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University of Tabuk Faculty of Applied Medical Science Department of Medical Laboratory Technology Medical Microbiology II (P)Identification of Gram Negative Bacilli (Enterobacteriaceae)Biochemical Tests Mr.AYMAN.S.YOUSIF M.SC IN Microbiology &IMMUNOLOGY Academic Year: 1434-1435 (2013-2014)
specimens Morphologic Identification Microscopy & Staining Biochemical tests ( Identification and Isolation ) Sub culture in the special types of media for confirmation Serological Test Susceptibility Testing ( to select the suitable antibiotics for treatment the pathogenic isolated bacteria from the specimen ) General Procedureof Bacteriological Diagnosis Cultivation in suitable types of media
Urea Hydrolysis (urea test) • Urea can be broken down with the help of the enzyme urease, producing the alkaline product of ammonia plus carbon dioxide. That causes the pH indicator phenol red to turn a beautiful shade of hot pink (pink-red) . OBJECTIVES: • Understand the reactions of bacteria in urea broth. THE PROCEDURE: • Inoculate the tube of urea broth with your unknown bacterium. • Incubate over night at 37 degrees C.
INTERPRETATION: • The alkaline reaction turns the pH indicator to hot pink or red colour . • A yellowish color is still a negative reaction, although acidic. • Some bacteria will produce a WEAK reaction, with a bit of pink in the tube. • This should be recorded as weak +. • It is a good idea to compare your tube with an uninoculated to make sure that you do not have a weak + result.
Triple sugar iron agar (TSI) OBJECTIVE: It is used to Differentiate Enterobacteriaceae based on the ability to • Reduce Sulfur • Ferment Carbohydrates.
Triple Sugar Iron (TSI) Agar Is a Differential medium that contains . • Yeast extract 0.3% (% = grams/100 mL) • Beef extract 0.3% • Peptone 1.5% • Proteose peptone 0.5% Total Protein = 2.6% • Lactose 1.0% • Sucrose 1 1.0% • Glucose 0.1% Carbohydrate = 2.1% 1Absent in Kligler Iron Agar
Triple Sugar Iron (TSI) Agar • Ferrous sulfate • Sodium thiosulfate • Sodium chloride • Agar (1.2%) • Phenol red • pH = 7.4
Triple sugar iron agar (TSI) THE PROCEDURE: • Inoculate the tube of TSI media with your unknown bacterium (stabbing and zigzag on the surface ). • Incubate over night at 37 degrees C. • If an organism can fermentany of the three sugars present in the medium, the medium will turn yellow. • If an organism can only ferment dextrose (Glucose) , the small amount of dextrose in the medium is used by the organism within the first ten hours of incubation. • If an organism can reduce sulfur, the hydrogen sulfide (H2S) which is produced will react with the iron to form iron sulfide, which appears as a black precipitate.
OXIDASE TEST • The oxidase test is a key test to differentiate between the families of Pseudomonadaceae (ox +) and Enterobacteriaceae (ox -) • Is useful for identification of many other bacteria, those that have to use oxygen as the final electron acceptor in aerobic respiration, and produce cytochrome oxidase enzyme. OBJECTIVE: • Test for the enzyme oxidase on your unknown isolates. Materials Needed: • Oxidase Reagent. (Tetramethyl-p-phenylenediamine dihydrochloride) • Wooden Rods. • Filter Paper .
OXIDASE TEST THE PROCEDURE: • A piece of filter paper in a clean Petri dish is soaked with a few drops of oxidase reagent. • Using a piece of stick or glass rod (not an oxidized wire loop) remove a colony of the test organism and smear it on the filter paper. • Look for the development of a blue-purple colour within a few seconds • When the organism is oxidase-producing, the phenylenediamine in the reagent will be oxidized to a deep purple colour. • Alternatively an oxidase reagent strip can be used.
OXIDASE TEST Result • Blue-purple colour - Positive oxidase test (within 10 seconds) Pseudomonas aeruginosa , N. gonorrhoeae , Vibrio cholerae • No blue-purple colour - Negative oxidase test (within10seconds) Escherichia coli