IMVIC Testing for Enterobacteriaceae

The IMViC test consists of 4 tests: Indole, methyl red, Voges-Proskauer, and citrate untilization and is used to identify and differentiate between different Gram (-) lactose fermenting coliform bacteria or Enterobacteriaceae.  The presence of coliform bacteria indicates fecal contamination.


SIM Three Tests In One: Hydrogen Sulfide, Indole, Motility



(A)                            (B)                               (C)                                (D)                                 (E)

                               

Sulfur-indole-motility test results from various microbes. From left to right:  (A) Escherichia coli, (B) Staphylococcus aureus, (C) Salmonella arizonae, (D) Enterobacter aerogenes, and (E) Proteus vulgaris. After addition of Kovács reagent, a pink ring at the top of the tube indicates a positive indole result (A and E). Blackening of the media indicates hydrogen sulfide production (C and E). Growth feathering away from the stab line creating a cloudy appearance in the media indicates motility (A, C, D, and E). Growth strictly along the stab line indicates a nonmotile organism (B).


(+): Pink ring at the top of the tube after the addition of 5-10 drops of Kovac’s reagent.  A pink layer at the top of the tube after addition of kovac's reagent is a by- product of tryptophan metabolism and indole production. 
(-): Clear or No black color indicates no Hydrogen sulfide production.
(+): Pink ring at the top of the tube after the addition of 5-10 drops of Kovac’s reagent.
(+): Black indicates Hydrogen sulfide production.  Sodium thiosulfate, source of sulfur, is provided in the tube.  If the organism has the enzyme thiosulfate reductase then it reduces the sodium thiosulfate to hydrogen sulfide gas which reacts with ferrous sulfate producing a black precipitate. 

(-):  Clear/yellow ring at top of tube after addition of 5-10 drops of Kovac’s reagent indicates no indole production.
(+): Black indicates Hydrogen sulfide production. 
(-):  Clear/yellow ring at top of tube after addition of 5-10 drops of Kovac’s reagent indicates no indole production.
(-): Clear or No black color indicates no Hydrogen sulfide production.















Methyl Red and Voges-Prosker

One medium used for two separate but related test, identifying butanediol (VP) or acid fermentation products (MR).  The organism being tested is inoculated into one tube of MR-VP liquid media and incubated for up to 10 days a 37 degree's C.  Once incubation is done 1 ml of broth is drawn and put into a separate tube to test for butanediol while the other tube is used to test for acid fermentation.


(A)                                (B)
(A)  Enterobacter aerogenes turns yellow indicating that it is (-)
for acid fermentation and that butanediol fermentation occurs.
(B)  E. coli turn red after methyl red addition indicating acid    
fermentation.
    Addition of 5-10 drops of Methyl Red, pH indicator that turn red when pH drops below 4.2, detects the ability of an organism to produce acidic fermentation products. Acidic products produce a red color in the presence of a pH-indicator methyl red.Yellow in the presence of methyl red indicates a neutral pH.




(A)                                            (B)
(A) Enterobacter aerogenes develops a rose/red color after addition of
Barrit's A and B indicating acetoin production
       (B)  E. coli remains brown after addition of Barrit's A and B indicating no
acetoin production

    After addition of 10 drops of  Barrit’s A (contains alpha-naphthol) and Barrit’s B (contains KOH) a rose color will develop if the organism is able to convert acidic fermentation products to the neutral compound 2,3 butanediol acetylmethylcarbinol (acetoin).









 

Simmon's Citrate

This media test for the presence of citrate permease which transports citrate into the cells and citrase which produces oxalacetic acid and acetate.  Citrate media distinguishes between certain Gram (-) organisms that are able to metabolize citrate during the Kreb;s cycle (citric acid cycle) using citrate as the sole source of carbon source and ammonium ions as the sole nitrogen source.  If citric acid is utilized carbon dioxide is produced causing the pH to rise above 7.6 causing the color indicator, bromothymol blue to change from green  (pH below 6.9) to blue(above 7.6).


             citric acid --citrase--->  oxalacetic acid + acetic acid  -------->  pyruvate + CO2


(A)                (B)
(A) E.coli grown on citrate media, (-) for citrate utilization.
 (B)  Enterobacter aerogenes grown on citrate media, (+) for
citrate utilization as seen by the media color change from
green to blue.




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All images copyright T. Sturm

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