Biochemical Test By: Mahmoud W El-Hindi 2013

1. General Microbiology Laboratory Biochemical Test By: Mahmoud W El-Hindi 2013

2. Exercise 13 Single Media/Multiple Tests

3. Introduction • Several media are designed to yield more than one biochemical reaction. • Among the more commonly used media in this category are SIM media, Triple Sugar Iron agar (TSI) and Kliger's Iron agar (KIA).

4. SIM medium • derives its name from three reactions: production of hydrogen sulfide from sulfur-containing amino acids, indole production and motility. • Check for motility by observing migration of the inoculumfrom the stab line through the semisolid medium.

5. Cont. • The liberation of hydrogen sulfide is indicated by a blackening of the medium. • Indole production is determined after the addition of Indole reagent. • The medium is used primarily for differentiation of gram negative enteric bacteria.

6. Kliger's iron agar (KIA) and triple sugar iron agar (TSI) • KIA and TSIA are widely used in the identification of gram negative bacteria particularly the Enterobacteriaceae. • The media TSI contains sucrose in addition to the dextrose and lactose found in KIA. • The media are poured as slants and are inoculated with a stab to the butt followed by a streak of the slant surface.

7. Cont. • The bacteria therefore are exposed to both an anaerobic environment (butt) and an aerobic one(slant). • Phenol red is present as an indicator. Do not tighten the cap on the tube. • If the bacteria are non fermenters, such as Pseudomonas, they can grow on the slant by the aerobic degradation of protein components in the medium.

8. Tube

9. Lysine Iron agar (LIA) • Here is another multipurpose medium in one tube. • Reactions for lysine decarboxylation (Anaerobic alkaline reaction –over neutralizing) (the acid formed from glucose fermentation), lysine deamination(formation of red- colored products at the top of the medium) and H2S production (black precipitate ).

10. Motility Indole Ornithine (MIO) Medium • The reactions in this medium are observed as follows: • Motility: Observe for cloudiness in the medium (growth away from the stab line ) . • For a non- motile organism be seen along cracks in the medium caused by gas production.

11. Cont. • OrnithineDecarboxylation: Observe the lower three-quarters ( anaerobic region ) of the medium for change in the color of the PH indicator; growth must be present in this part of the tube for correct analysis of result: • Gray, blue or purple color: positive reaction for Ornithinedecarboxylation- formation of a highly alkaline product, over-neutralizing the acid produced from glucose fermentation.

12. Cont. • Yellow color: Negative reaction. Yellow color is due to the default acid production from glucose fermentation. • Indole Production: About one-half dropper ful of Kovacs reagent is add to the medium. • A red ring indicates production of indole from the breakdown of tryptophan.

13. TRIPLE SUGAR IRON AGAR TRIPLE SUGAR IRON AGAR

14. PRINCIPLE: • TSIA detects three primary characteristics of a bacterium: • The ability to produce gas from the fermentation of sugars. • The ability ferment lactose and sucrose. • The production of large amounts of hydrogen sulfide.

15. SIGNIFICANCE: • This test is of a great value in the initial identification of the family Enterobacteriaceae

16. PROCEDURES: • Inoculate the test organism on TSIA slants by stabbing the butt and streaking the slant completely. • Incubate for 24hoursat 37 oC.

17. RESULTS : • Yellow=Acid=A • Red=Alkaline=K • Cracks in the butt=Gas=G • Blakining in the butt=+=hydrogen sulfide.

21. Cont. Good Luck By: Mahmoud W El-Hindi