IMVIC

IMVC TESTES&TSI TEST

Biochemical Reactions

IMVC Test

Indole, Methyl Red, Voges-Prosakaur, Citrate (IMVC) Tests The IMVC testes allows for the differentiation of the members of Enterobacteriaceae.

Indole test

Principle Certain microorganisms can metabolize tryptophan by tryptophanase
Tryptophane amino acids
Tryptophanase
Indole + Pyurvic acid + NH3
Kovac’s Reagent Red ring color in upper organic layer`

Indole test

Indole test
Result: Positive result: A bright red ring in the top layer indicates the presence of indole. Negative result The absence of color means that indole was not produced. Used to differentiate of genera e.g. E. coli (+) from Klebsiella (-).
Positive test e.g. E. coli
Negative test e.g. Klebsiella



2. Methyl red test : All enteric bacteria metabolize glucose for their energy needs ,however ,the end products vary depending on the enzyme pathways present in the bacteria . This test used for determine the ability of microorganism to ferment the glucose and produce acids such as lactic acid ,acetic and formic acids.


Procedure :1.Inoculate the tube of ( Peptone – glucose – phosphate water ) with the bacterial culture. 2.Inocubate the tub at37Cfor 24hr.3.Add a few drops of methyl red reagent. and observe the change of color

Result : If the bacteria ferment the glucose and produce acid this will be lower the PH of the media under PH 4.5 and the color will be change to the red color. positive result (+ Ve) red color negative result (- Ve) no change in color E.Coli (+),Klebsiella spp (-)

3. Voges – Proskauer – test : This test used to determine the ability of some microorganism to ferment glucose,leading to Acety methyl carbinol(ACETOIN) accumulated in the medium ,the addition of Barritts reagent will detect the presence of aceton(and will be oxidized to Diacetyl a cherry red to brown color).

Procedure :1. Inoculate the tube of peptone – glucose – phosphate water with the bacterial culture. 2. Incubate the tube at 37Co for 24hr. 3.Add few drops from Barritt reagent to the tube. and observe the change of color within 15 min.

Result : Observe the change of color. Positive result (+ Ve) Change of color cherry red to brown. Negative result (- Ve) no change in color -E.Coli (-),Klebsiella spp (+)

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

Glucose
Complete fermentation
Mixed acids  pH less than 4.5 Methyl Red indicator
Red color
Principle
MR positive E. coli
Or
Partial fermentation
Acety methyl carbinol (ACETOIN)
Barrit’s reagent cherry color
VP positive Klebsiella



4. Citrate test : This test use for determine the ability of microorganism to used the citrate as only source of carbon for their energy needs .this ability depends on the presence of a citrate permease that transport of citrate into the bacterium ,citrate is converted to pyruvic acid and CO2,


O`is necessary for citrate utilization .when bacteria oxidize citrate ,they remove it from medium and liberate CO2which will combined with sodium (supplied by sodium citrate )and water to form sodium carbonate—an alkaline product this raise the PH of medium and change the color of media by the indicator bromothaymol blue..Sodium citrate pyrovic acid+CO2Sodium from sodium citrate+CO2+H2O Na2CO3(alkaline)pH

Procedure : 1.Inoculate agar slant of simmon citrare medium to bacterial culture. 2.Inocubate the tube at 37Co for 24hr. 3.After Inoculation observe the color of tube . Positive result (+ Ve) blue color Negative result (- Ve) green color -E.Coli (-),Klebsiella spp (+)

Citrate Utilization Test

Growth on the medium and rise in pH to change the medium from its green color to deep blue
Result
Positive Klebsiella, Enterobacter
Negative E. coli

Triple Sugar Iron (TSI) Agar

TSI test is generally used for identification of enteric bacteria (Enterobacteriaceae).it is also used to distinguish the Enterobacteriaceae from other gram negative intestinal bacilli

Reaction on Triple Sugar Iron (TSI) Agar

TSI contains *Three sugars ( lactose 1% , Sucrose 1% , glucose 0.1% ) *liberate sulfides from ferrous ammonium sulfate or sodium thiosulfate to produce black precipitate (H2S)) *Phenol red as indicator for sugar fermentation ( the media before inoculation of bacteria will be red in color )


Reaction on TSI

Result 1.If there is fermentation the PH of the media will decrease, and the phenol red indicator will change to yellow color. 2. If there no fermentation the color of the media will remain red. 3. If bacteria ferment glucose = Yellow butt 4. If bacteria ferment lactose and or sucrose = Yellow slant

1. Alkaline slant / alkaline butt red / red Non carbohydrate fermentation Ex: pseudomonas areuginosa 2. Alkaline slant / Acid butt red / Yellow Glucose fermented, Lactose & sucrose not fermentation Ex: Shigella species 3. alkalin slant red Black , / butt acid Yellow red / yellow Glucose fermented, Lactose & sucrose not fermented, hydrogen sulfide produce. Ex: Salmonella sp., proteus sp. 4. Acid slant / acid butt Yellow / Yellow Glucose fermented, Lactose & or sucrose fermented Ex: E.Coli , Klebisella. 5. Gas formation noted by splitting of the agar. 6. Black precipitate (H2S) seen by blackening of agar.

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