glucose. Carbohydrate catabolism will be demonstrated in this exercise. These differential tests are important in identifying bacteria. Materials First Period Petri plate containing nutrient starch agar OF-glucose deeps (2) Mineral oil Second Period Gram's iodine OF-glucose deep BSL-1 Demonstration OF-glucose tubes inoculated with Pseudomonas aeruginosa Cultures (As Assigned) Bacillus subtilis Escherichia coli Alcaligenes faecalis Micrococcus luteus Pseudomonas aeruginosa BSL-2 Techniques Required Inoculating loop and needle technique (Exercise 4) Aseptic technique (Exercise 4) Procedure First Period Starch Hydrolysis With a marker, divide the starch agar into three sectors by labeling the bottom of the plate. Using a loop, streak a single line of Bacillus, Escherichia, and Pseudomonas BSL-2 in the appropriate sector. Incubate the plate, inverted, at 35°C for 24 hours. After growth occurs, the plate may be refrigerated at 5°C until the next lab period. OF-Glucose Using an inoculating needle, inoculate two tubes of OF-glucose media with the assigned bacterial culture (Escherichia, Micrococcus, Alcaligenes, or Pseudomonas BSL-2). Place about 5 mm of mineral oil over the medium in one of the tubes. Replace the cap. Incubate both tubes at 35°C until the next lab period. Procedure Second Period Starch Hydrolysis Record any bacterial growth, and then flood the plate with Gram's iodine (Figure 13.3). Areas of starch hydrolysis will appear clear, while unchanged starch will form a dark blue complex with the iodine. Record your results. Figure 13.3 Starch hydrolysis test. After incubation, add iodine to the plate to detect the presence of starch. The clearing around the bacteria indicates that the starch was hydrolyzed. OF-Glucose Compare the inoculated tubes and an uninoculated OF-glucose tube. Record the following: the presence of growth, whether glucose was used, and the type of metabolism. From your classmates' tubes, observe and record the results from the microorganisms you did not culture. Background Once a bacterium has been determined to be fermentative by the OF test, further tests can determine which carbohydrates, in addition to glucose, are fermented; in some instances, the end-products can also be determined. Many carbohydrates---including monosaccharides such as glucose, disaccharides such as sucrose, and polysaccharides such as starch---can be fermented. Many bacteria produce organic acids (for example, lactic acid) and hydrogen and carbon dioxide gases from carbohydrate fermentation (Figure 14.1). Figure 14.1 Fermentation. Bacteria are often identified by their enzymes. These enzymes can be detected by observing a bacterium's ability to metabolize specific compounds. For example, E. coli and Salmonella are distinguished because E. coli can ferment lactose, and typical Salmonella cannot. Fermentation can occur whether O2 is present or not present. Play Lab Technique Video with Pre-Lab Quiz \@MasteringMicrobiology Carbohydrate Catabolism: MRVP Fermentation Tubes A fermentation tube is used to detect acid and gas production from carbohydrates. The fermentation medium contains peptone, an acid--base indicator (phenol red), an inverted tube to trap gas, and 0.5--1.0% of the desired carbohydrate. In Figure 14.2, the phenol red indicator is red (neutral) in an uninoculated fermentation tube; fermentation that results in acid production will turn the indicator yellow (pH of 6.8 or below). Some bacteria produce acid and gas. When gas is produced during fermentation, some will be trapped in the inverted, or Durham, tube. Fermentation occurs with or without oxygen present; however, during prolonged incubation periods (greater than 24 hours), many bacteria will begin growing oxidatively on the peptone after exhausting the carbohydrate supplied, causing neutralization of the indicator and turning it red because of ammonia production. Figure 14.2 Carbohydrate fermentation tube. \(a) The phenol red indicator is red in a neutral or alkaline solution. (b) Phenol red turns yellow in the presence of acids. (c) Gases are trapped in the inverted tube while the indicator shows the production of acid. MRVP Broth Fermentation processes can produce a variety of end-products, depending on the substrate, the incubation, and the organism. In some instances, large amounts of acid may be produced, and in others a majority of neutral products may result (Figure 14.3A, page 111). The MRVP broth is used to distinguish organisms that produce large amounts of acid from glucose and organisms that produce the neutral product acetoin (Figure 14.4, page 111). MRVP medium is a glucose-supplemented nutrient broth used for the methyl red (MR) test and the Voges--Proskauer (V--P) test. If an organism produces a large amount of organic acid from glucose, the medium will remain red when methyl red is added in a positive MR test, indicating that the pH is below 4.4. Methyl red is orange-red between pH 4.4 and 6.0. If neutral products are produced, methyl red will turn yellow, indicating a pH above 6.0. The production of acetoin is detected by the addition of potassium hydroxide and α-naphthol. If acetoin is present, the upper part of the medium will turn red; a negative V--P test will turn the medium light brown. The chemical process is shown in Figure 14.3B. Figure 14.3 MRVP test. \(a) Organic acids, such as lactic acid, or neutral products, such as acetoin, may result from fermentation. (b) Potassium hydroxide (KOH) and α-naphthol are used to detect acetoin. Figure 14.4 Methyl red test. Some bacteria produce large amounts of acid from pyruvic acid (pH \< 5.5). Other bacteria make neutral products such as acetoin (pH \> 6.0). Citrate Agar The ability of some bacteria to ferment citrate can be useful for identifying bacteria. When citric acid or sodium citrate is in solution, it loses a proton or Na+ to form a citrate ion. Bacteria with the enzyme citrate lyase can break down citrate to form pyruvate, which can be reduced in fermentation. Simmons citrate agar (Table 14.1) is used to determine citrate use. When bacteria use citrate and ammonium, the medium is alkalized because of ammonia (NH3) produced from NH4+. The indicator bromthymol blue changes to blue when the medium is alkalized, indicating a positive citrate utilization test (Figure 14.5). Table 14.1 Simmons Citrate Agar Ingredient Amount Sodium citrate 0.2% Sodium chloride 0.5% Monoammonium phosphate 0.1% Dipotassium phosphate 0.1% Magnesium sulfate 0.02% Agar 1.5% Bromthymol blue 0.0008% Figure 14.5 Citrate test. Use of citric acid as the sole carbon source in Simmons citrate agar causes the indicator to turn blue (b). Tube (a) is citrate-negative. Materials First Period Glucose fermentation tube Lactose fermentation tube Sucrose fermentation tube MRVP broths (4) Simmons citrate agar slants (2) Second Period MRVP broth Glucose fermentation tube Simmons citrate agar slant Parafilm squares Safety goggles Empty test tube Methyl red V--P reagent I, α-naphthol solution V--P reagent II, potassium hydroxide (40%) Cultures (As Assigned) Escherichia coli Enterobacter aerogenes Alcaligenes faecalis Proteus vulgaris BSL-2 Techniques Required Inoculating loop technique (Exercise 4) Aseptic technique (Exercise 4) Procedure First Period Fermentation Tubes Use a loop to inoculate the fermentation tubes with the assigned bacterial culture. Incubate the tubes at 35°C. Examine them at 24 and 48 hours for growth, acid, and gas. Compare them to an uninoculated fermentation tube. Why is it important to record the presence of growth? MRVP Tests Using a loop, inoculate two MRVP tubes with Escherichia and two with Enterobacter. Incubate the tubes at 35°C for 48 hours or longer. Why is time of incubation important (Figure 14.4)? Citrate Test Using a loop, inoculate one citrate slant with Escherichia coli and the other slant with Enterobacter aerogenes. Incubate the tubes at 35°C until the next lab period.