How to Perform a Decarboxylase Test: Lysine, Ornithine, and Arginine

The decarboxylase test is a biochemical method used to detect the ability of bacteria to decarboxylate specific amino acids—lysine, ornithine, or arginine—through the action of amino acid decarboxylases or, in the case of arginine, the arginine dihydrolase pathway. This test is performed using Moeller decarboxylase broth, a differential medium containing a pH indicator (bromocresol purple) and a specific amino acid substrate. The test is useful for differentiating members of the Enterobacteriaceae family and other Gram-negative bacilli, as well as certain Gram-positive bacteria, based on their metabolic capabilities. A positive result is indicated by a color change from yellow to purple due to the production of alkaline amines, while a negative result remains yellow. An oil overlay is applied to create anaerobic conditions, which are required for decarboxylase enzyme activity. This protocol is designed for routine BSL-1 teaching laboratory settings and does not cover clinical or pathogenic applications.

At a Glance

Scientific Principle

The decarboxylase test relies on the enzymatic activity of amino acid decarboxylases, which are inducible enzymes produced by bacteria under acidic conditions and in the presence of the specific amino acid substrate. These enzymes remove the carboxyl group (-COOH) from the amino acid, producing a corresponding amine and carbon dioxide. For the three amino acids tested:

• Lysine decarboxylase converts lysine to cadaverine
• Ornithine decarboxylase converts ornithine to putrescine
• Arginine dihydrolase (not a true decarboxylase) converts arginine to citrulline, then to ornithine, which is subsequently decarboxylated to putrescine

The production of these alkaline amines raises the pH of the medium, causing the bromocresol purple indicator to change from yellow (acidic, pH below 5.2) to purple (alkaline, pH above 6.8). The initial acidification of the medium (from glucose fermentation) is necessary to induce decarboxylase enzyme production. The oil overlay creates anaerobic conditions that favor decarboxylase activity over oxidative deamination.

The biological significance of these pathways extends beyond laboratory identification. For instance, ornithine decarboxylase-mediated putrescine biosynthesis is essential for cell envelope integrity in certain bacteria, such as Fusobacterium nucleatum, where putrescine depletion leads to membrane disruption and cell lysis under hypoosmotic conditions [1]. In eukaryotic cells, putrescine transport via SLC45A4 facilitates GABA synthesis through the arginine/ornithine/putrescine pathway, highlighting the broader metabolic importance of these decarboxylation reactions [2]. Additionally, ornithine decarboxylase turnover is regulated by cullin-RING E3 ubiquitin ligases in trypanosomes, and this enzyme is a target of the trypanocide eflornithine [3].

Materials and Instrumentation

Moeller Decarboxylase Broth

Moeller decarboxylase broth is the standard medium for this test. It contains:

• Peptone and beef extract (nutrient base)
• Glucose (0.5%) as a fermentable carbohydrate
• Bromocresol purple (pH indicator)
• Pyridoxal (vitamin B6), which serves as a cofactor for decarboxylase enzymes
• The specific amino acid substrate (1% L-lysine, L-ornithine, or L-arginine)

The broth is typically prepared as a base medium without amino acids, which is then supplemented with the desired amino acid. A control broth without any amino acid should always be included to detect non-specific alkaline reactions.

Amino Acid Substrates

• L-lysine monohydrochloride: For lysine decarboxylase test
• L-ornithine monohydrochloride: For ornithine decarboxylase test
• L-arginine monohydrochloride: For arginine dihydrolase test

These substrates are added to the base medium at a final concentration of 1% (w/v). The pH is adjusted to 6.0–6.5 before sterilization.

Mineral Oil

Sterile mineral oil (light paraffin oil) is used to create an anaerobic environment. The oil is layered approximately 1 cm deep over the inoculated broth.

Inoculation Equipment

• Sterile inoculating loops or needles
• Sterile Pasteur pipettes or syringes for oil overlay
• Test tubes (13 × 100 mm or similar) with loose-fitting caps

Incubation Equipment

• Incubator set to 35–37°C
• Test tube rack

Quality Control Strains

Appropriate control strains are essential for validating test performance. Recommended strains include:

Controls

Positive Control

Inoculate a tube of Moeller decarboxylase broth containing the appropriate amino acid with a known positive control strain. For lysine decarboxylase, use E. coli or K. pneumoniae. For ornithine decarboxylase, use E. coli or E. cloacae. For arginine dihydrolase, use E. cloacae.

Negative Control

Inoculate a tube of Moeller decarboxylase broth containing the appropriate amino acid with a known negative control strain. For all three tests, P. mirabilis serves as a reliable negative control.

Uninoculated Control

Include an uninoculated tube of each amino acid broth to monitor for sterility and to provide a color reference for the initial pH.

Base Medium Control (No Amino Acid)

For each test organism, inoculate a tube of Moeller decarboxylase base broth without any amino acid. This control is critical because it demonstrates that any alkaline reaction observed is specifically due to decarboxylation of the added amino acid, not to non-specific alkalinization from peptone metabolism. The base medium control should remain yellow (acidic) throughout incubation.

Conceptual Workflow

Step 1: Prepare the Medium

1. Prepare Moeller decarboxylase base broth according to manufacturer instructions or standard formulation.
2. Dispense the base broth into test tubes (approximately 3–4 mL per tube).
3. Add the appropriate amino acid substrate to each tube (except the base medium control) to achieve a final concentration of 1%.
4. Adjust pH to 6.0–6.5 using 1N NaOH or 1N HCl.
5. Sterilize by autoclaving at 121°C for 15 minutes.
6. Allow tubes to cool to room temperature before use.

Step 2: Prepare the Inoculum

1. Select a pure, well-isolated colony from an 18–24 hour culture grown on a non-selective medium (e.g., Tryptic Soy Agar).
2. Emulsify the colony in 2–3 mL of sterile saline or nutrient broth to create a light suspension (approximately 0.5 McFarland standard).
3. Use this suspension within 30 minutes of preparation.

Step 3: Inoculate the Tubes

1. Using a sterile pipette, transfer 0.1–0.2 mL of the bacterial suspension into each test tube:
   • One tube with lysine
   • One tube with ornithine
   • One tube with arginine
   • One tube with base medium only (no amino acid)
2. Gently mix the tubes to distribute the inoculum.
3. Using a sterile Pasteur pipette or syringe, carefully layer approximately 1 mL of sterile mineral oil over the surface of each broth. The oil layer should be about 1 cm deep to effectively seal the medium from atmospheric oxygen.

Step 4: Incubate

1. Place the tubes in a test tube rack and incubate at 35–37°C.
2. Examine the tubes daily for up to 4 days.
3. Record the color of each tube at each reading.

Step 5: Interpret Results

1. Positive result: The broth turns purple (alkaline) due to amine production.
2. Negative result: The broth remains yellow (acidic) throughout incubation.
3. Delayed positive: Some organisms may require 2–4 days to produce a visible color change.
4. Base medium control: Should remain yellow throughout incubation.

Quality Checks

Pre-Test Quality Control

• Verify that the pH of the prepared medium is 6.0–6.5 before use.
• Confirm that the bromocresol purple indicator is functional by checking the color of uninoculated tubes (should be purple at the initial pH).
• Ensure that control strains are viable and pure.
• Verify that the mineral oil is sterile and free of contaminants.

During-Test Quality Control

• Monitor control strains daily. Positive controls should show a color change within 24–48 hours.
• Negative controls should remain yellow throughout the incubation period.
• The base medium control should remain yellow, confirming that any alkaline reaction in the amino acid-containing tubes is substrate-specific.

Post-Test Quality Control

• Record all results, including the time to color change.
• If positive controls fail to produce a color change, investigate possible causes (e.g., expired medium, incorrect pH, inactive inoculum).
• If negative controls show a color change, consider contamination or non-specific alkalinization.

Result Interpretation

Color Changes

Organism-Specific Patterns

The decarboxylase test is particularly useful for differentiating members of the Enterobacteriaceae family:

• Escherichia coli: Lysine (+), Ornithine (+), Arginine (-)
• Klebsiella pneumoniae: Lysine (+), Ornithine (-), Arginine (-)
• Enterobacter cloacae: Lysine (-), Ornithine (+), Arginine (+)
• Proteus mirabilis: Lysine (-), Ornithine (-), Arginine (-)
• Salmonella enterica serovar Typhi: Lysine (+), Ornithine (+), Arginine (-)
• Shigella sonnei: Lysine (-), Ornithine (+), Arginine (-)

Troubleshooting

Limitations

False Positives

• Non-specific alkalinization can occur if the base medium control turns purple, indicating that the organism is producing alkaline byproducts from peptone metabolism rather than from specific amino acid decarboxylation.
• Contamination with other bacteria can produce misleading color changes.
• Some organisms may produce weak alkaline reactions that are difficult to distinguish from true positives.

False Negatives

• Insufficient inoculum may delay or prevent enzyme induction.
• Incorrect pH of the medium (too alkaline) may prevent initial acidification required for enzyme induction.
• Expired or improperly stored medium may lack essential cofactors (pyridoxal).
• Some organisms require extended incubation (up to 4 days) to produce detectable color changes.
• The oil overlay may be insufficient to create anaerobic conditions if not applied correctly.

Organism-Specific Considerations

• Some bacteria may decarboxylate multiple amino acids, requiring a full panel of tests for accurate identification.
• The arginine dihydrolase test detects a two-step pathway (arginine → citrulline → ornithine → putrescine), not a single decarboxylation event. Results should be interpreted with this in mind.
• Certain Gram-positive bacteria (e.g., Enterococcus faecalis) may also show decarboxylase activity, but the test is primarily validated for Gram-negative organisms.

Test Limitations

• The decarboxylase test is a phenotypic method and may not correlate perfectly with genotypic identification.
• Results should be interpreted in conjunction with other biochemical tests (e.g., lysine iron agar, carbohydrate fermentation tests) for accurate organism identification.
• The test does not distinguish between constitutive and inducible decarboxylase activity.

Documentation

Record Keeping

For each decarboxylase test performed, document the following:

1. Organism identification: Source and strain designation
2. Date and time of inoculation
3. Medium lot number and expiration date
4. Control strains used and their results
5. Daily observations: Color of each tube (lysine, ornithine, arginine, base control)
6. Final interpretation: Positive or negative for each amino acid
7. Technician initials

Example Documentation Table

Reporting Results

Results should be reported as positive (+) or negative (-) for each amino acid tested. If the base medium control shows a color change, the test should be considered invalid and repeated.

Biosafety

BSL-1 Considerations

This protocol is designed for use with non-pathogenic teaching strains (e.g., E. coli K-12, K. pneumoniae ATCC 13883) under BSL-1 conditions. All work should be performed in a laboratory with standard microbiological practices as outlined in the Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition [4].

Standard Precautions

• Wear appropriate personal protective equipment (lab coat, gloves, safety glasses).
• Perform all work on a disinfected bench surface.
• Use aseptic technique to prevent contamination.
• Decontaminate all waste (including used tubes and oil) by autoclaving before disposal.
• Wash hands thoroughly after handling cultures.

Waste Disposal

• Used test tubes and oil should be autoclaved at 121°C for 30 minutes before disposal.
• Liquid waste (including used oil) should be treated with an appropriate disinfectant or autoclaved.
• Solid waste (gloves, pipette tips) should be disposed of in biohazard waste containers.

Special Considerations

• The mineral oil used for the overlay is flammable. Keep away from open flames and heat sources.
• If working with unknown or potentially pathogenic organisms, consult your institution's biosafety officer and follow appropriate BSL-2 practices.

Frequently Asked Questions

1. Why is an oil overlay necessary for the decarboxylase test?

The oil overlay creates anaerobic conditions by preventing atmospheric oxygen from diffusing into the broth. Decarboxylase enzymes are most active under anaerobic conditions, and the initial acidification of the medium (from glucose fermentation) is required to induce enzyme production. Without the oil overlay, oxidative deamination of amino acids could occur, leading to false-negative results or non-specific alkaline reactions. The oil layer should be approximately 1 cm deep to effectively seal the medium.

2. Can the decarboxylase test be used for Gram-positive bacteria?

While the decarboxylase test is primarily validated for Gram-negative enteric bacteria, some Gram-positive organisms (e.g., Enterococcus faecalis, Lactobacillus spp.) may also show decarboxylase activity. However, the test conditions (pH, incubation time, medium composition) are optimized for Gram-negative bacteria. For Gram-positive organisms, alternative methods or modified protocols may be more appropriate. Always include appropriate control strains when testing non-enteric organisms.

3. What should I do if the base medium control turns purple?

If the base medium control (without amino acid) turns purple, the test is invalid because it indicates non-specific alkalinization from peptone metabolism rather than specific amino acid decarboxylation. This can occur with organisms that produce alkaline byproducts (e.g., ammonia) from peptone. Repeat the test with fresh medium and ensure that the base medium control is included. If the problem persists, consider using a different medium formulation or reducing the inoculum size.

4. How long should I incubate the decarboxylase test before calling it negative?

Most positive reactions occur within 24–48 hours, but some organisms (e.g., Enterobacter cloacae for arginine dihydrolase) may require up to 4 days. The standard recommendation is to incubate for up to 4 days, reading daily. If no color change is observed after 4 days, the test can be considered negative. However, if the organism shows poor growth in the medium, consider extending incubation to 5–7 days or confirming the result with an alternative method.

References and Further Reading

1. Xu S, G C B, Phan A, Wu C. The gene encoding ornithine decarboxylase for putrescine biosynthesis is essential for the viability of Fusobacterium nucleatum. 2026. PubMed ID: 41347511. PubMed

2. Colson C, Wang Y, Atherton J, Dahiya NR, Kharaghani D, Su X. SLC45A4 encodes a peroxisomal putrescine transporter that promotes GABA de novo synthesis. 2025. PubMed ID: 41266324. PubMed

3. Del Pino RC, Zoltner M, Butterfield ER, Field MC. Evolution, composition and functions of cullin E3 ubiquitin ligases in trypanosomes. 2025. PubMed ID: 41413177. PubMed

4. CDC and NIH. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. U.S. Department of Health and Human Services, 2020. CDC

5. National Institutes of Health. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. NIH Office of Science Policy. NIH

6. National Center for Biotechnology Information. NCBI Bookshelf: Molecular Biology and Laboratory Methods. NCBI

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