Citrate utilization is a metabolic test used to determine an organism's ability to utilize citrate as its lone source of carbon. Simmons citrate agar is a synthetic medium containing citrate, ammonium ions, and the pH indicator bromthymol blue. This indicator is green below pH 7.6 and blue above pH 7.6. Once an unknown bacterium is isolated and diluted in liquified agar medium, citrate metabolism can be ascertained. An inoculating needle is sterilized and dipped into the bacterial medium. Using aseptic technique, this needle is plunged into the green simmons-citrate agar and zigzagged across the surface of the slant. If the bacterium is capable of utilizing citrate as its sole source of carbon, the enzyme citrate permease engages in active transport and is responsible for the entry of citrate into the cell. Citrate is hydrolyzed by citrase into two acids, acetic acid and oxaloacetic acid. Oxaloacetic acid is hydrolyzed into pyruvic acid and carbon dioxide. Carbon dioxide reacts with sodium ions and water to produce the basic compound sodium carbonate. Basisity moves the pH to a mark higher than 7.6, and the bromthymol blue pH indicator changes the color of the agar from green to blue after an incubation time of approximately 48 hours. Organisms typically produce citric acid at the beginning of the krebs cycle by combining acetyl CoA and oxaloacetic acid. The citrate utilization test is selective because only certain bacteria can utilize citrate in place of a fermentable carbohydrate.