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, tetramethylparaphenylenediamine dihydrochloride is oxidized, forming indophenol blue color.

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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.