Decarboxylase Test: Principle and Biochemical Basis
The Decarboxylase Test is a critical biochemical assay utilized primarily in clinical microbiology to differentiate bacteria, especially members of the *Enterobacteriaceae* family and *Pseudomonas* species, based on their ability to produce certain amino acid decarboxylase enzymes. Decarboxylases are substrate-specific, inducible enzymes that catalyze the removal of the carboxyl (-COOH) group from the amino acid molecule. This reaction, known as decarboxylation, results in the formation of an alkaline-reacting product: a corresponding amine, alongside carbon dioxide (CO2). The enzymatic production of these amines, such as cadaverine or putrescine, raises the pH of the medium, which is detected via a color change of the embedded pH indicators.
The fundamental principle relies on a two-step process. First, the test medium contains a small, modest amount of fermentable carbohydrate, typically dextrose (glucose). When inoculated with a glucose-fermenting bacterium, the resulting production of organic acids causes a rapid decrease in the pH of the medium, typically below pH 5.2. This acidification serves a dual purpose: it causes a color change in the pH indicator (e.g., bromcresol purple) from its initial purple color to yellow, and, critically, this acidic environment is necessary to induce or activate the organism’s production of the specific decarboxylase enzyme.
In the second step, if the organism possesses the necessary decarboxylase, the enzyme is activated and begins to attack the amino acid present in the medium. The removal of the carboxyl group generates a highly alkaline amine. This alkaline product neutralizes the previously formed acids from glucose fermentation, causing the pH of the medium to rise and revert from yellow back toward its original alkaline color, which is typically a turbid purple or deep violet. Organisms lacking the specific enzyme will not produce the alkaline amine, and the medium will remain yellow due to the stable acidic products of glucose fermentation.
Key Components of the Decarboxylase Medium
The basal medium used for the decarboxylase test, often based on Moeller’s formulation, is a nutrient broth designed to support bacterial growth while facilitating the two-step color change. Its composition includes essential ingredients that regulate the reaction environment.
First, **Meat Peptone and Beef Extract** supply the necessary nitrogenous nutrients for the test organism’s growth. Second, **Dextrose (Glucose)** acts as the fermentable carbohydrate, providing the initial energy source and, more importantly, the acidic environment required to induce the decarboxylase enzyme. Third, **pH Indicators** such as bromcresol purple and cresol red are present to visually track the pH changes; bromcresol purple is purple at neutral or alkaline pH and yellow at an acidic pH. Finally, **Pyridoxal (Pyridoxal-5-Phosphate)** is included as an essential enzyme co-factor, which significantly enhances the decarboxylase enzyme’s activity.
The specific amino acid being tested (L-lysine, L-ornithine, or L-arginine) is added to the basal medium at a final concentration, typically 0.5% or 1%. A crucial procedural step involves adding a layer of sterile mineral oil to the surface of the inoculated medium. This oil overlay prevents the entry of oxygen and creates an anaerobic or strictly fermentative environment. This anaerobic condition is vital because it locks in the acidic products of glucose fermentation and prevents false-positive results that could arise from oxidative deamination, which also produces alkaline byproducts on the surface.
Amino Acids and Their Products
Three amino acids are routinely tested due to their significance in bacterial identification:
– **Lysine Decarboxylase (LDC):** Decarboxylation of lysine produces the diamine **Cadaverine**.
– **Ornithine Decarboxylase (ODC):** Decarboxylation of ornithine produces the diamine **Putrescine**.
– **Arginine Decarboxylase / Dihydrolase Test (ADH):** The process for arginine is more complex and proceeds through the Arginine Dihydrolase pathway. Arginine is first hydrolyzed to citrulline via a dihydrolase reaction, which is then converted to ornithine, and finally, ornithine is decarboxylated to form **Putrescine**. The overall effect is a significant increase in alkalinity.
Procedure and Interpretation of Results
The standard procedure involves inoculating four tubes per test organism: a base control medium (containing glucose and indicators but no amino acid) and three test media tubes, each containing one of the specific amino acids (Lysine, Ornithine, and Arginine). Inoculation is performed from a pure, overnight culture. After inoculation, a 4-mm layer of sterile mineral oil is immediately added to each tube to ensure an anaerobic environment. The tubes are then incubated at 35-37°C and checked daily for up to four days.
Interpretation of the test is based on the color change relative to the control tube:
– **Control Tube:** The control tube must turn bright clear yellow due to glucose fermentation. If the control remains purple or turns purple/alkaline, the test is invalid, suggesting the organism cannot ferment glucose or has an issue with the medium integrity.
– **Positive Decarboxylase Test:** A positive reaction is indicated by the color of the medium reverting from yellow to a turbid **purple** or pale yellow-purple. This signifies that the organism fermented the glucose to acid (initial yellow), which induced the decarboxylase enzyme, and the subsequent production of alkaline amines (cadaverine, putrescine) raised the pH back into the alkaline range.
– **Negative Decarboxylase Test:** A negative reaction is indicated by the medium remaining a bright, clear **yellow** color. This means the organism fermented glucose to acid, but it lacks the specific decarboxylase enzyme, so no alkaline amines were produced to neutralize the acid. The yellow color, therefore, reflects a negative result for decarboxylation.
Uses and Clinical Significance
The Decarboxylase Test is an essential tool in the identification flow chart for the Gram-negative enteric bacteria (*Enterobacteriaceae*). It allows for the rapid and reliable differentiation of closely related species with similar morphological and physiological characteristics.
For instance, the **Lysine Decarboxylase Test (LDC)** is highly valuable in distinguishing between certain clinically important pathogens: most *Salmonella* species are LDC-positive, whereas *Shigella* species are typically LDC-negative. The **Ornithine Decarboxylase Test (ODC)** helps to differentiate between members of the *Klebsiella-Enterobacter-Serratia* group, as many *Enterobacter* and *Serratia* species are ODC-positive while *Klebsiella pneumoniae* is often ODC-negative. The **Arginine Dihydrolase (ADH)** reaction is used to help identify many *Pseudomonas* species and further differentiate enteric bacteria. In summary, the pattern of positive and negative results across these three tests forms a distinctive biochemical fingerprint critical for accurate bacterial identification in diagnostic laboratories.