The citrate test is a foundational biochemical method utilized in microbiology to differentiate between species of bacteria, particularly those belonging to the family Enterobacteriaceae. This test determines an organism’s metabolic capability to use citrate as its only source of carbon for growth and energy production. By isolating the carbon source in the growth medium, scientists identify which microbes possess the specific enzyme systems required to import and metabolize this organic acid. The result provides a powerful clue for distinguishing between clinically relevant organisms during the identification process.
The Citrate Utilization Metabolic Pathway
A bacterium must possess a specialized transport protein, often called Citrate Permease, to use citrate. This enzyme is situated in the cell membrane and actively shuttles the citrate molecule from the external medium into the cell’s cytoplasm, where metabolism begins. Without this uptake mechanism, the organism cannot access the carbon source and will not grow on the test medium.
Once inside the cell, the imported citrate is immediately acted upon by the enzyme Citrase, also known as Citrate Lyase. Citrase catalyzes the cleavage of the six-carbon citrate molecule into a four-carbon compound, oxaloacetate, and a two-carbon unit, acetate. This step releases the carbon from the citrate molecule, making it available for the cell’s energy and biosynthetic pathways.
Oxaloacetate is a molecule that is further metabolized by the bacterial cell to produce the three-carbon compound, pyruvate, along with the release of carbon dioxide (\(CO_2\)). Pyruvate is a central metabolite that can enter other pathways, such as the Krebs cycle, to generate cellular energy and building blocks. This entire cascade of reactions is only possible in organisms that can survive using citrate as their sole carbon source.
The most important metabolic consequence for the test result is the production of alkaline byproducts. The \(CO_2\) released during the breakdown of oxaloacetate reacts with water and sodium ions to form sodium carbonate, an alkaline salt. Furthermore, the test medium contains inorganic nitrogen as ammonium salts. The utilization of these ammonium salts releases ammonia into the medium, which also increases the environmental pH. This combined chemical shift toward alkalinity is the underlying mechanism that produces a positive test result.
Techniques for Performing the Citrate Test
The standard growth medium used for this procedure is Simmons Citrate Agar (SCA), a chemically defined medium specifically formulated to restrict nutrient availability. The medium contains sodium citrate as the single, limiting carbon source and ammonium dihydrogen phosphate as the only source of nitrogen. Other necessary components, like dipotassium phosphate and magnesium sulfate, are included to support growth and act as buffers or cofactors.
The medium is dispensed into tubes and allowed to solidify at an angle, creating a slant that provides a large surface area for aerobic growth. The initial pH of the uninoculated medium is approximately 6.9, giving it a characteristic deep forest green color. A pure culture of the bacterium being tested must be used to ensure result reliability.
Inoculation must be done with a light touch, using a sterile needle to streak the agar slant surface in a zig-zag pattern. A heavy inoculum must be avoided because it can accidentally transfer enough carbon-containing nutrients from the previous medium to cause a false-positive reaction. The test tube cap is then loosened to ensure an adequate supply of oxygen, since citrate utilization is an oxidative process.
The inoculated tube is incubated aerobically at a temperature between 35°C and 37°C, which mimics the optimal growth conditions for most human-associated bacteria. While positive results often appear within 18 to 24 hours, tubes should be held and checked daily for up to four to seven days before being considered a definitive negative result. The long incubation time is sometimes necessary because some organisms exhibit a delayed or weak ability to utilize the citrate.
Interpreting Results and Identifying Bacteria
Accurate reading of the citrate test relies on the inclusion of the pH indicator Bromothymol Blue in the Simmons Citrate Agar. This indicator is green at the medium’s initial neutral pH of 6.9. It changes color when the alkalinity of the medium increases, typically at a pH above 7.6, turning a deep, intense blue.
A positive result is identified by the presence of visible bacterial growth on the slant and a distinct color change of the medium from green to blue. The blue color confirms that the organism has successfully transported and metabolized the citrate, producing alkaline byproducts like sodium carbonate and ammonia that raise the pH. Even if only a small section of the slant turns blue, the result is recorded as positive, indicating the presence of the necessary metabolic pathway.
A negative result is characterized by a complete absence of growth or only a faint trace of growth, with the medium remaining the original green color. The lack of growth indicates that the bacterium was unable to produce the necessary Citrate Permease enzyme to utilize citrate as its carbon source. Without the metabolic activity of citrate utilization, the \(\text{pH}\) of the medium remains neutral, and the Bromothymol Blue indicator does not change color.
This differential test is routinely employed to distinguish between closely related Gram-negative bacteria, particularly within the Enterobacteriaceae family. For instance, species such as Klebsiella pneumoniae and Enterobacter aerogenes are reliably citrate-positive, making them easy to separate from the closely related Escherichia coli, which is typically citrate-negative. These results are often combined with other biochemical tests to definitively identify a pathogen, allowing clinicians to make informed decisions about patient treatment.