Bacterial Cultivation and Identification Media inoculation @ Use sterile loops ( wire, plastic, platinum) . @ Also spoon, swab, pipette, capillary tubes @ Make intersecting lines, for discrete colonies

Culture media (1). * Excess nutrients retard growth (sugars, fat, phosphorus). @ Types of Media: (1) Routine media :must be: a-Able to provide enough nutrients for a wide range of organisms. b-Cheap and easy to prepare. c-Contain no C source. Bacteria convert amino-acids in these media to energy. * Constituents are : a-Peptone: containing aminoacids & protein.

Culture Media (2) b-Inorganic salts & growth factors: add yeast extract, blood, serum, egg yolk C-Na Cl: added to balance osmotic pressure. (2) Synthetic media: Materials added to give a defined chemical composition. Used in research studies, very expensive. Contains: carbon source (glucose, lactose), nitrogen source (ammonium salt), salts. (3) Complex media: contain undefined nutrients. Not commonly used.

c-Indicator media: contain chemical Culture Media (3) 4- Special media: They include: a- Selective media: with substances that prevent growth of all organisms except the one selected. Isolating a pure strain e.g: TM medium-Neisseria. b-Enriched media: fluid media help one organism to multiply & prevent growth of other organisms-Selenite-F. c-Indicator media: contain chemical pigments (neutral red, methylene blue) changing colour on bacterial growth.

Anaerobic Culture Several techniques for anaerobic conditions: @ Sachets containing oxygen removing chemicals. Sachets do not produce hydrogen, hence do not require a catalyst. @ Copper coated steel wool to remove oxygen. Copper absorbs oxygen, hence air is removed from the jar. @ Reducing agents in culture media: * Thioglycollate broth (blood culture). * Cooked meat medium (Clostridium culture)

Control of anaerobiasis: The Anaerobic jar: @ Methylene blue as a control indicator. The Anaerobic jar: @ An anaerobic jar with pressure gauge, valves, etc is not needed. A plastic air- tight container is enough. Carbon dioxide generation: @ Sodium bicarbonate mixed with tartaric acid is moistened with water. @ Moisten Alka-Seltzer tablet with water. @ A candle jar @ Commercially produced sachets.

Reporting Cultures Sites normally sterile: @ Report bacteria up to genera & species Sites having a microbial flora: @ Report bacteria for which techniques was performed. If no pathogen isolated from stool on a selective medium, report No Salmonella, Shigella or Vibrio isolated NOT No pathogen or Normal flora isolated @ More quicker, easier, & economical to use selective media to isolate from a normal flora site.

Identification Biochemical Tests (1) @ Analytical Profile Index (API) for all tests. @ Bile solubility test: For S. pneumoniae @ Catalase test: For streptococci @ Citrate utilization test: For Entrobacteriace @ Coagulase test: For S. aureus @ DNA-ase test: For S. aureus @ Indole test: Mainly for E. coli

Identification Biochemical Tests (2) @ Litmus milk decolorization test: To help identification of Enterococcus & Clostridia @ Lysine decarboxylase test: To identify salrnonellae and shigellae (Pos. purple). @ Oxidase test: For Neisseria, Vibrio, Ps. @ Urease test: For Proteus, H.pylori @ Kligler iron agar test: For Salmonella @ Peptone water sugars: Fermentation tests

Rosco Identification Tablets (1) @ Diatabs - of Rosco Diagnostica-Denmark @ Have a stable, long shelf-life: 2-4 years, suitable for tropical climates, economical, and easy to use. @ Add tablet to a saline suspension of test organism in a test tube, and incubate. @ Reactions are read same day or after four hours incubation. @ Special reagents (supplied by Rosco) are required to read some reactions.

Rosco Identification Tablets (2) Some of the Rosco tablets tests are: @ Beta lactamase test: To detect beta lactamase-producing N. gonorrhoeae, etc @ Beta galactosidase (ONPG) test: To differentiate species of Enterobacteriacae. @ Lysine decarboxylase test: Differentiate between species of Enterobacteriaceae. @ Nitrate reduction test: Detect reduction of nitrate by Mycobacteria, Brucella, etc. @ Ornithine decarboxylase test: Differentiates species of Enterobacteriace

Rosco Identification Tablets (3) @ Aesculin hydorolysis test: For E. faecalis @ Factors X and V test: For H. influenzae @ Hippurate hydrolysis test: To identify Campylobacter, S. agalactiae @ Voges-Proskauer (VP) test: Differentiate between species of Enterobacteriaceae.