Closely related organisms can be differentiated using a series of biochemical tests that identify various metabolic properties of different bacterial species.

A single biochemical test is usually not sufficient to identify an unknown bacteria, but biochemical tests can be critically combined to successfully identify a bacterial species. Kits that are designed to identify common pathogens are commercially available. Biochemical tests may also be critically combined with other methods of bacterial identification, including differential or selective media and staining. The main drawback of biochemical identification methods is they can only be used for organisms that can be cultivated in the laboratory. Nucleic acid-based assays overcome this problem.

Examples of biochemical tests

Catalase test

The enzyme catalase catalyzes the reaction of hydrogen peroxide into oxygen and water (H₂O₂ -> H₂O + O₂). Hydrogen peroxide is a harmful byproduct of respiration. Catalase serves to protect the cell from oxidative damage. It is present in most aerobic bacteria. The presence of catalase can be determined by the production of bubbles of gas when bacterial culture is added to hydrogen peroxide. No bubbles are produced in catalase-negative organisms.

Oxidase test

Cytochrome c oxidase is an enzyme present in many aerobic bacteria. It catalyzes the transport of electrons from electron donors donor compounds to electron acceptors in the energy-producing electron transport chain. In the oxidase test, the enzyme oxidases reagents such as N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-dimethyl-p-phenylenediamine (DMPD) that are colorless (or light pink) in the reduced state and blue in the oxidized state. A blue color, therefore, indicates the presence of cytochrome c oxidase. Note that bacteria may use other enzymes in the electron transport chain, so there is no direct correlation between aerobes and a positive oxidase test.

Urease Test

The enzyme urease catalyzes the reaction of urea into ammonia, water, and carbon dioxide. The presence of the enzyme can be determined by adding urea to the medium and detecting ammonia through the associated increase in pH.

Indole test

The indole test determines the ability of bacteria to produce indole from tryptophan by various enzymes. Indole can be detected in a bacterial culturing by adding it to Kovac’s reagent, which results in a pink to red color or p-dimethylaminocinnamaldehyde (DMACA) which results in a blue color.

Figure 1. Positive (left) and negative (right) catalase, oxidase, and indole tests.