1. University of Tabuk Faculty of Applied Medical Science Department of Medical Laboratory Technology Mr.AYMAN.S.YOUSIF M.SC IN Microbiology &IMMUNOLOGY Academic Year: 1434-1435 (2013-2014)

2. 2 specimens General Procedure of Bacteriological Diagnosis Morphologic Identification Sub culture in the special types of media for confirmation Biochemical tests ( Identification and Isolation ) Susceptibility Testing ( to select the suitable antibiotics for treatment the pathogenic isolated bacteria from the specimen ) Serological Test Microscopy & Staining 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: 1. Inoculate the tube of urea broth with your unknown bacterium. 2. 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 extract0.3% (% = grams/100 mL) Beef extract0.3% Peptone1.5% Proteose peptone0.5% Total Protein = 2.6% Lactose 1.0% Sucrose 1 1.0% Glucose0.1% Carbohydrate = 2.1% 1 Absent 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: 1. Inoculate the tube of TSI media with your unknown bacterium (stabbing and zig zag on the surface ). 2. Incubate over night at 37 degrees C. If an organism can ferment any 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 (H 2 S) which is produced will react with the iron to form iron sulfide, which appears as a black precipitate.

9. InterpretationSymbolResults (slant/butt) Glucose fermentation only; Peptone catabolized K/A Red/yellow Glucose and lactose and/or sucrose fermentation A/AYellow/yellow No fermentation; Peptone catabolized K/KRed/red No fermentation; Peptone used aerobically K/NCRed/no color change Glucose and lactose and/or sucrose fermentation; Gas produced A/A,GYellow/yellow with bubbles Glucose fermentation only; Gas produced K/A,GRed/yellow with bubbles Glucose fermentation only; Gas produced; H2S produced K/A,G, H2SRed/yellow with bubbles and black precipitate Glucose fermentation only; H2S produced K/A, H2SRed/yellow with black precipitate Glucose and lactose and/or sucrose fermentation; H2S produced A/A, H2SYellow/yellow with black precipitate A=acid production; K=alkaline reaction; G=gas production; H2S=sulfur reduction

10. InterpretationSymbolResults (slant/butt) Shigella, ProvidenciaK/A Red/yellow Serratia marcescens 2 Yersinia enterocolitica 2 A/A Yellow/yellow Nonfermenters such as PseudomonasK/K Red/red Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter aerogenes Enterobacter cloacae, Serratia marcescens 1, 2 A/A,G Yellow/yellow with bubbles Salmonella serotype Paratyphi AK/A,G Red/yellow with bubbles Salmonella (most serotypes). Proteus mirabilis. Edwardsiella tarda. K/A,G, H2S Red/yellow with bubbles and black precipitate Citrobacter freundii Proteus vulgaris1 1Non-lactose, sucrose fermenter A/A, H2S Yellow/yellow with black precipitate 2 Non-lactose, sucrose fermenter

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