Decarboxylase Broth Protocol

Decarboxylase Broth

Moeller introduced the decarboxylase media to detect the ability of bacteria to decarboxylate amino acid. They were used for the first time to detect lysine, ornithine, and arginine decarboxylases among Enterobacteriaceae bacteria.
Basal media for the Moeller included peptone and beef extract, bromocresol purple and cresol red, pyridoxal as well as glucose.
To measure decarboxylase activity, 1 % of the appropriate amino acids was added. After the medium it was placed in narrow tubes as a column measuring approximately 2 cm in height, and autoclaved. A layer of approximately 5 mm of sterile paraffin oil was then poured into each tube.
An indicator was used to measure the decarboxylase activity, which was measured on the basis of a pH rise of the amino acid reagent.
The tubes’ color changes were monitored for up to 10 consecutive days after incubation.
Calquist used the lysine-decarboxylase reaction to create a medium for Salmonella arizonae differentiation from Citrobacter.
Falkow created a medium(lysine decarboxylase medium) for lysine-decarboxylase differentiation to identify and differentiate Salmonella and Shigella. Falkow’s medium was extended to investigate ornithine-arginine decarboxylase reactions.
Gilardi suggested that these tests could also be used to identify non-fermentative Gram negative bacilli. The above methods required at least 4 days incubation to detect ornithine descarboxylase activity.
Fay & Barry (1972), modified the Moeller decarboxylase media by removing glucose and decreasing pH to 5.5. The test volume was also decreased to 1 ml. This modified ornithine medium was used to obtain highly reliable results. It took only 2-4 hours to incubate at 35-37o C, as opposed with the Moeller 4-day time.
This rapid ornithine decarboxylase test differentiated Klebsiella from the Enterobacter-Serratia group of organisms (K. pneumoniae cells are non-motile and ornithine decarboxylase negative, Enterobacter species are motile and usually ornithine decarboxylase positive except for Enterobacter agglomerans.
It also helped to separate Proteus species (About 93% of P. mirabilis and 100% of P. morganii strains are ornithine decarboxylase positive, whereas P. vulgaris and P. rettgeri are ornithine decarboxylase negative).
Brooker et. al. modified Fay & Barry’s medium slightly. Brooker et al. detected Lysine Decarboxylase in several members of Enterobacteriaceae.
Brooks & Sodeman (1974), a similar to Fay & Barry’s ornithine-decarboxylase media, published a method to quickly assay for lysine, arginine descarboxylase and arginine hydrolase. The rapid test of Fay & Barry was compared with the Moeller method for testing several species of Enterobacteriaceae as well as Gram-negative, nonfermenting rod-shaped bacteria. They found a close match between the two methods.
Oberhofer et al. Oberhofer et al. evaluated the rapid dihydrolase and decarboxylase tests and found them extremely sensitive and specific for differentiation of a broad range of non-fermentative bacteria.

Principle of Decarboxylase Broth

The decarboxylase test is used for determining decarboxylase production and amino acid decarboxylation in bacteria, especially in the family of Enterobacteriaceae species.
This simple test can be used to determine if bacteria is capable of producing dihydrolase or decarboxylase. These enzymes are used to remove the carboxyl group of amino acids and form amines.
Decarboxylases are used to remove the carboxyl groups from amino acids such as lysine, ornithine, while arginine decarboxylation occurs with the help of dihydrolase in addition to decarboxylase.
The Decarboxylase medium is made up of beef extract and peptic digestions of animal tissues, which provide nitrogenous nutrients to the organisms.
Medium contains glucose, a fermentable carbohydrate.
Bromocresol purple, and cresol red are pH indicators that change color under acidic or alkaline conditions. They help to detect glucose fermentation and amino acid descarboxylation.
Pyridoxal is the cofactor for decarboxylase.
To promote fermentation, decarboxylation or dihydrolation are anaerobic processes. Therefore, each tube is coated with sterile mineral oils to protect the medium from oxygen.
Inoculating medium with a bacterium that can ferment glucose produces acid, which results in a drop in pH. The pH indicator also changes the color of the medium from yellow to brown.
Decarboxylase is also stimulated by acid production.
Decarboxylase is a process that decarboxylates an added amino acid, resulting in the formation amines.
The pH indicator will change from yellow to purple due to amine formation.
The medium will remain yellow or acidic if the organism that is being tested ferments glucose but not decarboxylase does so.

Decarboxylation of lysine, ornithine, and arginine occur as follows:

Lysine decarboxylase, which yields cadaverine (Fig. 1), removes the carboxyl group from lysine.
Ornithine can be decarboxylated using ornithine’s decarboxylase, giving rise to putrescine
Arginine decarboxylation requires a combination of decarboxylase and dihydrolase to achieve the complete decarboxylation of arginine to putrescine. Arginine decarboxylase decarboxylates the amino acid arginine into agmatine (Fig. 3). Enterobacteriaceae members are capable of converting agmatine to putrescine or urea through agmatinase. Urea can be further broken down by bacteria using urease.

Moeller’s Decarboxylase Broth Composition (g/liter)

Base broth	(g/liter)
Peptone	5g
Beef Extract	5g
Glucose	0.5g
Bromocresol Purple	0.01g
Cresol Red	0.005g
Pyridoxal	0.005g
Distilled water	1L

To prepare different decarboxylase broths with one of the following three L-amino acid

Arginine	10g (1%)
Lysine	20g (2%)
Ornithine	10g (1%)

Decarboxylase Broth Preparation

Sterilized Mineral Oil for overlaying the inoculated broth 200 ml:

Protocol For Decarboxylase Broth

Inoculation

As an inoculation source, use a fresh (18-to 24-hour) culture of test bacteria.
Aseptically transfer one isolated colony to the decarboxylase broth tube along with one of the amino acids.
Innoculate a tube of control decarboxylase soup base (without any amino acid) and the tube containing decarboxylase bone broth (as a test).
Inoculate the tubes with 1 ml of sterile oil. This will separate the medium from oxygen. It will promote fermentation of glucose, decarboxylation reactions, and seal the tubes.

Incubation

Incubate tubes in an incubator at 37°C.
For color changes lasting up to four days, make sure you check the tubes each day.
After 4 days of inoculation, final readings should take place.
Some microorganisms may require an increase incubation time of up to 10 days.

Decarboxylase Test Interpretations:

For signs of fermentation or decarboxylation, check the medium’s color every day for at least 10 days. Uninoculated Moeller’s media base and media with an amino acid are light brown.

Decarboxylase negative: A medium that is not colored changes indicates that it does not ferment glucose. This is called decarboxylase negative (-).
Decarboxylase negative: If the medium changes to yellow color (but remains yellow and does not change to purple) it indicates that the organism ferments glucose and acidic by products are formed but the organism is decarboxylase negative (-) for that amino acid. The appearance of yellow broth indicates fermentation of glucose, but it is not indicative of Decarboxylation.
Decarboxylase positive: A media that changes to purple indicates that an organism has decarboxylated the amino acid and formed amine (alkaline side-products). This is called decarboxylase (+). If the media does not have a purple hue, it means that the amino acid has not yet been decarboxylated. The organism also did not produce decarboxylase enzyme.

Media color	Bacterial reaction
No Change (Media remains light brown)	Decarboxylase negative (-)
Changes to yellow but does not change to purple	Decarboxylase negative (-)
Changes to purple	Decarboxylase positive (+)