Decarboxylase Test- Principle, Procedure, Results, Uses

Decarboxylase Test Definition

Decarboxylase test is a biochemical test performed to differentiate members of Enterobacteriaceae on the basis of their ability to produce the enzyme decarboxylase.

• The metabolism of amino acids might differ in aerobic and facultatively anaerobic bacteria as well as in gram-negative organisms.
• Metabolism of amino acid might occur either by decarboxylation, hydrolysis, or deamination, depending on the amino acid being metabolized and the organism.
• Decarboxylation occurs in the presence of a decarboxylase enzyme that catalyzes the breaking of the bond that binds the carboxylic group to the rest of the amino acid.
• Deamination occurs in the presence of the deaminase enzyme that catalyzes the breaking of the bond that binds the amino group to the rest of the amino acid.
• Three different decarboxylase enzymes are produced by organisms that catalyze the metabolism of amino acid; ornithine decarboxylase, arginine decarboxylase, and lysine decarboxylase.
• The production of these enzymes is taken as an important parameter for the differentiation of bacteria present in the Enterobacteriaceae family.
• Besides, in the identification of Enterobacteriaceae, the ornithine decarboxylase test is of paramount importance, especially for separating members of the Klebsiella-Enterobacter-Serratia group and for identifying species of Proteus.

Objectives of Decarboxylase Test

• To test the ability of an organism to produce a decarboxylase enzyme.
• To differentiate the members of the Enterobacteriaceae family on the basis of their ability to produce decarboxylase enzyme.

Principle of Decarboxylase Test

• Arginine, lysine, and ornithine decarboxylase media are used to detect an organism’s ability to decarboxylate or hydrolyze an amino acid, forming an amine that produces an alkaline pH.
• The basal medium is usually Moeller’s formula and contains meat peptones and beef extract, which supply nitrogenous nutrients to support bacterial growth.
• The media contains glucose as the fermentable carbohydrate and pyridoxal, which is an enzyme cofactor that enhances decarboxylase activity. The pH indicators are bromcresol purple and cresol red.
• Amino acids like arginine, lysine, and ornithine are singly added to the basal medium to detect the production of enzymes that decarboxylate or hydrolyze these substrates.
• After the fermentation of glucose in the medium, acids are produced which lower the pH, resulting in a change in color from purple to yellow.
• If the organism produces decarboxylase, decarboxylation, or hydrolysis of the amino acid occurs in response to the acid pH.
• Decarboxylation results in alkaline end products (amines), causing the medium to revert to its original color (purple).
• In the case of the organism that does not ferment glucose, the medium does not turn yellow; however, the test can still be performed, but by including a control without amino acids for comparison.
• The decarboxylation of lysine yields cadaverine, decarboxylation of ornithine produces putrescine, and decarboxylation of arginine results in agmatine, which is hydrolyzed by a dihydrolase to form putrescine.
• In another reaction, arginine dihydrolase catalyzes the conversion of arginine to citrulline, which is further converted to ornithine, finally producing putrescine.
• Decarboxylation is an anaerobic reaction, and thus, the contents of each tube must be sealed with oil or paraffin.

Microorganisms Tested

• Enteric Gram-negative rods and Vibrio, Plesiomonas, and Aeromonas for identification to the species level
• Probable Stenotrophomonas and Burkholderia (lysine and arginine) Fluorescent Pseudomonas (arginine)
• Coagulase-negative staphylococci (ornithine)
• Viridans group streptococci (arginine)
• Miscellaneous non-glucose-fermenting, Gram-negative rods (arginine)
• Spreading indole-negative Proteus (ornithine)

Media, Reagents, and Supplies Used

Media

• Decarboxylase Test Medium Base (Moeller’s is used for testing amino acid decarboxylase activity. Other media like Motility-indole-ornithine medium (MIO) and Lysine iron agar can also be used.
• The composition of the decarboxylase medium base is given below:

Reagents Used

• Mineral oil
• Vaspar, liquid paraffin, or petroleum jelly, maintained at 56°C in liquid form

Supplies Used

• Sterile sticks or inoculating loops
• Incubator at 35°C

Procedure of Decarboxylase Test

A. Preparation of the media

• In a beaker, 9.02 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of pure distilled or deionized water.
• The solution is then heated to bring it to boil in order to dissolve the medium completely.
• The media is then divided into four equal parts. One part is tubed without the addition of any amino acid, and the tube is labeled as ‘Control’.
• The remaining three parts are dispensed onto three tubes, to which L-lysine hydrochloride, L-arginine hydrochloride, and L-ornithine hydrochloride are added separately to a final concentration of 0.5%.
• About 3-4 ml of the media are dispensed in screw-capped tubes and sterilized by autoclaving at 10 lbs pressure (115°C) for 20 minutes.
• To avoid false alkalinization at the surface of the medium, it is recommended to add liquid paraffin to a height of about 5 mm before sterilization.

B. Decarboxylase test

For Glucose-Fermenting Organisms

• A drop of 18-24 hour brain heart infusion broth culture is added to each of the three decarboxylase broths (arginine, lysine, and ornithine).
• A control tube is not required for glucose-fermenting organisms.
• A 4 mm layer of sterile mineral oil is added to each of the tubes.
• The tubes are then incubated for 4 days at 35-37°C in ambient air.
• The tubes are then observed for color change at 24, 48, 72, and 96 hours.

Glucose-Nonfermenting Organisms

• A suspension (> McFarland No. 5) in brain-heart infusion broth is prepared from an overnight culture (18-24 hours old) growing on 5% sheep blood agar.
• Each of the four decarboxylase broths is inoculated with four drops of the prepared suspension.
• A 4 mm layer of sterile mineral oil is added to each of the tubes.
• The tubes are then incubated for 4 days at 35-37°C in ambient air.
• The tubes are then observed for color change at 24, 48, 72, and 96 hours.

Control organisms

Result Interpretation of Decarboxylase Test

• A positive test is a turbid purple to faded-out yellow-purple color (alkaline).
• A negative test is a bright clear yellow color (acid) or no change (nonfermenting rods).
• The control tube must retain its original color or turn yellow. The turbidity and alkaline or purple color in the control tube invalidate the test. The questionable results are compared to the control tube.

Figure: Decarboxylase tests (Moeller’s method) – Positive, Negative, and Uninoculated tube. Image Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier.

Uses of Decarboxylase Test

• Decarboxylase test is used to differentiate the members of the Enterobacteriaceae family with closely related physiological characteristics.
• Arginine decarboxylase is useful in the identification of Enterococcus to the species level; Enterococcus faecalis and Enterococcus faecium are arginine positive but, Enterococcus avium is arginine negative.
• Lysine decarboxylase is used to differentiate between Salmonella (+) and Shigella (-).

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Limitations of Decarboxylase Test

• Mineral oil or a similar barrier to gas release must be applied to the surface of each inoculated broth medium. Oil on the surface reduces the possibility of an alkaline shift occurring in the medium due to oxidation.
• Result interpretation should not be made prior to 18 to 24 hours of incubation. Earlier interpretation may lead to erroneous results. Glucose fermentation occurs within the first 10 to 12 hours of incubation. The acidic environment from fermentation is necessary for the production of decarboxylase.
• Glucose-Nonfermenting microorganisms might display weak decarboxylase activity, thereby resulting in insufficient production of amines necessary to convert the pH indicator system. Some non-fermenters, however, will produce sufficient amines to result in deeper purple color as compared to the uninoculated tube.
• Grey color in the tube may indicate a reduction of the indicator, instead of the production of alkaline end products. To aid in reading the reaction, additional bromcresol purple should be added.
• If two layers of different colors appear, the tube should be shaken gently before interpreting the reaction.
• Glucose-Nonfermenting bacteria that are arginine positive must be lysine and ornithine negative.

References and Sources

• Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook, 3.17.1.1–3.17.48.3.
• Fay, G. D., & Barry, A. L. (1972). Rapid ornithine decarboxylase test for the identification of enterobacteriaceae. Applied microbiology, 23(4), 710–713.
• Pilsucki, R. W., N. W. Clayton, V. J. Cabelli, and P. S. Cohen. 1979. Limitations of the Moeller lysine and ornithine decarboxylase tests. Appl. Environ. Microbiol. 37:254-260.
• Decarboxylase Test Medium Base. M912S. HiMedia Laboratories.
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