1. Isolation and identification of Enteric Bacteria
Experiment five
Isolation and identification of Enteric Bacteria

2. The family Enterobacteriaceae includes a group of bacteria that inhabit the intestinal tracts of humans and other animals. Some species are normal flora and others are pathogens of certain diseases. Commonly used techniques for their isolation and identification include biochemical test, antigens present on their surfaces and motility. Among the important genera included as enterics are Escherichia, Salmonella, Shigella, Klebsiella, Proteus and Yersinia.

3. Dimidiation of the enterobacteria according to the fermentation of lactose
Lactase fermenters: saprophytic and commensal
Escherichia Klebsiella Citrobater
Enterobacter
Non lactase fermenters: pathogens
Salmonella Shigella some Citrobacter
Proteus Serratia

4. Isolation culture and identification procedure
Colonial characteristic observation
Specimens isolation Gram Staining
(SS/EMB plate) Serological identification
TSI
Biochemical reaction

5. Specimens
Different specimens should be taken depending on the kind and the process of the disease.
blood
bone marrow
Urine
stool

6. Isolation Culture medium: S.S agar Method: streak plate Result:
Non-pathogenetic colonies: middle size, red
Suspect colonies: colorless, small, opaque

7. Biochemical reactions of Salmonella, etc
Species bottom slope H2S motility
E.coli AG AG
Salmonella A /
Shigella A /
bottom: ferment dextrose slope: lactose
A:acid AG: acid and gas

8. Items of the experiment
(1) Eosin-Methylene Blue Agar (EMB)
Reagents: Eosin and Methylene-blue
Carbohydrate Source: Lactose
Principle: a precipitate can be formed at acidic pH
Purpose: differentiate lactose fermenters from
non-lactose fermenters.
Interpretation:
Lactose fermenter: purple
Lactose non-fermenter: colorless

9. EMB agar
E.coli: black and typically have a metallic green sheen
negative

10. EMB agar
E.coli
S.typhi
S.dysenteriae
( ++ )
( - )

11. Items of the experiment
(2) Salmonella-Shigella Agar(S.S agar )
Reagents:
Lactose (Carbohydrate Source)
Neutral red( as an indicator),
Bile salt, Ferric ammonium citrate (Provides for inhibition of normal flora coliforms and differentiationof stool pathogens (e.g. Salmonella anal Shigella)
Interpretation:
Lactose non-fermenter: colorless
Lactose fermenter: pink to red colonies
Principle:

12. S.S agar

13. S.S agar
E.coli
S.typhi
S.dysenteriae
pink
colorless

14. Items of the experiment
(3) Indole Broth
purpose: Distinguish Enterobacteriaseae based on the ability to produce indole from tryptophan.
principle:
Interpretation
Positive Test - red ring
Negative Test - no color development
tryptophanase
Kovac’s reagent
indole
tryptophan
rosindole
(red)

15. Indole Broth - +

16. Indole Broth
E.coli
S.typhi
S.dysenteriae
( + )
( - )

17. (4) Double sugar iron slant
Items of the experiment
(4) Double sugar iron slant
main component:
Glucose: 0.1%
Lactose: 1%
Phenol red: as an indicator
Ferrous sulfate(FeSO4) :FeSO4+H2S →FeS(black)+H2SO4
purpose: It provides information about carbohydrate
fermentation.
Principle:

18. Only glucose fermenting
Slant: oxidized →neutral →red
Butt: not oxidized →yellow

19. Lactose fermenting
yellow both on the slant
and in the butt

20. H2S production
FeSO4+H2S
FeS(black)+H2SO4

21. Gas production

22. Double suger iron slant

23. Double suger iron slant
E.coli
S.typhi
S.dysenteriae
Slant
yellow
red
butt
Gas
( + )
( - )
H2S