Decarboxylase Test- Principle, Procedure, Results, Uses

Decarboxylase test is a biochemical test that determines the ability of bacteria to produce decarboxylase enzymes that break down amino acids. Decarboxylase enzymes are important for the metabolism of amino acids in bacteria, and they also play a role in the production of various biogenic amines that have physiological effects on humans and animals. Decarboxylase test is mainly used to differentiate members of the Enterobacteriaceae family, which are gram-negative rods that inhabit the intestinal tract of humans and animals. Some members of this family are pathogenic and cause diseases such as typhoid fever, dysentery, and urinary tract infections. Decarboxylase test can also be used to identify other bacteria that produce decarboxylase enzymes, such as Vibrio, Plesiomonas, Aeromonas, Stenotrophomonas, Burkholderia, Pseudomonas, and Streptococcus.

The main objectives of the decarboxylase test are:

• To test the ability of an organism to produce a decarboxylase enzyme that can break down an amino acid into an amine and carbon dioxide.
• To differentiate the members of the Enterobacteriaceae family on the basis of their ability to produce decarboxylase enzyme for specific amino acids such as arginine, lysine, and ornithine.
• To identify some other bacteria that can produce decarboxylase enzyme for certain amino acids, such as Enterococcus, Pseudomonas, Aeromonas, Vibrio, and Plesiomonas.

The principle of the decarboxylase test is based on the ability of some bacteria to produce decarboxylase enzymes that catalyze the removal of a carboxyl group from an amino acid, resulting in the formation of an amine and carbon dioxide. The amine is an alkaline compound that raises the pH of the medium, causing a color change in the pH indicator.

Decarboxylase test is mainly used to test the ability of enteric Gram-negative rods to produce decarboxylase enzymes. These bacteria belong to the family Enterobacteriaceae and include genera such as Escherichia, Salmonella, Shigella, Klebsiella, Enterobacter, Serratia, Proteus, and others. The test helps to differentiate these bacteria based on their ability to decarboxylate or hydrolyze specific amino acids.

Some other bacteria that can also be tested by decarboxylase test are:

• Vibrio, Plesiomonas, and Aeromonas: These are Gram-negative rods that are associated with waterborne infections and gastroenteritis. They can be identified by their ability to decarboxylate lysine and ornithine.
• Stenotrophomonas and Burkholderia: These are Gram-negative rods that are opportunistic pathogens and can cause respiratory infections. They can be identified by their ability to decarboxylate lysine and arginine.
• Fluorescent Pseudomonas: These are Gram-negative rods that produce pigments and can cause infections in immunocompromised patients. They can be identified by their ability to decarboxylate arginine.
• Coagulase-negative staphylococci: These are Gram-positive cocci that are part of the normal flora of the skin and mucous membranes. They can cause infections in patients with indwelling devices or prosthetic implants. They can be identified by their ability to decarboxylate ornithine.
• Viridans group streptococci: These are Gram-positive cocci that are part of the normal flora of the oral cavity and upper respiratory tract. They can cause dental caries, endocarditis, and abscesses. They can be identified by their ability to decarboxylate arginine.
• Miscellaneous non-glucose-fermenting Gram-negative rods: These are Gram-negative rods that do not ferment glucose and have diverse metabolic characteristics. They can cause infections in various sites of the body. They can be identified by their ability to decarboxylate arginine.
• Spreading indole-negative Proteus: These are Gram-negative rods that produce a characteristic swarming motility on agar plates. They can cause urinary tract infections, wound infections, and septicemia. They can be identified by their ability to decarboxylate ornithine.

Media

The media used for the decarboxylase test are based on Moeller`s formula, which contains meat peptones and beef extract as sources of nitrogen, glucose as a fermentable carbohydrate, pyridoxal as an enzyme cofactor, and bromcresol purple and cresol red as pH indicators. The media are supplemented with one of the three amino acids: arginine, lysine, or ornithine. The media are dispensed in screw-capped tubes and sterilized by autoclaving. Before inoculation, a layer of mineral oil or liquid paraffin is added to create an anaerobic environment.

Some alternative media that can also be used for the decarboxylase test are:

• Motility-indole-ornithine medium (MIO): This is a semi-solid medium that contains peptone, glucose, ornithine, bromcresol purple, and agar. It can be used to test for ornithine decarboxylase as well as motility and indole production.
• Lysine iron agar (LIA): This is a solid medium that contains peptone, glucose, lysine, ferric ammonium citrate, sodium thiosulfate, and phenol red. It can be used to test for lysine decarboxylase as well as hydrogen sulfide production and lysine deamination.

Reagents

The only reagent required for the decarboxylase test is mineral oil or liquid paraffin, which is used to seal the surface of the inoculated media and prevent oxygen diffusion. The reagent should be sterile and maintained at 56°C in liquid form.

Supplies

The supplies needed for the decarboxylase test are:

• Sterile sticks or inoculating loops
• Incubator at 35°C
• Brain heart infusion broth
• 18-24 hour pure culture of the test organism
• Control organisms (optional)

The procedure of the decarboxylase test varies depending on whether the organism is a glucose-fermenter or a glucose-nonfermenter. The following steps describe the general procedure for both types of organisms:

1. Prepare the decarboxylase media by adding the appropriate amino acid (arginine, lysine, or ornithine) to the basal medium. Sterilize the media by autoclaving and dispense about 3-4 ml into screw-capped tubes. Label the tubes according to the amino acid added. For glucose-nonfermenting organisms, prepare a control tube without any amino acid.
2. Inoculate each tube with a drop of 18-24 hour brain heart infusion broth culture of the test organism. For glucose-nonfermenting organisms, use a suspension of the organism in brain-heart infusion broth that is equivalent to McFarland No. 5 turbidity standard.
3. Add a 4 mm layer of sterile mineral oil to each tube to create an anaerobic environment. Screw the caps tightly and incubate the tubes at 35-37°C for 4 days in ambient air.
4. Observe the tubes for color change at 24, 48, 72, and 96 hours. Compare the results with the control tube if present.

The procedure of the decarboxylase test is simple and straightforward, but it requires careful attention to details such as adding mineral oil, sealing the tubes properly, and reading the results at the correct time intervals. The following section will explain how to interpret the results of the decarboxylase test.

The result of the decarboxylase test is based on the color change of the medium after incubation. The color change is due to the pH indicator system of bromcresol purple and cresol red, which are sensitive to the production of alkaline amines from the decarboxylation or hydrolysis of amino acids.

A positive test is indicated by a turbid purple to faded-out yellow-purple color in the medium. This means that the organism has produced decarboxylase enzyme and has decarboxylated or hydrolyzed the amino acid, resulting in alkaline end products (amines) that raise the pH of the medium. The purple color may be more intense at the bottom of the tube where anaerobic conditions are more favorable for decarboxylation. A positive test can be confirmed by adding a drop of 10% ferric chloride solution to the medium, which will produce a dark green color in the presence of amines.

A negative test is indicated by a bright clear yellow color in the medium. This means that the organism has not produced decarboxylase enzyme and has not decarboxylated or hydrolyzed the amino acid, resulting in acidic end products (organic acids) that lower the pH of the medium. A negative test can be confirmed by adding a drop of 40% sodium hydroxide solution to the medium, which will produce no color change in the absence of amines.

A no change in color or a purple color in the control tube (without amino acid) indicates that the organism does not ferment glucose and does not produce decarboxylase enzyme. This means that the organism is neither acidogenic nor alkaligenic and has no effect on the pH of the medium. A no change can be confirmed by adding a drop of 10% ferric chloride solution to the medium, which will produce no color change in the absence of amines.

The result interpretation should be done after 18 to 24 hours of incubation for glucose-fermenting organisms and after 4 days for glucose-nonfermenting organisms. The control tube must retain its original color or turn yellow for valid results. If the control tube turns purple, it indicates that there is an error in the preparation or inoculation of the medium, and the test should be repeated.

The table below summarizes some examples of decarboxylase test results for different microorganisms:

• Decarboxylase test is used to differentiate the members of the Enterobacteriaceae family with closely related physiological characteristics. Enterobacteriaceae are a large group of gram-negative bacteria that inhabit various environments and cause various infections in humans and animals. The ability to produce decarboxylase enzymes is one of the biochemical characteristics that can help to identify and classify these bacteria.
• Arginine decarboxylase is useful in the identification of Enterococcus to the species level. Enterococcus are gram-positive cocci that are part of the normal flora of the gastrointestinal tract, but can also cause opportunistic infections such as endocarditis, urinary tract infections, and bacteremia. Enterococcus faecalis and Enterococcus faecium are arginine positive, but Enterococcus avium is arginine negative.
• Lysine decarboxylase is used to differentiate between Salmonella and Shigella. Salmonella and Shigella are two genera of gram-negative rods that cause gastroenteritis and dysentery in humans and animals. Salmonella are lysine positive, but Shigella are lysine negative.
• Ornithine decarboxylase is used to differentiate between Proteus and other indole-negative enteric bacteria. Proteus are gram-negative rods that are part of the normal flora of the intestinal tract, but can also cause urinary tract infections, wound infections, and septicemia. Proteus are ornithine positive, but other indole-negative enteric bacteria such as Morganella, Providencia, and Edwardsiella are ornithine negative.
• Decarboxylase test can also be used to identify other bacteria such as Vibrio, Plesiomonas, Aeromonas, Stenotrophomonas, Burkholderia, Pseudomonas, and viridans group streptococci based on their ability to produce decarboxylase enzymes for different amino acids.

• The decarboxylase test has some limitations that should be considered before performing and interpreting the results. Some of these limitations are:
• The test requires a strict anaerobic environment for the decarboxylation reaction to occur. Therefore, it is essential to seal the tubes with mineral oil or paraffin to prevent oxygen from entering the medium. If the tubes are not sealed properly, false-negative results may occur due to the lack of decarboxylase activity.
• The test also requires an acidic pH for the induction of decarboxylase enzymes. Therefore, it is important to inoculate the tubes with a sufficient amount of glucose-fermenting organisms that can lower the pH by producing acids. If the inoculum is too small or the organisms are not glucose-fermenters, false-negative results may occur due to the absence of decarboxylase induction.
• The test should not be read before 18 to 24 hours of incubation, as glucose fermentation may not be completed by then. Reading the results too early may lead to false-positive results due to the presence of residual glucose in the medium. Similarly, reading the results too late may lead to false-negative results due to the exhaustion of glucose and amino acids in the medium.
• The test should be performed with pure cultures of organisms, as mixed cultures may produce conflicting results due to the presence of different decarboxylase enzymes. For example, if a culture contains both lysine-positive and lysine-negative organisms, the medium may turn purple due to the production of cadaverine by the lysine-positive organisms, masking the negative reaction of the lysine-negative organisms.
• The test should be performed with fresh cultures of organisms, as old cultures may lose their decarboxylase activity due to genetic or environmental factors. For example, some strains of Proteus may lose their ornithine decarboxylase activity after prolonged storage or subculturing.
• The test should be interpreted carefully by comparing the color change in the inoculated tubes with that in the control tube. A positive result is indicated by a purple color that is darker than or similar to the original color of the medium. A negative result is indicated by a yellow color that is brighter than or similar to the control tube. A gray color may indicate a reduction of the indicator rather than an alkaline shift and should be confirmed by adding more bromcresol purple.
• The test should be performed with specific media and reagents that are designed for detecting decarboxylase activity. Different media and reagents may have different compositions and pH indicators that may affect the results. For example, some media may contain other amino acids or carbohydrates that may interfere with the decarboxylation reaction or cause false-positive or false-negative results.
• The test should be performed with appropriate control organisms that can verify the quality and performance of the media and reagents. Positive control organisms should produce a clear positive result in all three amino acid media, while negative control organisms should produce a clear negative result in all three amino acid media. If the control organisms do not show the expected results, the test should be repeated with fresh media and reagents.

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