University of Tabuk Faculty of Applied Medical Science Department of Medical Laboratory Technology

1. 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)

2. 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

3. 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.

4. 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.

5. Triple sugar iron agar (TSI) OBJECTIVE: It is used to Differentiate  Enterobacteriaceae  based on the ability to • Reduce Sulfur • Ferment Carbohydrates.

6. 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

7. Triple Sugar Iron (TSI) Agar • Ferrous sulfate • Sodium thiosulfate • Sodium chloride • Agar (1.2%) • Phenol red • pH = 7.4

8. 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.

12. 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 .

13. 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.

14. 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