Lab 19 IMViC

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Biology

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Feb 20, 2024

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IMViC & Water Analysis III OBJECTIVES: Students should be able to 1. describe the biochemical basis for each of the IMViC tests 2. perform each of the IMViC tests 3. interpret results from each of the IMViC tests INTRODUCTION IMViC is a series of four tests that can be used together to help determine the identities of certain species of Gram negative bacilli among the Enterobacteriaceae – enteric bacteria. Individual tests can also be used to identify certain species within other bacterial groups (e.g. Bacilli). It is the final step in the water analysis that identifies individual coliforms. The initials stand for I ndole, M ethyl Red, V oges-Proskauer, C itrate. Indole : this test determines whether the bacterium can produce the enzyme tryptophanase and utilize tryptophan (an amino acid) as a carbon source producing pyruvate and producing indole as a waste product. The test organism is inoculated into a tube of Tryptone Broth and after incubation the broth is tested for the presence of indole by adding 10-20 drops of Kovacs Reagent. A cherry red color in the upper phase of the tube is a positive result, yellow is negative. This is the formulation for one liter of Tryptone broth . Tryptone = 10.0g Sodium Chloride = 5.0g Methyl Red : this test determines the presence of acidic by-products of glucose fermentation. (mixed acid fermentation). The test organism is inoculated into a tube of MRVP broth , which is composed of peptone, glucose, and phosphate buffer. After incubation it is tested for a pH below 4.4 by adding the pH indicator Methyl Red . A bright red color indicates a positive result ( pH below 4.4 ), whereas a negative result shows orange to yellow (pH above 4.8).
Voges-Proskauer : this test determines the ability of bacteria to produce non-acidic byproducts from glucose. The test actually determines the presence of acetoin, an intermediate of 2,3- butanediol fermentation. The test organism is inoculated into a tube of MRVP broth , allowed to grow and then tested for the presence of acetoin by adding alpha naphthol and 40% KOH . A dull brick red color indicates a positive result; this result may require vigorous agitation and application of heat (place the tube in the incubator) and may take up to 30 min to develop. No change in the color of the tube indicates a negative result. Citrate : this test determines the ability of an organism to use citric acid when present as a sole source of carbon. The organism is inoculated into an agar slant of Simmons Citrate Agar allowed to grow and changes of color in the agar are noted. If the organism can metabolize citrate it will produce pyruvate as well as byproducts such as ammonia and bicarbonates that will increase pH. The agar contains a pH indicator ( bromothymol blue ) which changes from green to blue as a result of an increased pH when citrate is metabolized.
Ingredients per liter of deionized water: Simmons Citrate Agar Grams Magnesium sulphate 0.200 Ammonium dihydrogen phosphate 1.000 Dipotassium phosphate 1.000 Sodium citrate 2.000 Sodium chloride 5.000 Bromothymol blue 0.080 Agar 15.000 Final pH (at 25°C) 6.8±0.2 https://www.micro.iastate.edu/video/microbiology-011-imvic-series MATERIALS Tryptone broth MRVP broth Simmons citrate agar slant EMB agar from water quality II Kovac’s reagent Methyl red dye 40% KOH Alpha naphthol Escherichia coli Enterobacter aerogenes METHODS 1. Inoculate E. coli and Enterobacter aerogenes into each medium. 2. Inoculate from a colony positive for lactose fermentation on the EMB plate. 3. Incubate for 48 hours at 37C. 4. After incubation, add the following reagents: a. Indole: Add 10- 20 drops of Kovac’s reagent. b. MR: Add several (~5) drops of methyl red dye. c. VP: Add 12 drops of alpha naphthol, THEN 4 drops of 40% KOH d. Citrate: observe the color change directly
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RESULTS 1. After adding the appropriate reagents to the Indole, Methyl Red, and VP tubes, Record your results in the table. Indole Methyl red VP Citrate E coli Ent. aerogenes EMB isolate DISCUSSION QUESTIONS 1. What is tryptone? Is this a chemically defined or complex medium? Explain. 2. What other medium can be used to detect indole? 3. Why can we use the same medium for both the MR and VP tests? 4. What does it mean if an organism is negative for both tests? positive for both tests? 5. Is Simmons citrate agar a chemically defined or complex medium? Why is it necessary to be that way? 6. Is Citrate agar differential? Explain . 7. Describe the test results pictured below for one of the organisms and indicate what these results mean metabolically. Based on the data, is this the result for Escherichia coli or Enterobacter aerogenes ?

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