Decarboxylase Test- Principle, Procedure, Results, Uses (2022)

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.

(Video) Decarboxylase test results

  • 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:
S.NIngredients Gram/liter
1.Peptic digest of animal tissue5.0
2.Yeast extract0.3
3.Dextrose1.0
4.Bromo cresol purple0.1
5.Cresol red0.005
6.Pyridoxal0.005
Final pH at 25°C: 6.7 ±0.2

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

ControlColour of the control tubeResults
ArginineLysineOrnithine
Klebsiella pneumoniaeYellow+
Enterobacter cloacaeYellow++

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.

Decarboxylase Test- Principle, Procedure, Results, Uses (1)

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

(Video) BIO 2192 Unit 10 Lysine Decarboxylase Broth

Test organismResults
Lysine decarboxylationArginine decarboxylationOrnithine decarboxylation
Enterobacter aerogenesPositive reaction; change in color to purpleNegative reaction; yellow-colored brothPositive reaction; change in color to purple
Escherichia coliVariableVariableVariable
Klebsiella pneumoniaePositive reaction; change in color to purpleNegative reaction; yellow-colored brothNegative reaction; yellow-colored broth
Proteus vulgarisNegative reaction; yellow-colored brothNegative reaction; yellow-colored brothNegative reaction; yellow-colored broth
Pseudomonas aeruginosa Negative reaction; yellow-colored brothPositive reaction; change in color to purpleNegative reaction; yellow-colored broth
Salmonella TyphiPositive reaction; change in color to purpleDelayed positive reaction or negative reactionNegative reaction; yellow-colored broth
Serratia marcescensPositive reaction; change in color to purpleNegative reaction; yellow-colored brothPositive reaction; change in color to purple
Shigella flexneriNegative reaction; yellow-colored brothThe delayed positive or negative reactionNegative reaction; yellow-colored broth
Vibrio cholerae Negative reaction; yellow-colored brothPositive reaction; change in color to purplePositive reaction; change in color to purple

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 (-).

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.
  • 3% – http://universe84a.com/decarboxylase-test/
  • 2% – https://microbenotes.com/decarboxylase-test-principle-procedure-and-result-interpretation/
  • 1% – https://www.coursehero.com/file/p64senn/II-MICROORGANISMS-TESTED-A-Enteric-gram-negative-rods-and-Vibrio-Plesiomonas/
  • 1% – https://www.austincc.edu/microbugz/decarboxylation_test.php
  • 1% – https://bio.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_I/40%3A__Decarboxylation_of_Amino_Acids
  • 1% – https://assets.thermofisher.com/TFS-Assets/MBD/Instructions/IFU60750.pdf
  • 1% – http://www.jbc.org/content/241/10/2228.full.pdf
  • <1% – https://www.sigmaaldrich.com/catalog/product/sial/53286
  • <1% – https://www.sciencedirect.com/science/article/pii/S246801251930001X
  • <1% – https://www.sciencedirect.com/science/article/pii/S0015736868704473
  • <1% – https://www.chegg.com/homework-help/questions-and-answers/think-effect-mineral-oil-affect-specificity-test-explain–would-omitting-sterile-mineral-o-q25396919
  • <1% – https://stattrek.com/statistics/dictionary.aspx?definition=Interquartile%20range
  • <1% – https://quizlet.com/135005841/micro-lab-exam-3-flash-cards/
  • <1% – https://quizlet.com/1340262/lysine-and-ornithine-decarboxylase-flash-cards/
  • <1% – https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/423601/ID_16i4.pdf
  • <1% – http://www.jbc.org/content/145/1/131.full.pdf
  • <1% – http://shodhganga.inflibnet.ac.in/bitstream/10603/12640/11/11_appendix.pdf
(Video) Decarboxylase test by Dr. V
(Video) Amino Acid Decarboxylation Test | Microbiology Lab

FAQs

What is the principle of decarboxylase test? ›

The test thus measures the enzymatic ability (decarboxylase) of an organism to decarboxylate (or hydrolyze) an amino acid to form an amine. Decarboxylation, or hydrolysis, of the amino acid results in an alkaline pH.

What is the purpose of lysine decarboxylase test? ›

What is the purpose of the test? The purpose is to see if the microbe can use the amino acid lysine as a source of carbon and energy for growth. Use of lysine is accomplished by the enzyme lysine decarboxylase.

What color indicates a positive decarboxylation test what causes this color change? ›

A positive decarboxylase result is indicated by the development of a purple to pale yellow-purple color. A negative decarboxylase result is indicated by the development of a bright yellow color for glucose-fermenting microorganisms.

What is a decarboxylase enzyme? ›

Decarboxylases are a group of enzymes that remove carboxyl groups (CO2H) from acidic substrates and require pyridoxal phosphate or pyruvate as a co-factor. They are known for their various roles in metabolic pathways, non-oxidative decarboxylation of α- and β-keto acids and carbohydrate synthesis.

What is the principle of citrate test? ›

The citrate test screens a bacterial isolate for the ability to utilize citrate as its carbon and energy source (3, 7). A positive diagnostic test rests on the generation of alkaline by-products of citrate metabolism. The subsequent increase in the pH of the medium is demonstrated by the color change of a pH indicator.

How does lysine decarboxylase media work? ›

Lysine decarboxylase (CadA) removes carbon dioxide from lysine, thus producing cadaverine, an amine which protonates to generate OH−.

What is the purpose of the mineral oil in the decarboxylation test? ›

Sterile mineral oil creates an anaerobic environment for fermentation to occur and thus the low pH induces the decarboxylase positive MO to produce the enzyme.

What is the basic principle of LDC? ›

Principle and Interpretation:

Lysine decarboxylase test is based on the ability of some bacteria to cleave L-lysine to cadaverine under the liberation of carbon dioxide (1). Decarboxylase media were first described by Moeller (2-4) for detecting lysine and ornithine decarboxylase and arginine dihydrolase.

What does a positive and negative reaction for decarboxylase test mean? ›

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.

What color indicates a positive decarboxylation test what causes this color change quizlet? ›

What color indicates a positive decarboxylation test? What causes this color change? yellow, it means the organism is a glucose fermenter but does not produce the appropriate decarboxylation.

Why must the control tube in the decarboxylase test be yellow in order for the results to be accurate? ›

7. Why does the decarboxylase-positive tube have to turn yellow before it can turn purple? a low pH is needed for the bacteria to work. Fermentation of sugar causes acid, lowers the pH and turns the pH indicator from purple to yellow.

What is the function of decarboxylation? ›

Decarboxylation, the removal of carbon dioxide from organic acids, is a fundamentally important reaction in biology. Numerous decarboxylase enzymes serve as key components of aerobic and anaerobic carbohydrate metabolism and amino acid conversion.

What enzyme causes decarboxylation? ›

The key decarboxylation enzyme is PEP carboxykinase, which is found not only in the bundle sheath cytoplasm, but BSC mitochondria also have NAD-malic enzyme activity.

What do you mean by decarboxylation reaction? ›

Decarboxylation reaction is defined as a chemical reaction that eliminates a carboxyl group and liberates carbon dioxide (CO2). Decarboxylation mostly refers to a reaction of carboxylic acids erasing a carbon atom from a chain of carbons.

What is the principle of oxidase test? ›

Principle of Oxidase Test

Cytochrome containing organisms produce an intracellular oxidase enzyme. This oxidase enzyme catalyzes the oxidation of cytochrome c. Organisms which contain cytochrome c as part of their respiratory chain are oxidase-positive and turn the reagent blue/purple.

Which medium is used for citrate utilization test? ›

Citrate agar is used to test an organism's ability to utilize citrate as a source of energy. The medium contains citrate as the sole carbon source and inorganic ammonium salts (NH4H2PO4) as the sole source of nitrogen.

What is the principle of indole test? ›

Principle of Indole Test

Indole is generated by reductive deamination from tryptophan via the intermediate molecule indolepyruvic acid. Tryptophanase catalyzes the deamination reaction, during which the amine (-NH2) group of the tryptophan molecule is removed.

What is amino acid decarboxylation? ›

Decarboxylation is the reduction of carbon, while transamination is the exchange within the amino group of an amino acid to a keto acid (the introduction or removal of nitrogen).

What is the principle of methyl red test? ›

Principle of Methyl Red (MR) Test

The type of acid produced differs from species to species and depends on the specific enzymatic pathways present in the bacteria. The acid so produced decreases the pH to 4.5 or below, which is indicated by a change in the color of methyl red from yellow to red.

Is L lysine an amino acid? ›

Lysine, or L-lysine, is an essential amino acid, meaning it is necessary for human health, but the body cannot make it. You have to get lysine from food or supplements.

What is a reagent for decarboxylation of L ornithine to putrescine? ›

Introduction. Ornithine decarboxylase (ODC, EC 4.1. 1.17) is a pyridoxal-5′-phosphate-dependent enzyme, catalyzing the decarboxylation of L-ornithine to the diamine putrescine as the precursor of polyamine biosynthesis [1].

What does lysine decarboxylase do to lysine? ›

Lysine decarboxylase (CadA) removes carbon dioxide from lysine, thus producing cadaverine, an amine which protonates to generate OH−.

What color will the solution turn if the bacteria can decarboxylate the amino acid? ›

If the inoculated medium is yellow, or if there is no color change, the organism is decarboxylase-negative for that amino acid. If the medium turns purple, the organism is decarboxylase-positive for that amino acid.

What is LDC test microbiology? ›

A test for lysine decarboxylase (LDC) is used for the identification of Salmonella spp. as typical nontyphoidal salmonellae are positive for LDC (Farmer, 2003). The mechanism of LDC activity has been studied in Escherichia coli and is considered to depend on the cad locus.

What is the indicator for phenylalanine deaminase test? ›

What is the indicator of the Phenylalanine deaminase test? What does a positive test result look like in the Phenylalanine deaminase test? A positive result will cause the Ferric Chloride indicator to turn a green color, after it has been added to the inoculated and incubated slant.

Is ornithine an amino acid? ›

Ornithine is a chemical called an amino acid. It is made in the body. It can also be made in a laboratory.

What is the basic principle of LDC? ›

Principle and Interpretation:

Lysine decarboxylase test is based on the ability of some bacteria to cleave L-lysine to cadaverine under the liberation of carbon dioxide (1). Decarboxylase media were first described by Moeller (2-4) for detecting lysine and ornithine decarboxylase and arginine dihydrolase.

What is the principle of methyl red test? ›

Principle of Methyl Red (MR) Test

The type of acid produced differs from species to species and depends on the specific enzymatic pathways present in the bacteria. The acid so produced decreases the pH to 4.5 or below, which is indicated by a change in the color of methyl red from yellow to red.

What is the purpose of the mineral oil in the decarboxylation test? ›

Decarboxylation and dihydrolation are anaerobic reactions therefore a layer of sterile mineral oil is overlaid in each tube to separate the medium from external oxygen and promote fermentation.

What is the purpose of the phenylalanine test? ›

Serum phenylalanine screening is a blood test to look for signs of the disease phenylketonuria (PKU). The test detects abnormally high levels of an amino acid called phenylalanine.

What is amino acid decarboxylation? ›

Decarboxylation is the reduction of carbon, while transamination is the exchange within the amino group of an amino acid to a keto acid (the introduction or removal of nitrogen).

What is LDC test microbiology? ›

A test for lysine decarboxylase (LDC) is used for the identification of Salmonella spp. as typical nontyphoidal salmonellae are positive for LDC (Farmer, 2003). The mechanism of LDC activity has been studied in Escherichia coli and is considered to depend on the cad locus.

Why must the control tube in the decarboxylase test be yellow in order for the results to be accurate? ›

7. Why does the decarboxylase-positive tube have to turn yellow before it can turn purple? a low pH is needed for the bacteria to work. Fermentation of sugar causes acid, lowers the pH and turns the pH indicator from purple to yellow.

What is the principle of oxidase test? ›

Principle of Oxidase Test

Cytochrome containing organisms produce an intracellular oxidase enzyme. This oxidase enzyme catalyzes the oxidation of cytochrome c. Organisms which contain cytochrome c as part of their respiratory chain are oxidase-positive and turn the reagent blue/purple.

What is the principle of indole test? ›

Principle of Indole Test

Indole is generated by reductive deamination from tryptophan via the intermediate molecule indolepyruvic acid. Tryptophanase catalyzes the deamination reaction, during which the amine (-NH2) group of the tryptophan molecule is removed.

Why is methyl red used as an indicator? ›

Methyl red displays pH dependent photochromism, with protonation causing it to adopt a hydrazone/quinone structure. Methyl Red has a special use in histopathology for showing acidic nature of tissue and presence of organisms with acidic natured cell walls.

What do you mean by decarboxylation reaction? ›

Decarboxylation reaction is defined as a chemical reaction that eliminates a carboxyl group and liberates carbon dioxide (CO2). Decarboxylation mostly refers to a reaction of carboxylic acids erasing a carbon atom from a chain of carbons.

Why is mineral oil added to lysine decarboxylase? ›

Why must the decarboxylation medium be converted with sterile mineral oil? Sterile mineral oil creates an anaerobic environment for fermentation to occur and thus the low pH induces the decarboxylase positive MO to produce the enzyme.

What is a reagent for decarboxylation of L ornithine to putrescine? ›

Introduction. Ornithine decarboxylase (ODC, EC 4.1. 1.17) is a pyridoxal-5′-phosphate-dependent enzyme, catalyzing the decarboxylation of L-ornithine to the diamine putrescine as the precursor of polyamine biosynthesis [1].

What type of indicator is used for the phenylalanine deamination test? ›

What is the indicator of the Phenylalanine deaminase test? What does a positive test result look like in the Phenylalanine deaminase test? A positive result will cause the Ferric Chloride indicator to turn a green color, after it has been added to the inoculated and incubated slant.

Which of the following shows positive result for phenylalanine deaminase test? ›

Positive test: development of light to dark green color within 1-5 minutes after applying ferric chloride reagent. Negative test: absence of a green color reaction or yellow coloration due to the color of the ferric chloride.

What is the specific reagent used in the phenylalanine deaminase test? ›

Our Ferric Chloride Reagent is used to detect the presence of the enzyme phenylalanine deaminase produced by some bacteria. Organisms possessing the enzyme, phenylalanine deaminase, can convert phenylalanine to the α-keto acid phenylpyruvic acid.

Videos

1. BIO 2192 Unit 10 Lysine Decarboxylase Broth
(cnmbioprof)
2. Decarboxylase Test | Amino Acid Decarboxylase Test | Decarboxylase Deaminase Test |
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3. Laboratory: Decarboxylation of Amino Acids Test
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4. Urease, Citrate, Decarboxylase, PDA, BEA tests
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5. Amino Acid Decarboxylation Test
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6. Decarboxylase deaminase test (Amino acid metabolism)
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