Gelatinase Sugar Fermentation Test

1. Gelatinase Sugar Fermentation Test  Lab #20

2. Experiment Gelatinases comprise a family of extracellular enzymes produced by some microorganisms to hydrolyze gelatin The smaller amino acids can then be taken up by the bacterial cell for metabolic purposes Bacteria is positive for the enzyme and will liquefy the media Bacteria does not secrete the enzyme and the media will remain hard E. coli S. aureus

3. HYDROLYTIC ENZYMESGelatinase Test Teams of 4 Organisms Escherichia coli Staphylococcus aureus Media: 2 gelatin tubes Put a heavy loopful of the organism all the way down to the bottom of the tube

4. Using Gelatin This media is used to test if bacteria can digest the protein gelatin. To digest gelatin, the bacteria must make an enzyme called gelatinase. To inoculate this media, use a transfer needle to stab the gelatin. After incubating the inoculated media for at least 48 hrs, transfer the tube into a refrigerator. The tube should be completely chilled prior to observation. If the media is solid after refrigeration then the test is negative (the bacteria did not digest gelatin). If the media is liquified even after refrigeration, then the test result is positive�the bacteria is able to digest gelatin.

5. Example results Results -examples The 'Serratia marcescens' on the left is positive for gelatinase production, as evidenced by the liquidation of the media.� The 'Salmonella typhimurium' on the right is negative, as evidenced by the solidity of the media.�

6. Sugar Fermentation Metabolic process by which organic molecules act as electron donors (become oxidized) and one or more of its organic products acts as a final electron acceptor. Bacteria use protein or carbohydrate as a carbon and energy source and catabolize carbohydrate and produce acid, the pH indicator changes color come produce gas as well as acid.

7. Phenol Red Broth for Sugar Fermentation Lactose fermenters are organisms that split lactose into glucose and galactose and ferments the monosaccharides (hydrolysis reactions) Glucose fermenters produce pyruvate (glycolysis)and ferment pyruvate to products alchohol and CO2 and H. Phenol Red is an acid indicator Yellow below 6.8, Pink above 7.4, and red from 6.8 to 7.4. Durham tube is and indicator for gas production. Differentiation of Enterobacteraciae from other Gram � rods

8. Tests results Acid Gas - produces yellow bubble in the tube Acid no Gas- produces yellow with no bubble Red Broth control pH 7.3 Degradation of peptone produces alkaline media with NH3 as the end product and will be pink/reddish E.Coli S. Aureus Alcaligenes faecalis

9. BIOCHEMICAL TESTSPhenol Red Broth for Sugar Fermentation Reading results Yellow color in the phenol red pH indicators shows acid hot pink shows alkaline Gas production will show as a bubble in the durham tube

10. UN Bacterial Unknowns Check your streak plates � if you have isolated colonies, put them on nutrient slants � if not, re-streak your unknown If you have growth on your slant� -Gram Stain -Inoculate into Sugars and Gelatin Media: 1 � 2 Nutrient agar slants or 1 Nutrient Agar plate