IMViC (Biochemical test)

Each of the letters in “IMViC” stands for one of these tests. “I” is for indole; “M” is for methyl red; “V” is for Voges-Proskauer, and “C” is for citrate,  lowercase “i” is added for the ease of pronunciation. IMViC is an acronym that stands for four different tests
Indole test
Methyl red test
Voges-Proskauer test
Citrate utilization test

1. Pooja Anothra Asst. Professor IEC group of institutions IMViC

2. INTRODUCTION Each of the letters in “ IMViC ” stands for one of these tests. “I” is for indole ; “M” is for methyl red; “V” is for Voges-Proskauer , and “C” is for citrate,  lowercase “ i ” is added for the ease of pronunciation.  IMViC is an acronym that stands for  four different tests Indole test Methyl red test Voges-Proskauer test Citrate utilization test

3. To obtain the results of these four tests, three test tubes are inoculated: tryptone broth ( indole test), methyl red – Voges Proskauer broth (MR-VP broth), and citrate(Simmons citrate agar).   IMViC tests are   employed in the identification/differentiation of members of family  enterobacteriaceae. General procedure for performing IMViC Tests and their interpretations: Cultures of any members of enterobacteriaceae have to grow for 24 to 48 hours at 37°C and the respective tests can be performed:

4. Indole test It is performed on sulfide- indole -motility (SIM) medium or in tryptophan broth, or in motility urease indole (MIU) medium. Result is read after adding Kovac’s reagent. The positive result is indicated by the   slightly pink layer at the top of the tube  after the addition of Kovács reagent. A negative result is indicated by the lack of color change at the top of the tube after the addition of Kovács reagent.

5. Indole Test Reaction Indole test is a commonly used biochemical test (e.g. in IMVIC   test). Indole test helps to differentiate Enterobacteriaceae  and other genera . Two methods are in use; a conventional tube method requiring  overnight incubation, which identifies weak indole producing organisms and a spot indole test, which detects rapid indole producing organisms

6. Procedure of  Conventional Tube method for Indole Test A. Inoculate the tryptophan broth with broth culture or emulsify isolated colony of the test organism in tryptophan broth. b. Incubate at  37°C for 24-28 hours in ambient air. c. Add 0.5 ml of Kovac’s reagent to the broth culture.

7. Expected results Positive : Pink colored rink after the addition of an appropriate reagent Negative : No color change even after the addition of an appropriate reagent. e.g.  Klebsiella pneumoniae

8. Procedure of Spot Indole Test Saturate a piece of filter paper with the 1% paradimethylaminocinnamaldehyde reagent. Use a wooden stick or bacteriologic loop to remove a small portion of a bacterial colony from the agar surface and rub the sample on the filter paper.

9. Results Positive:  Development of a blue color within 30 seconds. Most indole positive organisms turn blue within 30 seconds. Negative:  No color development or slightly pink color.

10. Methyl red test (MR test) Methyl red test and Voges-Proskauer test  both are done in methyl red– Voges-Proskauer (MR-VP) broth, but the reagents that are added varies according to the  test Positive methyl red test is indicated by the development of red color after the addition of methyl red reagent. A negative methyl red test is indicated by no color change after the addition of methyl red reagent

11. Methyl Red (MR)  test determines whether the microbe performs mixed acids fermentation when supplied glucose. Types and proportion of fermentation products produced by anaerobic fermentation of glucose are one of the key taxonomic characteristics which help to differentiate various genera of enteric bacteria.

12. MR Positive : When the culture medium turns red after the addition of methyl red, because of a pH at or below 4.4 from the fermentation of glucose. MR Negative : When the culture medium remains yellow, which occurs when less acid is produced (pH is higher) from the fermentation of glucose.

13. Procedure MR-VP broth is used for both MR Test and VP test. Only the addition of reagent differs, and both tests are carried out consecutively. 1. Inoculate two tubes containing MR-VP Broth with a pure culture of the microorganisms under investigation. 2. Incubate at 35 °C for up to 4 days. 3. Add about 5 drops of the methyl red indicator solution to the first tube ( for Voges-Proskauer test , Barrit’s reagent is added to another tube). 4. A positive reaction is indicated if the color of the medium changes to red within a few minutes.

14. Expected results Escherichia coli : MR test positive- the appearance of red color after the addition of methyl red reagent. Klebsiella  (formerly  Enterobacter )  aerogenes : MR test negative- the lack of color change after the addition of methyl red.

15. Voges-Prokauer test Negative test is indicated by a lack of color change after the addition of Barritt’s A and Barritt’s B reagents. A positive Voges-Proskauer test is indicated by the development of red-brown color after the addition of Barritt’s A and Barritt’s B reagents.

16. Procedure Inoculate a tube of MR/VP broth with a pure culture of the test organism. Incubate for 24 hours at 35°C At the end of this time, aliquot 1 mL of broth to clean test tube. Add 0.6mL of 5% α- naphthol , followed by 0.2 mL of 40% KOH.  (Note: It is essential that the reagents be added in this order.) Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain undisturbed for 10 to 15 minutes.

17. Results and Interpretation A positive test is represented by the development of a red color 15 minutes or more after the addition of the reagents indicating the presence of diacetyl , the oxidation product of acetoin . The test should not be read after standing for over 1 hour because negative Voges-Proskauer cultures may produce a copper like color, potentially resulting in a false positive interpretation.

18. Citrate test The test is performed on Simmons citrate agar: Negative citrate utilization test is indicated by the lack of growth and color change in the tube  A positive citrate result as indicated by growth and a blue color change.   Citrate utilization test is used to determine the ability of bacteria to utilize sodium citrate.

19. Procedure 1. Inoculate  Simmons citrate agar  lightly on the slant by touching the tip of a needle to a colony that is 18 to 24 hours old. 2. Incubate at 35°C to 37°C for  18 to 24 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium. 3. Observe the development of blue color; denoting alkalinization .

20. Expected results in Citrate Utilization Test Citrate positive:   growth will be visible on the slant surface and the medium will be an  intense Prussian blue . Citrate negative:   trace or no growth will be visible. No color change will occur; the medium will remain the deep forest green color of the uninoculated agar.

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