Oxidase Test


What is the purpose of the test? 


A vital part of a bacterium’s ability to generate ATP is a group of membrane-bound proteins called cytochromes. Their function is to take the energetic electrons removed during metabolism of foods and transfer them to oxygen during oxidative metabolism. The last of the cytochromes is called terminal oxidase. This test is used to determine whether the oxidase cytochrome is present and functional.



How is oxidase activity determined? 


A nutrient medium is streaked with bacteria. After colonies have arisen, individual colonies are removed using a sterile, non-metallic instrument (pre-sterilized plastic loop or sterile wooden splint).  The cells are rubbed into a moistened strip impregnated with oxidase reagent. This chemical takes the place of oxygen as a recipient for the electrons from the oxidase cytochrome. The additional electrons turn the oxidase reagent from colorless to purple. If oxidase is not present, no color change is observed.



What medium is used?  


No medium is required for this test in the software, as it is a video-based test.



How is the test performed?  

The microbe being tested is streaked for isolation on an appropriate growth medium, such as nutrient agar.  After 24h incubation, the cells can be tested in one of two ways.  The simplest way is to simply flood the growth with oxidase reagent and observe for the development of a deep indigo blue in and around the colonies.  The clear oxidase reagent turns blue when cytochrome oxidase adds electrons to the chemical instead of to oxygen, as it normally does.  Development of the blue color is positive for presence of the enzyme cytochrome oxidase.  The color change does not occur in a negative test.   


Because of the expense and instability of oxidase reagent, this test is rarely performed in that manner.  Instead, more reliable and inexpensive methods have become the standard.   Common in the clinical lab is the use of paper strips impregnated with oxidase reagent.  Fresh growth is removed from the agar plate using a non-metallic instrument such as a sterile plastic inoculating loop or a sterile swab or wooden splint.  The oxidase test strip is moistened slightly with sterile water and the growth is rubbed into the moistened paper of the strip.  If the microbe has cytochrome oxidase, it will add electrons to the reagent, changing it from its colorless appearance to a deep indigo blue in a matter of 10-20 seconds.  Waiting any long than this increases the likelihood that the reagent turns blue due to natural chemical changes caused by exposure to air.  If the color does not turn blue within 20 seconds, the test is negative for the presence of oxidase.


What reagents are added?


Oxidase reagent is provided in the form of impregnated paper strips.



To view the oxidase test in VirtualUnknown™ Microbiology, complete the following steps:


1. Select View -> Oxidase Video from the Main Menu.


2. Press the Play button to watch the video clip showing performance of an oxidase test for the unknown organism.  Growth will be removed from a colony of the unknown and rubbed into a moistened paper strip impregnated with oxidase reagent.

ox1.jpg   ox2.jpg

3. Observe the results. If the unknown bacterial cells possess oxidase, a deep indigo blue color will develop where the growth was smeared on the oxidase test strip.  This is a positive test.  If the bacteria lack oxidase, there will be no perceptible change in the appearance of the smear on the oxidase test strip.


4. Record the result by pressing the Record Result button on the oxidase video screen and selecting the appropriate result.