Enterobacteriaceae

Enterobacteriaceae

Biochemical Reactions

IMViC Test

Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests: The following four tests comprise a series of important determinations that are collectively called the IMViC series of reactions The IMViC series of reactions allows for the differentiation of the various members of Enterobacteriaceae.

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`

IMViC: Indole test

IMViC: 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

IMViC testMethyl Red-Voges Proskauer (MR-VP) Tests

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)
Barrit’s ABarrit;s B Pink color
VP positive Klebsiella

IMViC test: MRVP test

Inoculate the tested organism into One tube of MRVP brothIncubate the tubes at 37°C for 24 hoursAFTER INCUBATION: Pour 1/3 of the suspension into a clean nonsterile tube: Run the MR test in the tube with 2/3, and the VP test in the open tube with 1/3. For methyl red: Add 6-8 drops of methyl red reagent. For Voges-Proskauer: Add 12 drops of Barritt's A (-naphthol), mix, 4 drops of Barritt's B (40% KOH), mix Let sit, undisturbed, for at least 1hour Method

IMViC test: MR/VP test

Results
Methyl Red test
Voges-Proskauer test
Red: Positive MR (E. coli)
Yellow or orange: Negative MR (Klebsiella)
Pink: Positive VP (Klebsiella)
No pink: Negative VP (E. coli)


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

Citrate Utilization Test

Method
Streak a Simmon's Citrate agar slant with the organism

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

Urease Test

Urea agar contains urea and phenol redUrease is an enzyme that catalyzes the conversion of urea to CO2 and NH3Ammonia combines with water to produce ammonium hydroxide, a strong base which ↑ pH of the medium. ↑ in the pH causes phenol red r to turn a deep pink. This is indicative of a positive reaction for urease Urea
Urease
CO2 + NH3
H2O
NH4 OH
↑ in pH Phenol Red
Pink Positive test

Method

Principle

Urease Test

If color of medium turns from yellow to pink indicates positive test. Proteus give positive reaction after 4 h while Kelebsiella and Enterobacter gave positive results after 24 h
Result
Positive test
Negative test



Reaction on Triple Sugar Iron (TSI) Agar
TSI contains Three different types of sugars Glucose (1 part) Lactose (10 part) Sucrose (10 part) Phenol red (acidic: Yellow) TSI dispensed in tubes with equal butt & slant Principle To determine the ability of an organism to attack a specific carbohydrate incorporated into a basal growth medium, with or without the production of gas, along with the determination of possible hydrogen sulphide production.

Reaction on TSI

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



EMB
SS
MacConkey
O/F
Nitrate reductase
Oxidase
Gram stain
Metallic sheen
LF
LF
O+/F+
+ve
-ve
-ve rod
E. coli
Dark
LF
LF
O+/F+
+ve
-ve
-ve rods
Citrobacter
Dark
LF
LF
O+/F+
+ve
-ve
-ve rods
Klebsiella
Dark
LF
LF
O+/F+
+ve
-ve
-ve rods
Enterobacter
Colorless
NLF/H2S
NLF
O+/F+
+ve
-ve
-ve rods
Salmonella
Colorless
NLF
NLF
O+/F+
+ve
-ve
-ve rods
Shigella
Colorless
NLF/H2S
NLF
O+/F+
+ve
-ve
-ve rods
Proteus
Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae

Motility

Urease
Citrate
VP
MR
Indole
TSI
Motile
-ve
-ve
-ve
+ve
+ve
A/A/-
E. coli
Motile
-ve
+ve
-ve
+ve
+ve
A/A/-
Citrobacter freundii
Non motile
+ve
+ve
+ve
-ve
-ve
A/A/-
Klebsiella pneumoniae
Motile
+ve
+ve
+ve
-ve
-ve
A/A/-
Enterobacter cloacae
Motile
-ve
+ve
-ve
+ve
-ve
A/Alk/+
Salmonella typhi
Non motile
-ve
-ve
-ve
+ve
-ve
A/Alk/-
Shigella boydii
Motile Swarwing
+ve
+ve
-ve
+ve
-ve
A/Alk/+
Proteus mirabilis
Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae

Oxidase Test

Positive
Negative
Pseudomonas
Enterobacteriaceae
Nitrate test: +ve further reduction to N2
Growth on cetrimide agar: Pale colonies with green pigmentation
MacConkey’s agar& TSI Lactose fermenter
Pink colonies on MacConkey & acidic butt and slant on TSI
colorless colonies on MacConkey & acidic butt alkaline slant onTSI
Lactose non-fermenter
IMViC test & EMB
IMViC ++ - - & black colonies with metalic shines on EMB
E.coli
IMViC - - ++
Klebsiella
No H2S production (no blacking in TSI)
H2S production (blacking in TSI)
Shigella
Urease production
+ve
Proteus
-ve
SS agar
colorless colonies with black centers
Salmonella
O/F test: O+/F-
Motility
Not motile
Motile
Enterobacter

Practical Work

Reaction on TSI Indole Test MR test VP test Citrate Utilization test Urease test