Ornithine Decarboxylase Test - Principle, Procedure, Result

Q: how is ornithine decarboxylase test used for identification of enterobacteriaceae?

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. Increases the acidity of the solution, thereby changing colour of indicator from yellow to purple. If the organism fails to produce ornithine-decarboxylase, the medium is acid (yellow).

Q: What is the basis for the key test for ornithine or lysine decarboxylase?

production of decarboxylase enzyme by the sleeted organisms.

What is the ornithine decarboxylase test?

In this test we will use ornithine decarboxylase broth.

Medium is nutritional broth , to which 0.5 percent ornithine is added.
A key component of this medium small amount of glucose, which is required in order to continue the process.

The indicator for pH, bromocresol purple is purple at pH 5.2. It turns yellow at pH <5.2.
A purified culture will be moved to a sterilized tube containing ornithine decarboxylase broth.
The tube that has been inoculated is then incubated at 35 to 37 C for 24 hours, and the preliminary results are analyzed.

The microbe first needs to use glucose to make the pH drop. This can be seen by a change in color from purple to yellow.
After the medium is acidified after which the enzyme ornithine decarboxylase is activated.
The incubation period is over the next 24 hours at 35 to 37 C to allow the microbe to make use of ornithine.

The final results are discovered by watching the tube after 48 hour. The change back to purple after yellow is a sign of an Positive test result for the ornithine degradation.
Failure to turn yellow within the end of 24 hours, or change to purple after 48 hours is negative results.

Purpose of Ornithine decarboxylase test

The aim is to find out whether the microbe is able to utilize ornithine, an amino acid that is the source of carbon and energy to grow. Ornithine utilization is achieved by the enzyme called ornithine decarboxylase.

Principle of Ornithine decarboxylase test

Peptone and extracts of meat provide nitrogenous as well as other nutrients required for bacterial growth.
Pyridoxal is an enzyme that acts as the cofactor of ornithine decarboxylase.
Dextrose is a carbohydrate that can be fermented. Bromocresol purple and cresol red are pH indicators.

The amino acid used to measure the ornithine decarboxylase production.
In the event that you inoculate the media bacteria that are able to produce dextrose through fermentation, acid are created which lower its pH. This can alter the color of the indicator, changing it from purple to yellow.
Decarboxylases eat specific amino acids. They then remove carboxyl groups and transform the amino acids to aliphatic amines creating CO2 as a secondary product.

The decarboxylation of ornithine result to an amine formation, resulting in an acidic pH shift that alters how the indicator appears.
The acidic environment also triggers decarboxylase activities. When the organism produces ornithine-decarboxylase, the ornithine present the medium is converted into putrescine.
Its production increases the acidity of the solution, thereby changing colour of indicator from yellow to purple.

If the organism fails to produce ornithine-decarboxylase, the medium is acid (yellow).

Principle of Ornithine decarboxylase test | Image Source: openi.nlm.nih.gov

Requirement

Test organisms
Ornithine decarboxylase broth

Bunsen burner, inoculating loop, incubator, test tubes

Composition of Ornithine decarboxylase broth

Peptone	5.0g
Meat Extract	5.0g
Pyridoxal	0.005g
Dextrose	0.5g
L-Ornithine	10.0
Bromocresol Purple	0.010
Cresol Red	0.005

Final pH 6.0 ± 0.2

Preparation of Ornithine decarboxylase broth

Add 21,0 grams into 1 liter of distillate water.

Stir until completely dissolved.
Dispense into containers for final use.
Autoclave at 121°C in 15 minutes.

Procedure of Ornithine decarboxylase test

A. Inoculation of Medium

Select the Ornithine Decarboxylase broth medium.
Start your Bunsen burner.
Select the loop to inoculate.

The inoculating loop is smuggled with flames for sterilization.
Caps off the test tube.
Flaming in the test tube’s mouths.

Use the sterile inoculating tool for inoculation to collect an inoculum from the tube of culture of the unknown bacteria.
The inoculum should be immediately transferred directly into the clean and non-sterile medium.
Flame The tube’s mouths once again.

Replace the cap of your test tube.
Re-flame the inoculating tool.

B. Incubation of the Inoculated Medium

Place the tube with the inoculation into the 35 to 37 C incubator.

Click The New Day button to move ahead 24 hours.

C. Determination of Test Results

Let it incubate over the required amount in time. For this test, the color has to be recorded at both the 24 hours and at 48 hours in order to find out the results. (i.e. you must repeat steps 12-16 twice in order to finish this testing).
Get desired incubated cultures out of the incubator.

Observe test results.
The color changes to yellow in the presence acid and stays purple in neutral pH or when there are bases or alkali. For this test, observing the color after 24 hours is required for accurate interpretation of the results, however, an additional 24 hrs of incubation are required to evaluate the results of the complete test. After the color of the tube has been seen, place the tube back within the incubator one additional period of time. (Perform steps 12-16 over again).
Record test result.

Get rid of the culture.

Ornithine decarboxylase test results

Positive ornithine decarboxylase test: The change back to purple from yellow signifies a positive test result for ornithine degradation.
Negative ornithine decarboxylase test: The failure of the test to change color after 24-hours or change back to purple after 48 hours, indicates the result is negative.

Ornithine decarboxylase test results of some organisms

Quality Control

Positive Control

Proteus mirabilis (4 – 18 hours)

Staphylococcus lugdunensis (30 minutes-2 hours)

Negative control

Proteus vulgaris (hold 18 hours)

Staphylococcus epidermidis (2 hours)

Uses

It is utilized for the biochemical differentiation of enteric bacilli that are gram-negative that is based on the production of ornithine-decarboxylase.

FAQ

how is ornithine decarboxylase test used for identification of enterobacteriaceae?

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. Increases the acidity of the solution, thereby changing colour of indicator from yellow to purple.
If the organism fails to produce ornithine-decarboxylase, the medium is acid (yellow).

What is the basis for the key test for ornithine or lysine decarboxylase?