1. Mic 224 Lab 10 IMViCs

2. IMViC Tests The IMViC tests are useful for differentiating the Enterobacteriaceae, especially when used alongside the urease test. When used alone, the IMViC tests are particularly useful for differentiating Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae The IMViC series includes four tests: Indole production (for which we use SIM medium) the methyl red test the Voges-Proskauer test and citrate production These 4 tests combined with those of the TSI/Carbohydrate tests are used to identify an unknown species of bacteria

3. The Indole Test: Tryptophane Metabolism The indole test screens for the ability of an organism to degrade the amino acid tryptophan and produce indole. Tryptophan is an amino acid that can undergo deamination and hydrolysis by bacteria that express tryptophanase enzyme. tryptophan + water = indole (end product)+ pyruvic acid + ammonia

4. Indole Protocol Inoculate the tube of tryptone broth with a small amount of a pure culture. You will need 4 indol tubes. Inoculate the following organisms into a single tube: E.coli, Enterobacter aerogenes, Proteus rettgeri, and Psuedomonas aeruginosa Incubate at 35°C (+/- 2°C) for 24 to 48 hours. To test for indole production, add 5 drops of Kovács reagent directly to the tube. A positive indole test is indicated by the formation of a pink to red color ("cherry-red ring") in the reagent layer on top of the medium within seconds of adding the reagent If a culture is indole negative, the reagent layer will remain yellow or be slightly cloudy

5. Indole Results Positive Indole testNegative Indole Test

6. Methyl Red Test Utilizes MR-VP broth containing glucose, casein, digested animal tissue, and potassium phosphate. The substrate is glucose-peptone Many different Gram negative organisms can be identified by their metabolic end products produced by the fermentation of glucose in the MR-VP broth Proteus sp, E. coli, Salmonella, and Aeromonas ferment the glucose and produce large amounts of acid byproducts These products include formic acid, succinic acid, acetic acid, and lactic acid –These acids accumulate in the media and lower the pH below 7.0 Other enteric produce more neutral end products When the methyl red agent is added to the test tube after incubation the tube will turn a deep red if the pH drops to 4.6 or below –This indicates mixed acid fermentation

7. Methyl Red Test Protocol You will need 4 MR-VP tubes. Label these MR + the organism name Inoculate the following organisms into a single tube: E.coli, Enterobacter aerogenes, Proteus rettgeri, and Psuedomonas aeruginosa

8. Methyl Red Test Results After incubation add 10 drops of methyl red reagent to each tube labeled MR –Red- positive for mixed acid fermentation –Yellow- negative for fermentation

9. Voges-Proskauer Test This test is used to determine if a MR negative organism is using the butanediol pathway thus producing acetoin The media is MR-VP broth The substrate is glucose-peptone End products testing for: acetoin Reagents: alpha napthol and KOH- 10 drops each –Positive Test: reddish color –Negative Test: brown/yellow

10. VP Protocol You will need 4 MR-VP tubes. Label these VP + the organism name Inoculate the following organisms into a single tube: E.coli, Enterobacter aerogenes, Proteus rettgeri, and Psuedomonas aeruginosa

11. VP Results Left: VP positive, Right: VP negative

12. Citrate Test The purpose of the citrate test is to determine whether an organism can utilize sodium citrate as their only carbon source Fecal coliform bacteria such as E.coli cannot utilize citrate and will not grow Other types of enteric bacteria such as Citorbacter, Pseudomonas, and Enterobacter can utilize the citrate and produce a positive reaction and alkaline conditions (pH greater than 7.0)

13. Citrate Test The medium for this test is simmon’s citrate The substrate is sodium citrate The end product is sodium carbonate The reagent is bromothymol blue –Positve: blue color –Negative: green color

14. Citrate Protocol You will need 4 citrate slants Inoculate the following organisms onto a single slant: E.coli, Enterobacter aerogenes, Proteus rettgeri, and Psuedomonas aeruginosa –You do not need to stab to the bottom of the tube, just inoculate the slant portion

15. Citrate Results