Decarboxylase Test

Lysine Positive Test Result:

A positive lysine result will produce a very bright purple broth.

Ornithine Positive Test Result:

A positive ornithine test result will produce a very bright, dark purple that is much darker than the control.

Lysine Negative Test Result:

A negative lysine result will produce a broth that is very close to the control broth color, if not lighter in color.

Ornithine Negative Test Result:

A negative ornithine test result will produce a very light purple broth that is lighter than the control, and almost pink.

Arginine Negative Test Result:

For a negative arginine test result, the broth will stay the same color as the original broth or turn a pale to bright yellow color.

Arginine Positive Test Result:

For a positive arginine test result, the purple color of the broth will become more pronounced.

After inoculation, all broths were sealed with mineral oil. Why is this necessary?

To seal the medium from external oxygen and promote fermentation. Fermentation will lower the pH turning (yellow) and with the presence of the specific amino acid will induce the production of the enzyme decarboxylase to perform decarboxylation resulting in alkaline end products changing the pH more basic (darker purple).

Goal

Goal: To aseptically inoculate three different types of decarboxylase broth, to learn how to determine the results of those tests, and to learn what the results of those tests mean.

Decarboxylase Broth Inoculation Procedure

Ensure none of the broths have contamination in them. Set up a Bunsen burner. Aseptically obtain a sample of your unknown using the inoculating loop. Aseptically inoculate the arginine broth with the collected sample. Repeat steps 8-9 for the lysine and ornithine broth. Take the tube of mineral oil, remove the cap (do not set the cap on the bench) and flame the lip. Flame the lip of your inoculated arginine broth, and carefully pour the mineral oil into it. Only pour enough so that the top of the broth is covered with the mineral oil (about 1 cm). Repeat steps 12-13 for lysine and ornithine inoculated broths. Incubate the tubes at 30 degrees Celsius for at least seven days.

Methods

Methods: Sterilize the workbench with the Lysol cleaning solution. Wash hands and obtain a pair of gloves. Collect all necessary materials. Collectively label all sterile media including your name, section number, the date, the type of broth, and what unknown you are using. Arrange all the materials needed for the experiment to allow easy and safe accessibility.

Materials

Materials: Lysol cleaning solution Paper towels Sharpie Rubber Gloves Inoculating Loop Bunsen Burner Decarboxylase Lysine Broth Decarboxylase Arginine Broth Decarboxylase Ornithine Broth Arginine Broth Control Ornithine Broth Control Lysine Broth Control Unknown Culture Mineral Oil Tape (optional)