cytochrome oxidase test

The cytochromes are iron-containing hemoproteins that act as the last link in the chain of aerobic respiration by transferring electrons (hydrogen) to

oxygen, with the formation of water. Cytochrome oxidase or Warburge’s respiratory

enzyme – the enzyme which reduces molecular oxygen to water to compete the last link in the chain of aerobic respiration

The test is most helpful in screening colonies suspected of being one of the Enterobacteriaceae (all negative) and in identifying colonies suspected of being a pseudomonas specie or a neisseria specie which are all positive. (Pseudomonas, Vibrio, Aeromonas, Neisseria and Comamonas)

The cytochrome oxidase test utilize certain reagent dyes, such as the “redux dye: tetramethyl-paraphenylenediamine dihydrochloride”, that substitute for oxygen as artificial electron acceptors.

In the reduced state the dye is colorless, however, in the presence of cytochrome oxidase and atmospheric oxygen, tetramethyl-paraphenylenediamine dihydrochloride is oxidized, forming indophenol blue color.

Materials:

Culture plate of organism Cytochrome oxidase paper strip (Pathotec oxidase strip) (tetra-methyl-para-phenylene diamine dihydrochloride)

Procedures:

The test is commonly performed by one of the two methods:

The direct plate technique in which 2 to 3 drops reagent are directly poured into the isolated bacteria colonies growing on the plate medium.

The indirect paper strip procedure. In which a few drops of the reagent are either added to a filter paper trip or commercial disk or strips impregnated which dried reagent are used.

In either method, a loopful of suspected colony is smeared into the reagent zone of the filter paper.

Observations:

Bacterial colonies having cytochrome oxidase actively will develop a deep blue color to purple to black at the inoculation site within seconds.