This document discusses methods for identifying Enterobacteriaceae bacteria, including biochemical tests like IMViC and the Triple Sugar Iron (TSI) agar test. IMViC tests examine indole production, methyl red reaction, Voges-Proskauer reaction, and citrate utilization. The TSI agar test detects glucose, lactose and sucrose fermentation, gas production, and hydrogen sulfide production. These tests can identify bacteria such as E. coli, Klebsiella, and Proteus based on their biochemical characteristics and reactions in the assays.

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