Biochemical tests. 1-IMViC tests. 2-TSI test. = KIA test

2. Suspected case of Diphtheria ,the
presence of polyphosphate inclusions
called metachromatic granules.
(volutin granules )
Corynebacterium spp.
(Albert stain)

3. Identification of the Enterobacteriaceae is based
on: * Biochemical tests.
1)IMViC tests.
2)TSI test. = KIA test
1) IMViC reactions: (principle)
I = test for production of Indole from tryptophane.
M = Methyl red test for acid production from
glucose.
V = Voges-Proskauer test for production of
acetone from glucose.
C = test for the use of Citrate as carbon source.

4. Indole
**Reagent: Kovac’s reagent
**Media: Peptone water
+ve: pink ring
-ve :yellow ring
broth
Methyl red
**Reagent: Methyl red
**Media: MR-VP medium
(Glucose phosphate peptone
water)
+ve: Red color
-ve :Yellow color

5. Voges -Proskauer
**Reagent:
Barritt’s reagent A (5% alpha-naphthol)
+
Barritt’s reagent B (40% KOH)
**Media: MR-VP medium
(Glucose phosphate peptone water)
+ve: Dark-red color
-ve: no change in color

6. Citrate utilization
**Reagent: Bromothymol blue
**Media: Simmon’s Citrate Agar
+ve: Blue
-ve : Green
Escherichia coli
+ + - -
Klebsiella
- - + +

7. Triple sugar iron agar (TSI )
Principle:
This test is used to determine the ability to:
1- Reduce sulfur into H2S.
2- Lactose & / or Sucrose fermentation.
3- Glucose fermentation and Gas production.
Media Used: Triple sugar-iron agar
TSI medium contains:
 1% lactose , 1% sucrose & 0.1% glucose.
Sodium thiosulfate & ferrous sulfate (iron).
 Phenol red as indicator (to detect acid production from
carbohydrate fermentation ).

8. Procedure:
Inoculate TSI medium with an organism by inoculating needle by stabbing
the butt & streaking the slant, incubate at 37°C for 24 hours.

9. Reading Results:
 A/A = yellow slant, yellow butt (all sugar fermentation).
 K/A = red slant, yellow butt (Glucose fermentation only).
 K/K = red slant, red butt (no sugar fermentation).
 Gas production = bubbles or cracks in medium.
 Black precipitate = hydrogen sulfide (H2S) production.
NOTE:
There is no way to get a A/K reaction when
using a G-rod on this medium.
IF YOU do, it means that
1) Incorrect inoculation with a stab and& streak
2) Inoculated something other than a G- rod.

10. Slant Red K Red K Yellow A Yellow A Yellow A Red K
Butt Red K Yellow A Yellow A Yellow A Yellow A Yellow A
Gas production No No No Yes No No
H2S production No No Yes No No Yes
Sugar
fermentation
Non glucose all all all Glucose
Symbol K/K K/A A/A,H2S A/A, Gas A/A K/A,H2S

11. E. coli Klebsiella Proteus
Results