General Microbiology Laboratory 1.  Among the many enzymes that bacteria may produce are exoenzymes (those that are excreted) used to degrade large polymers.

Presentation on theme: "General Microbiology Laboratory 1.  Among the many enzymes that bacteria may produce are exoenzymes (those that are excreted) used to degrade large polymers."— Presentation transcript:

1 General Microbiology Laboratory 1

2  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

3  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

4  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

5  Most enzymes are endoenzymes.  They are produced in the cell and catalyze intracellular reactions. 5

6  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

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8  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

9  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

10  POSITIVE CONTROL: Bacillus subtilits  NEGATIVE CONTROL: E.coli 10

11  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

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16 16 Gelatin Liquefaction

17  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

18  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. 18

19  Gelatin polypeptide Amino acid “Liquid” 19

20  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

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24  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

25  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

26  Figure (1): Result of Gelatin Liquefaction: The tubes to the right depict a gelatinase negative (A) and elatinase positive (B and C) reactions. 26

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29 29 Good Luck