What is Decarboxylase Test?

Decarboxylase test is one of the biochemical tests. This test is commonly used to differentiate the members of the family Enterobacteriaceae on the basis of their ability to produce the enzyme decarboxylase.

However, the metabolism of amino acids differs depending on their characteristic as aerobic or anaerobic facultative anaerobes and as well as in gram-negative organisms.

Metabolism of amino acids occurs either by decarboxylation, deamination or hydrolysis depending on the metabolization of amino acid in an organism.

Decarboxylation often occurs in the presence of an enzyme deaminase and catalyzation occurs by breaking the bonds and it also binds to the rest of the amino acid in the amino group.

Three different decarboxylase enzymes are produced by the organisms, which catalyze the metabolism of amino acids, ornithine, arginine decarboxylase, decarboxylase, and lysine decarboxylase.

The production of these enzymes is taken as an important parameter for differentiating the bacteria present in the family of Enterobacteriaceae.

Apart from differentiating and identifying the Enterobacteriaceae, Ornithine decarboxylase teat is considered as one of the paramount importance especially in separating the members of the Klebsiella-Enterobacter Serratia group and also in identifying the species Proteus.

Decarboxylase Test Objective

 To detect the ability of an organism to produce an enzyme known as decarboxylase.

 To differentiate the members of the family Enterobacteriaceae on the basis of their ability to produce an enzyme decarboxylase.

Decarboxylase Test Principle

 The medias of Arginine, lysine and orthenine decarboxylase are used to detect the ability of an organism to decarboxylate or hydrolyze the amino acid and forms an amine which produces an alkaline ph.

 The basal medium is formed by using Moeller’s formula.

 The medium contains meat peptones and beef extracts which supplies nitrogenous nutrients to support the growth of bacteria.

 The media is composed of glucose which is a fermentable carbohydrate.

 The enzyme pyridoxal acts as a cofactor and enhances the decarboxylase activity. Bromocresol purple and cresol red is used as a pH indicator.

 Amino acids namely arginine, Lysine and ornithine are added once in the basal medium and it also helps in detecting the production of enzyme which decarboxylate or hydrolyze these substrates.

 After fermenting the glucose in the medium, acids are produced. These acids have the capability to lower the pH in the medium which results in change in color from purple to yellow.

 If the organism produces an enzyme decarboxylase, it results in decarboxylation or hydrolysis of amino acids, due to the response in the acid pH.

 Decarboxylation thus results in alkaline end products known as amines, which causes the medium to get back its original color, purple.

 In case if the organism does not ferment glucose, then the medium does not change into yellow, but in these cases too, the tests are performed by including a control without amino acids for comparison.

Decarboxylase Test Requirements

Media: Decarboxylase test medium Base is generally used for testing the amino acid decarboxylase activity. Where as the other medias like Motility-indole-ornithine medium and Lysine iron agar can also be used.

Reagents:

 Mineral oil

 Vaspar

 Liquid paraffin

 Petroleum jelly, which is maintained at 56ºC in the liquid form.

Supplies:

 Sterile sticks

 Inoculating loops

 Incubator

Decarboxylase Test Procedure

1. Preparation of Media

 Initially 9.02 grams of dehydrated powder is filled into a beaker containing 1000 milli liters of pure distilled or deionized water.

 The prepared solution is then heated till it boils, so that the medium dissolves completely.

 The media is initially divided into four equal parts. When one part is tube without adding any amino acid and the tube is labelled as control.

 Then the remaining three parts are dispensed into each of the tubes and L-lysine hydrochloride, L-arginine hydrochloride, and L-ornithine hydrochloride are separately added to bring a final concentration of about 0.5%.

 About 3 to 4 ml of the media is dispensed in a screw capped tubes and they are sterilized by autoclaving them at a 10 lbs. pressure in 115ºC for about 20 minutes.

 In order to avoid the false alkalinization on the surface of the medium, liquid paraffin is added to height of about 5mm before sterilizing.

2. Decarboxylase Test

 For Glucose-fermenting organisms

 A drop of 18-24hour brain heart infusion broth culture is add in each of the three decarboxylase broths.

 Here the control tube is not used for glucose-fermenting organisms.

 4mm layer of sterile mineral oil is added to each of the tubes and the tubes are incubated for 4 days at a temperature of 35 to 37ºC in an ambient air.

Glucose-Nonfermenting Organisms

 A suspension is prepared using brain heart infusion broth and it is prepared from an overnight culture on a sheep blood agar.

 Each of the four-decarboxylase broth is inoculated with four drops of the prepared suspension.

 Then a sterile oil is added to a height of about 4mm.

 The tubes are then incubated for 4 days at a temperature of about 35 to 37ºC in an ambient air.

 The tubes are then observed for color change for 24, 48, 72 and 96 hours.

Decarboxylase Test Results

 A positive test is found out by change in color of the medium from turbid purple to faded out yellow purple color.

 A negative test is detected by a bright yellow color or no change in color.

 The control tube must retain, its original color or sometimes it turns yellow. If turbidity or alkalinity of the purple color is seen in the control tube, then the test is considered as invalid one.