Biochemical testing in microbiology is founded on observing the specific metabolic capabilities of bacteria, allowing scientists to differentiate between various types of microorganisms. The citrate test is a classic example of this methodology, determining if a bacterium possesses the necessary machinery to metabolize citrate. A positive result demonstrates that an organism can rely on citrate as its sole carbon source for energy and growth.
The Core Mechanism of Citrate Utilization
For a bacterium to register a positive citrate test, it must first acquire the citrate molecule from the surrounding medium. This capability is conferred by a specialized transport protein known as citrase, or citrate permease, embedded within the cell membrane. The permease transports the citrate into the bacterial cell cytoplasm.
Once inside the cell, the transported citrate molecule is enzymatically broken down by the organism. This breakdown involves cleaving the citrate into oxaloacetate and acetate. The oxaloacetate is immediately metabolized further, yielding pyruvic acid and carbon dioxide (\(CO_2\)).
The generation of carbon dioxide is consequential for the visual indication of a positive test. This \(CO_2\) dissolves in the aqueous component of the testing medium, reacting with the sodium and water present. This reaction results in the formation of an alkaline compound, specifically sodium carbonate.
Interpreting a Positive Result
The test is performed using a specialized medium called Simmon’s Citrate Agar, which is formulated to be selective. The composition features sodium citrate as the only available carbon source and ammonium dihydrogen phosphate as the sole source of nitrogen. This ensures that only bacteria capable of utilizing citrate can grow.
The agar also contains the pH indicator bromothymol blue, which makes the result visually interpretable. In its uninoculated state, the medium has a neutral pH, typically around 6.9, causing the bromothymol blue to display a dark green color. A positive result occurs when the bacterium grows and metabolizes the citrate, leading to the accumulation of alkaline byproducts.
The accumulation of these alkaline substances causes the pH of the medium to increase significantly. When the pH rises above 7.6, the bromothymol blue indicator undergoes a color transformation. The medium changes from its original green color to a vibrant, deep blue, which is the definitive visual indication of a positive test. A negative result, where the organism cannot utilize citrate, will show no significant growth and the agar will remain green.
Practical Application in Bacterial Identification
The citrate test is a standard diagnostic tool used to differentiate closely related species of bacteria, particularly those belonging to the family Enterobacteriaceae. The test is often performed as the final component of a four-part testing battery known by the acronym IMViC, which stands for Indole, Methyl Red, Voges-Proskauer, and Citrate.
For instance, certain species consistently test positive for citrate utilization, such as Klebsiella pneumoniae and Enterobacter aerogenes. This positive result helps distinguish them from other closely related organisms, such as Escherichia coli, which typically tests negative for citrate utilization.
For example, Salmonella species are typically citrate-positive. This helps separate them from Shigella species, which are consistently citrate-negative.