Enterobacteriaceae

Family: Enterobacteriaceae

E. coli
Microscopical appearance Gram negative bacilli Motile Non-spore former Non- capsulated

Cultural characteristics

Optimum temp. is 37 C˚.Can grow on simple mediaSmall (2-4 mm) in diameter, convex, opaque, smooth with entire edge.On MacConkey 's agar: Pink colonies due to lactose fermentation

E. coli

Eosin Methylene Blue Agar
E.coli produce black colonies with a metallic green sheen on EMB agar.

Genus Klebsiella

Gram negative bacilli Non motile Non spore-former capsulated


Cultural characteristics
Large colonies, mucoid, convex. On MacConkeys agar, pink, mucoid.

IMViC Test

Indole, Methyl Red, Voges-Prosakaur, Citrate

The IMViC formula is used in differentiation of the various members of Enterobacteriaceae.

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IMViC: Indole test

Principle Certain microorganisms can metabolize tryptophan by tryptophanase The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia The presence of indole is detected by addition of Kovac's reagent.
Tryptophane amino acids
Tryptophanase
Indole + Pyurvic acid + NH3
Kovac’s Reagent Red color in upper organic layer`
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Indole test

Indole test
Result: A bright pink color in the top layer indicates the presence of indole The absence of color means that indole was not produced i.e. indole is negative Special Features: Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-).
Positive test e.g. E. coli
Negative test e.g. Klebsiella


Glucose
Acidic pathway
Mixed acids  pH less than 4.4 Methyl Red indicator
Red color
Principle
MR positive E. coli
Or
Neutral pathway
Acety methyl carbinol (ACETOIN)
solution A solution B
Pink color
VP positive Klebsiella
* Methyl Red-Voges Proskauer (MR-VP) Tests
diacetyl

MR-VP test

Inoculate the tested organism into tubes of MR-VP broth (Glucose phosphate peptone water).Incubate the tubes at 37°C for 24 hours For methyl red: Add 6-8 drops of methyl red reagent. For Voges-Proskauer: Add 3 ml of Barritt's A (5% -naphthol), mix, 1ml of Barritt's B (40% KOH), mix . Method

Methyl Red test

Voges-Proskauer test
Red: Positive MR (E. coli)
Yellow : Negative MR (Klebsiella)
Pink: Positive VP (Klebsiella)
Green : Negative VP (E. coli)


Citrate Utilization Test
Principle:
Citrate
Na2CO3
Alkaline,↑pH Blue colour
Bromothymol blue
Simmone’s Citrate media CO2 + Na + H2O
Pyruvate
Positive test
Negative test: E. coli
Contains Citrate as a sole of C source

Citrate Utilization Test

Method

Citrate Utilization Test

Examine for growth (+) Growth on the medium is accompanied by a rise in pH to change the medium from its initial green color to deep blue
Result
Positive Klebsiella, Enterobacter
Negative E. coli

Sugar Fermentation

Determines the ability of M.O. to ferment sugar.Acid production: Yellow colour.Gas production: Bubble in the Durhams’ tube.