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Learn about the role of exoenzymes in bacterial metabolism, including amylase, gelatinase, caseinase, and lipase. Discover methods to test for amylase production and gelatin liquefaction in lab settings.
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General Microbiology Laboratory Biochemical Test By: Mahmoud W El-Hindi 2013
Introduction • 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.
Cont. • 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). • Caseinase is an enzyme that hydrolyzes casein, the major protein component in milk.
Cont. • 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.
Cont. • Most enzymes are endoenzymes. • They are produced in the cell and catalyze intracellular reactions.
Cont. • 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.
Exercise 5 Amylase Production
Principle • Amylase activity is demonstrated using starch agar, a medium containing starch as the carbohydrate source. • 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.
Cont. • Amylase production is tested by growing organisms on starch agar. • After incubation, the starch agar is floodedwith Gram's iodine. • The iodine reacts with starch to produce a dark purple or brown color. • If amylase is present, clear zones will appear in the starch agar where hydrolysis has occurred.
Controls • POSITIVE CONTROL: Bacillus subtilits • NEGATIVE CONTROL: E.coli
Procedures • 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.
Exercise 6 Gelatin Liquefaction
PRINCIPLE • 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.
Cont. • Normally, gelatin produces a gel in water below 25°C. • 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.
Cont. • Gelatin polypeptide Amino acid “Liquid”
SIGNIFICANCE • 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.
PROCEDURES • 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.
RESULTS • Positive: Strong: Liquefaction occurs within 3 days. • Positive: weak: Liquefaction occurs in 4-30 days. • Negative: No liquefaction after 30 days.
Figure (1): Result of Gelatin Liquefaction: The tubes to the right depict a gelatinase negative (A) and elatinase positive (B and C) reactions.
Cont. Good Luck
