Enterobacteriaceae
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.
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.
Tryptophanase
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 (-).
Glucose
Acidic pathway
Or
Neutral pathway
Barrits A Barrit;s B
Red color
MR positive E. coli
VP positive Klebsiella
Pink color
Method
Inoculate the tested organism into One tube of MRVP broth Incubate the tubes at 37C for 24 hours
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
Results
Principle:
Citrate
Pyruvate
CO2 + Na + H2O
Bromothymol blue
Positive test
Blue colour
the organism
Incubate at 37C for 24 hours.
is accompanied by a rise in pH to change the medium from its initial green color to deep blue
Positive Klebsiella, Enterobacter
Negative E. coli
Principle
Urease Test
Urea agar contains urea and phenol red Urease is an enzyme that catalyzes the conversion of urea to CO2 and NH3 Ammonia 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
Urease H2O
Urea
CO2 + NH3
NH4 OH
in pH Phenol Red
Method
Streak a urea agar tube with the organism
incubate at 37C for 24 h
Urease Test
Result
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
Positive test Negative test
Reaction on TSI
Method:
Inoculate TSI medium with an organism by inoculating needle by stabbing the butt and streaking the slant Incubate at 37C for 24 hours
Result
Reaction on TSI Butt color Red Slant color Red H2 S Negative Negative Yellow Red Positive black in butt Result Non fermenter e.g. Pseudomonas LNF e.g. Shigella LNF e.g. Salmonella & Proteus LF e.g. E. coli, Klebsiella, Enterobacter Example
Alk/Alk/(No action on sugars) A/Alk/(Glucose fermented without H2S) A/Alk/+ (Glucose fermented with H2S) A/A/(three sugars are fermented)
Yellow
Red
Yellow
Yellow
Negative
+ve +ve
+ve
-ve -ve
-ve
+ve -ve
+ve
-ve -ve
+ve
A/Alk/+ -ve
Oxidase Test
Negative Positive Pseudomonas
Enterobacteriaceae
MacConkeys agar & TSI
Pink colonies on MacConkey colorless colonies on MacConkey & acidic butt and slant on TSI & acidic butt alkaline slant onTSI
reduction to N2
Lactose fermenter
IMViC test & EMB
IMViC ++ - & black colonies with metalic shines on EMB IMViC - - ++
Growth on cetrimide agar: Lactose non-fermenter Pale colonies with green pigmentation
H2S production (blacking in TSI) Urease production
Shigella
Motility
+ve
-ve
SS agar Motile
E.coli
Not motile
Proteus
Salmonella
Practical Work
Reaction on TSI Indole Test MR test VP test Citrate Utilization test Urease test