IMViC biochemical tests

IMViC biochemical tests

1. SHITAL TRIVEDI ASSISTANT PROFESSOR, Department of pharmaceutical technology, L.J.INSTITUTE OF PHARMACY PRESENTED BY Subject: Pharmaceutical Microbiology SEMESTER 5 IMViC -BIOCHEMICAL TESTS

2. BIOCHEMICAL TESTS Biochemical tests are the tests to identify chemical reaction/metabolic reaction occurring in biological organisms. Biochemical tests are based on the various metabolic pathways involving specific enzyme systems in biological organisms. Biochemical tests are the important methods for the microbial identification.

3. IMViC Tests IMViC is an acronym that stands for four different tests. Each of the letters in “ IMViC ” stands for one of these tests. I- Indole Test, M - Methyl red, V - Voges-Proskauer , and C – Citrate Lowercase “ i ” is added for the ease of pronunciation.  IMViC tests are employed in the identification/differentiation of members of family enterobacteriaceae .

4. Indole Test Principle: + Kovac’s or Ehrlich’s reagent ( 4 (p)- dimethylamino benzaldehyde ) Red-colored compound Test Organism

5. Indole Test Procedure: Two methods are in use: Conventional tube method requiring  overnight incubation, which identifies weak indole producing organisms. Spot indole test, which detects rapid indole producing organisms

6. Procedure of  Conventional Tube method for Indole Test ( 1) (2 ) (3) Test organism (Inoculation) Kevok’s reagent, 0.5 ml Incubation ( At  37°C for 24-48 hours in ambient air.) Tryptone broth Culture broth Observation

7. Spot Indole Test Principle: Indole + C innamaldehyde Blue-green compound.   Procedure of Spot Indole Test: 1% paradimethylaminocinnamaldehyde Inoculation of test organism 30 seconds Piece of filter paper Observation & Interpretation.

8. Indole positive organisms : Most strains of E.coli, Proteus vulgaris , Morganella morganii and Providenicia species . Morphological characteristics, gram stain result, colony characteristics and biochemical tests can assist in the rapid identification of test organisms. Example: Gram negative, a flat, dry lactose fermenting (pink) colony on MacConkey agar that is also spot indole positive and oxidase negative can be reported presumptively as E.coli .

9. In Diagnostic laboratories, Indole test can is performed using multitest agar. : Three most commonly used agar medium are: Sulfide-indole-motility (SIM) medium : The SIM medium is a multitest agar used to test for indole production while simultaneously determining motility and hydrogen sulfide producing abilities of the isolate. Motility-indole-urease (MIU) medium: MIU medium is used to test for indole and urease producing characteristics of the organism along with a test for motility. Motility-indole-ornithine (MIO) medium: In addition to testing for indole production, MIO agar is used to test for motility and ornithine decarboxylase.

10. Methyl red Test Principle: Methyl Red (MR)  test determines whether the microbe performs mixed acids fermentation when supplied with glucose. Embden –Meyerhof– Parnas (EMP pathway )/ Glycolysis Glucose Pyruvic acid Mixed acids pH ≤ pH 4.4 Methyl red Yellow Red (+ve test)

11. Methyl red test Media and Reagents: Methyl red- Voges - Proskauer (MR/VP) broth (formulated by Clark and Lubs ) is used in this test. The composition of MR/VP broth is as follows: Ingredient MR/VP broth (g/L) Polypeptone 7 g Glucose 5 g Dipotassium phosphate 5 g Distilled water q.s.. 1 L Final pH 6.9 Methyl Red pH Indicator: Methyl red: 0.1 g in 300 mL of 95% ethyl alcohol. Distilled water: 200 mL

12. Quality Control Positive and negative controls should be run after the preparation of each lot of medium and after making each batch of reagent. Suggested controls include the followings: Positive control: Escherichia coli Negative control: Klebsiella (formerly Enterobacter ) aerogenes

13. Methyl red Test Procedure: ( 1) (2 ) (3) Test organism (Inoculation) Methyl red indicator, 5 drops Few minutes Incubation ( At  35 °C for 48 hours.) MR-VP broth Culture broth Observation

14. Methyl-Red (MR) positive organisms Escherichia coli Shigella species Salmonella species Citrobacter species Proteus species Yersinia species Methyl-Red (MR) negative organisms Enterobacter species Hafnia species Serratia marcescens Klebsiella pneumoniae

15. Voges-Proskauer Test Voges and Proskauer , in 1898, first observed the production of a red color after the addition of potassium hydroxide to cultures grown on specific media . Harden later revealed that the development of the red color was a result of acetyl-methyl carbinol production . In 1936 Barrit made the test more sensitive by adding alpha-naphthol to the medium before adding potassium hydroxide . The Voges-Proskauer test identifies bacteria that are able to metabolize pyruvic acid to form acetyl-methyl carbinol (acetoin ). Reference: https :// catalog.hardydiagnostics.com/cp_prod/content/hugo/rapidanginosusidkit.html

16. Voges-Proskauer Test Principle:

17. Voges-Proskauer Test Media and Reagents: 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. Reagents : 1. Barritt's A reagent : Alpha-naphthol , 5% color intensifier α – naphthol 5 g Absolute ethyl alcohol q.s. to 100 mL 2. Barritt's B reagent : Potassium Hydroxide, 40%, oxidizing agent Potassium hydroxide 40g Distilled water q.s. to 100 mL

18. Quality Control Positive and negative controls should be run after preparation of each lot of medium and after making each batch of reagent. Suggested controls include the following: Positive Control: Klebsiella (formerly Enterobacter ) aerogenes Negative Control: Escherichia coli

19. Voges-Proskauer Test:-Procedure : ( 1) (2 ) (3) (4) First add 0.6mL of 5% α- naphthol , ( Inoculation) followed by 0.2 mL of 40% KOH. Test Organism 1 ml aliquot Incubation ( At  37 °C for 24 - 48 hours.) Shake the tube gently, then undisturbed for 10-30 min. Clean test tube MR-VP broth Culture broth Observation (The test should not be read after standing for over 1 hour because negative Voges-Proskauer cultures may produce a copper like color(reaction between KOH & α- naphthol ), potentially resulting in a false positive interpretation .)

20. Voges-Proskauer (VP) Positive Organisms of Enterobacteriaceae family are: Klebsiella species Enterobacter species Hafnia species Serratia species Voges-Proskauer (VP) Positive Organisms Citrobacter species Shigella species Yersinia species Salmonella species E.coli

21. Citrate utilization test:- Principle: Citrate utilization test is used to determine the ability of bacteria to utilize.. Sodium citrate as its only carbon source and Inorganic ammonium dihydrogen phosphate as the sole fixed nitrogen source. CO 2 + water + Sodium ions Sodium bicarbonate and sodium carbonate pH ≥ 7.6 Deep forest green to blue color change

22. Citrate utilization test Media: The composition of Simmons citrate agar is as follows: The medium is poured into a tube on a slant. Ingredient Simmons Citrate Agar (g/L) Ammonium dihydrogen phosphate 1 g Dipotassium phosphate 1 g Sodium chloride 5 g Sodium citrate 2 g Magnesium sulfate 0.20 g Agar 15 g Bromothymol blue 0.08 g Distilled water 1 L Final pH 6.9

23. Citrate utilization test Procedure: ( 1) ( 2 ) Test organism (Inoculation by streaking/stabbing) No growth Growth Incubation ( At  35°C to 37°C for  18 to 24 hours ) or up to 7 days Simmons citrate agar slant Observation Bromothymol blue

24. Quality Control Positive control: Klebsiella pneumoniae Negative control : Escherichia coli

25.   List of Bacteria which gives positive citrate utilization test Klebsiella pneumoniae Enterobacter species (minority of strains gives negative result) Citrobacter freundii Salmonella other than Typhi and Paratyphi A Serratia marcescens Proteus mirabilis (minority of strains gives negative result) Citrate Test: variable (different strains give different results) Proteus vulgaris Vibrio cholerae Vibrio parahaemolyticus

26. Citrate test: negative Escherichia coli Shigella spp Salmonella Typhi Salmonella Paratyphi A Morganella morganii Yersinia enterocolitica Although uncommon, natural E. coli variants that are citrate positive have been isolated.  Citrate-negative strains of E. aerogenes have also been found . Content and images used in this presentation may be subject to copyright

27. References: https://microbeonline.com/imvic-tests-principle-procedure-and-results / https://catalog.hardydiagnostics.com/cp_prod/content/hugo/rapidanginosusidkit.html