1. Biochemical Test biology & biotechnology department
General Microbiology
Laboratory
Biochemical Test biology & biotechnology department

2. Single Media/Multiple Tests
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 inoculum from 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. TRIPLE SUGAR IRON AGAR
TRIPLE SUGAR IRON AGAR

10. 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.

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

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

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

14. Example Result Reaction on TSI H2S Butt color Slant color
Non fermenter e.g. Pseudomonas
Alk/Alk/-
(No action on sugars)
Negative
Red
NLF
e.g. Shigella
K/A-
(Glucose fermented without H2S)
Yellow
e.g. Salmonella & Proteus
K/A+
(Glucose fermented with H2S)
Positive
black in butt LF
e.g. E. coli, Klebsiella, Enterobacter
A/A/-
(three sugars are fermented)

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