About this test
What is the purpose of the test?
The purpose is to see if the microbe can use the amino acid ornithine as a source of carbon and energy for growth. Use of ornithine is accomplished by the enzyme ornithine decarboxylase .
How is ornithine decarboxylase activity determined?
A medium containing ornithine and a pH indicator is used. When ornithine is used, the pH of the medium rises and the indicator changes color.
What medium is used?
The medium used is ornithine decarboxylase broth. The medium is a nutrient broth to which 0.5% ornithine is added. An important component of the medium is a modest amount of glucose, necessary for the process to proceed. The pH indicator brom cresol purple is purple at neutral or alkaline/basic pH but turns yellow at pH <5.2.
How is the test performed?
An inoculum from a pure culture is transferred aseptically to a sterile tube of ornithine decarboxylase broth. The inoculated tube is incubated at 35-37 C for 24 hours and the preliminary results are determined. The microbe must first use the glucose present to cause the pH to drop. This is indicated by a change from purple to yellow . Once the medium has been acidified, the enzyme ornithine decarboxylase is activated. The culture is incubated an additional 24 hours at 35-37 C to allow the microbe to now use the ornithine . The final results are then obtained by observing the tube at 48 hours. Change back to purple from yellow indicates a positive test for ornithine decarboxylase . Failure to turn yellow at 24 hours or to revert back to purple at 48 hours indicates a negative result.
What reagents are added?
Performing this test in the VUMIE Online lab
Inoculation of Medium
1. Select the ornithine decarboxylase broth medium.
2. Complete the process of a tube-to-plate aseptic transfer to inoculate the medium. Forgotten how to do these things? Watch the “Show Me How To” videos.
Incubation of the Inoculated Medium
3. Place the inoculated tube into the 35-37 C incubator.
Determination of Test Results
5. Incubate this test for 24 hours. Make note of the color, then incubate for an additional 24 hours.
6. Retrieve the incubated culture from the incubator.
7. Observe test result. If the test protocol was followed as described above, the culture will have changed to yellow due to acids produced in the first 24 hours and then reverted to purple at the 48 hour mark as the amino acid is used. Both color changes must occur for the test to be positive. Any other result is considered negative.
8. Record test result.