General Microbiology Laboratory 1 Among the many enzymes that bacteria may produce are exoenzymes (those that are excreted) used to degrade large polymers into smaller compounds. For example, starch digestion results from the action of amylase released into the surrounding medium. The starch is a polysaccharide that cannot pass across the cell membrane. 2 Amylase breaks starch into smaller sugar residues that can enter the cell and be processed by respiration or fermentation. Gelatinase is another exoenzyme. It can cause the liquefaction of media solidified by gelatin (rather than agar). the major protein component in milk Caseinase is an enzyme that hydrolyzes casein, the major protein component in milk. 3 Lipase production Lipase production is common to bacteria that grow in foods rich in fats such as butter and mayonnaise. This enzyme breaks fats into its components glycerol and fatty acids. Agar which contains lipids prepared from egg yolks is used in identifying lipolytic activity. 4 Most enzymes are endoenzymes. They are produced in the cell and catalyze intracellular reactions. 5 Among the kinds of reactions that are used as evidence in identification of unknown bacteria are: a) The breakdown of toxic wastes such as hydrogen peroxide or urea. b) The reduction of nitrate or oxygen. c) The degradation of specific amino acids. 6 7 Amylase activity is demonstrated using starch agar, a medium containing starch as the carbohydrate source. Amylase hydrolyzes starch Amylase hydrolyzes starch. Starch is a polysaccharide--a long chain of glucose molecules linked by glycosidic bonds. Amylase breaks the glycosidic bonds, producing small oligosaccharides and free glucose. 8 Amylase production is tested by growing organisms on starch agar. After incubation, the starch agar is flooded with Gram's iodine. The iodine reacts with starch to produce a dark purple or brown color. clear zones hydrolysis has occurred. If amylase is present, clear zones will appear in the starch agar where hydrolysis has occurred. 9 POSITIVE CONTROL: Bacillus subtilits NEGATIVE CONTROL: E.coli 10 Streak each organism across a small portion of the agar surface. Incubate at 37 oC for 48 hours. Cover the surface with iodine. Rotate to distribute the iodine into a thin layer. Do not flood the plate. Iodine will turn blue when it reacts with starch. A clear zone will be seen where starch has been digested. 11 12 13 14 15 16 Gelatin Liquefaction Gelatin is liquefied by the virtue of the production of an enzyme called gelatinase. Gelatin is an incomplete protein; it lacks tryptophan. However, the ability to hydrolyze gelatin is a well established bacterial classification characteristic. Gelatin is produced by the hydrolysis of collagen, a large protein found in the connective tissues of animals. 17 Normally, gelatin produces a gel in water below 25C. When gelatin is hydrolyzed, it loses its ability to form a gel. In this experiment, you will use nutrient gelatin in place of nutrient agar. If a given microbe produces gelatinase, the nutrient gelatin will liquefy. 18 Gelatin polypeptide Amino acid Liquid 19 This test is used to differntiate Gram- egative species. Serratia, Pseudomonas, and Vibrio are positive for this test. The practicality of this test was not appreciated until, the development of the rapid procedures. The gelatin stab method employs nutrient gelatin deep tubes that contain 12% gelatin. 20 21 22 23 1- Inoculate gelatin deeps using bacteriological needle for up to 30 days. 2- To determine whether liquefaction has occurred, place the tube in the refrigerator for 30 minutes. Remove and check the tube for liquefaction. If negative, continue incubation until liquefaction occur. 24 Positive: Strong Positive: Strong: Liquefaction occurs within 3 days. Positive: weak Positive: weak: Liquefaction occurs in 4-30 days. Negative Negative: No liquefaction after 30 days. 25 Figure (1): Result of Gelatin Liquefaction: The tubes to the right depict a gelatinase negative (A) and elatinase positive (B and C) reactions. 26 27 28 29 Good Luck