Simmon’s Citrate Agar & Spirit Blue agar

Simmon’s Citrate Agar

This is a defined medium used to determine if an organism can use citrate as its sole carbon source. It is often used to differentiate between members of Enterobacteriaceae. In organisms capable of utilizing citrate as a carbon source, the enzyme citrase hydrolyzes citrate into oxaoloacetic acid and acetic acid. The oxaloacetic acid is then hydrolyzed into pyruvic acid and CO₂. If CO₂ is produced, it reacts with components of the medium to produce an alkaline compound (e.g. Na₂CO₃). The alkaline pH turns the pH indicator (bromthymol blue) from green to blue. This is a positive result (the tube on the right is citrate positive). Klebsiella pneumoniae and Proteus mirabilis are examples of citrate positive organisms. Escherichia coli and Shigella dysenteriae are citrate negative.

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Spirit Blue agar

This agar is used to identify organisms that are capable of producing the enzyme lipase. This enzyme is secreted and hydrolyzes triglycerides to glycerol and three long chain fatty acids. These compounds are small enough to pass through the bacterial cell wall. Glycerol can be converted into a glycolysis intermediate. The fatty acids can be catabolized and their fragments can eventually enter the Kreb’s cycle. Spirit blue agar contains an emulsion of olive oil and spirit blue dye. Bacteria that produce lipase will hydrolyze the olive oil and produce a halo around the bacterial growth. The Gram-positive rod, Bacillus subtilis is lipase positive (pictured on the right) The plate pictured on the left is lipase negative.