Shaylyn Robison

Lab Report #4

Micro Lab BIOL 2065

W 10 am-11:50 am

Report #4: IMViC Test

Introduction

The IMViC Test is a series of four experiments that help differentiate between the groups of Enterobacteriacaea according to their biochemical properties and reactions. The IMViC test stands for Indole, Methyl-red, Voges-Proskauer, Citrate Utilization tests. The four series of tests include the Indole Production Test, Methyl Red test, Voges-Proskauer Test, and the Citrate Utilization test.

Part 1: Indole Production Test

This test will “determine the ability of microorganisms to degrade the amino acid tryptophan.” (Rudolph 121)

Materials and Methods

  • 24 to 48-hour Trypticase soy broth cultures of Escherichia coli, Proteus vulgaris and Enterobacter aerogenes
  • 4 SIM agar deep tubes
  • Kovac’s reagent
  • Bunsen burner
  • Inoculating needle
  • Test tube rack
  • Marker

1.)Label 3 of the 4 tubes with one organism name on each tube.

2.)Use sterile technique to inoculate each organism into its corresponding labeled deep tube. Use a stabbing inoculating technique. The last tube is used as the control and will not contain any organism.

3.)Incubate tubes for 24 to 48 hours at 37 degrees Celsius.

4.)After incubation, add 10 drops of Kovac’s reagent to all tubes and agitate gently.

5.) Examine the tubes and record results.

Results

Organisms / Color of Reagent layer / Trytophan Hydrolysis (+) or (-)
E. coli / Red / +
E. aerogenes / Yellow / -
P. vulgaris / Red / +

Discussion

The organism E. coli and P. vulgaris yielded positive results for the Indole test, meaning they degrade tryptophan using tryptophanase to produce indole. The contents of the tube containing E. aerogenes turned yellow which means that E. aerogenes does not degrade tryptophan.

Extension

The Indole production test would be useful in determining and differentiating between multiple microorganisms in a patient’s sample. It would differentiate between pathogens and normal intestinal flora, such organisms as indole producing E. coli ( normal intestinal flora) and E. aerogenes.

Part 2: Methyl Red Test

The Methyl Red test will determine microorganisms’ ability to ferment glucose and “differentiate between all glucose-fermenting enteric organisms.” (Rudolph 123)

Materials and Methods

  • 24 to 48-hour Trypticase soy broth cultures of Escherichia coli, Proteus vulgarisand Klebsiella pneumoniae.
  • MR-VP broth
  • Methyl red indicatior
  • Bunsen burner
  • Inoculating loop
  • 4 test tubes
  • Marker

1.)Label tubes with organisms’ names, one organism per tube. The fourth tube will serve as a control.

2.)Use sterile technique to inoculate each organism into the corresponding tube. Do not inoculate the last tube.

3.)Incubate cultures for 24 to 48 hours at 37 degrees Celsius.

4.)After incubation, transfer one-third of each culture into an empty test tube and set aside for the Voges-Proskauer test.

5.)Add five drops of methyl red indicator to the remaining cultures.

6.)Examine cultures and record results.

Results

Methyl Red Test
Organisms / Color of Medium / (+) or (-)
E. coli / Pink/rose / +
E. aerogenes / yellow / -

*Note: The results of the third tube containing K. pneumoniae was not recorded.

Discussion

The Methyl red test differentiated E. coli from E. aerogenes. The results showed that E. coli does have the ability to oxidize glucose into acid end-products. The color red was result of a pH < 4.4. The color of E. aerogenes showed that its pH was at or around 6.2. This means that E. aerogenes does not produce acid.

Extension

The Methyl red test can be used to differentiate between acid-producing microorganisms and non-acid-producers. This differentiation would be useful when narrowing down a list of potential pathogens.

Part 3: Voges-Proskauer Test

This test will help differentiate between enteric organisms such as E. coli and E. aerogenes.

Materials and Methods

  • 24 to 48-hour Trypticase soy broth cultures of E. coli and E.aerogenes.
  • Barritt’s reagents A and B
  • Bunsen burner
  • Inoculating loop
  • Marker

1.)Obtain aliquots from the Methyl Red test.

2.)Add 10 drops of Barritt’s reagent A to the aliquots of each broth culture separated during the methyl red test. Shake cultures. Immediately add 10 drops of Barritt’s reagent B and shake. Re-shake cultures every 3 to 4 minutes.

3.)After 15 minutes of the addition of Barritt’s reagent, examine and record the color of the cultures.

Results

Voges-Proskauer Test
Organisms / Color of Medium / (+) or (-)
E. coli / Yellow / -
E. aerogenes / Pink / +

Discussion

E. aerogenes turned pink after the addition of the Barritt’s reagent which means the organism produces non-acidic end products. The detection of the non-acidic product, such as acetylmethylcarbinol will occur due to the presence of a-naphthol catalyst and guanidine group in the MR-VP medium. A yellow color means that the test proved the organism does not produce non-acidic end products and therefore was a negative result.

Extension

The Voges-Proskauer Test could be compared next to the Methyl red test to determine non-acid and acid producers and differentiate between the two. One test could serve as a control or evidence that will support the other test’s results.

Part 4: Citrate Utilization Test

The Citrate Utilization test will help further differentiate between enteric microorganisms based of the ability to ferment citrate.

Materials and Methods

  • 24 to 48-hour Trypticase soy broth cultures of E. coli and E.aerogenes.
  • 4 Simmons citrate agar slants
  • Bunsen burner
  • Inoculating needle
  • Test tube rack
  • Marker

1.)Label all tubes with one organism each. The fourth tube will be the control.

2.)Use sterile technique to inoculate each organism into its corresponding tube. Do not inoculate the fourth tube.

3.)Incubate all cultures for 24 to 48 hours at 37 degrees Celsius.

4.)After incubation, examine the agar slant cultures for growth and coloration of the medium. Record the results.

Results

Organism / Presence or Absence of Growth (+) or (-) / Color of Medium / Citrate Utilization (+) or (-)
E.coli / + / Green / -
E. aerogenes / + / Blue / +

Discussion

The blue color of growth of E. aerogenes showed a positive result for citrate utilization as a carbon source. The green color of growth of E. coli showed a negative result for citrate utilization. The results show that E. aerogenes is capable of using citrate as a carbon source for energy. They also mean that E. coli may ferment glucose or lactose; further tests could prove this theory.

Extension

The Citrate Utilization test would be helpful in figuring out if organisms are able to ferment citrate. This would help differentiate between organisms who require different nutrients but are similar in morphology, or those who are alike in biochemistry in other terms.

References

Rudolph, Dr. Jane. Symbiosis. Boston: Pearson, 2010. 121-130. Print.