10.1: Oxidase Test
- Demonstrate an understanding of the principle behind the oxidase test, including the role of cytochrome c oxidase in the electron transport chain.
- Perform the oxidase test, including the inoculation of the test organism onto the oxidase reagent.
- Develop skills in interpreting the results of the oxidase test, including recognizing the color change indicating a positive or negative result.
- Understand the significance of the oxidase test in differentiating between bacteria that possess cytochrome c oxidase and those that do not.
- Correlate the results of the oxidase test with the biochemical characteristics and classification of microorganisms.
- Demonstrate proficiency in recording and reporting the results of the oxidase test accurately.
Cytochrome c Oxidase
Aerobic respiration is an O 2 -requiring process that uses energy from nutrient molecules to produce ATP molecules to provide for the cell's energy needs. During aerobic respiration, the electron transport chain transfers high-energy electrons from protein to protein and uses that energy to build up a H + gradient that is utilized by ATP synthase to make ATP. At the end of the aerobic electron transport chain, an enzyme transfers the electron from the electron transport chain to O 2 . In some bacteria capable of aerobic respiration, that enzyme that transfers electrons to O 2 , the final electron acceptor for aerobic respiration, is called cytochrome c oxidase . (Fig.1.)
A test called the oxidase test can be used in the laboratory to determine if a bacterial species has cytochrome c oxidase. Those species that have cytochrome c oxidase are called oxidase positive and those species that do not have cytochrome c oxidase are called oxidase negative . The ability of a bacterial species to produce cytochrome c oxidase is coded in its DNA. If the cytochrome c oxidase gene is present in the bacterial species, it will produce this enzyme. If the gene is absent, that bacterial species cannot produce cytochrome c oxidase. The oxidase test is therefore useful for characterizing bacterial species and differentiating bacterial species from each other for identification purposes. For example, the gram-negative opportunist Pseudomonas aeruginosa and the gram-negative cholera-causing Vibrio cholerae use cytochrome c oxidase, which can be detected by the oxidase test, whereas other gram-negative Enterobacteriaceae, like E. coli , are negative for this test because they produce different cytochrome oxidase types.
Bacterial species that contain cytochrome c oxidase are capable of aerobic respiration. Oxidase-positive bacterial species are all aerobic.
However, oxidase-negative species are not necessarily anaerobic. Oxidase-negative species may still be aerobic species or facultative anaerobes, but they produce and use a different enzyme, other than cytochrome c oxidase, to transfer electrons from the electron transport chain to O 2 .
The Oxidase Test
The oxidase test is a key test to differentiate between the bacterial families Pseudomonadaceae (oxidase positive species) and Enterobacteriaceae (oxidase negative species).
The oxidase test utilizes a special reagent called an oxidase reagent . This reagent is colorless. However, in the presence of cytochrome c oxidase, the oxidase reagent will transfer its electrons to cytochrome c oxidase and exhibit a bluish or purplish color in 20 seconds or less . (Fig.2.)
- oxidase-positive bacteria contain cytochrome c oxidase and produce a change in color of the reagent from colorless to bluish or purplish in less than 20 seconds.
- oxidase-negative bacteria do not contain cytochrome c oxidase and do not change the color of the oxidase reagent in less than 20 seconds.
The Diagnosis of Gonorrhea
Neisseria gonorrhoeae, a gram-negative diplococcus, is an obligate human pathogen known to cause gonorrhea. Often transferred through sexual intercourse, its symptoms include abnormal bleeding, discharge, and soreness in the genitalia. If left untreated, gonorrhea can result in severe complications, such as pelvic inflammation, ectopic pregnancy, infertility, and long term pelvic/abdominal pain. Hence, it is essential for doctors to diagnose and treat this infection.
The Oxidase test is one of many that are used identify N. gonorrhoeae. This bacterium is oxidase positive, so the presence of oxidase positive gram-negative diplococci in a patient sample strongly suggests an infection with N. gonorrhoeae. It is important to note that the oxidase test is not often the sole test used when performing laboratory diagnosis. Other tests, such as the catalase test, are also used to help verify the diagnosis.
Laboratory Instructions
Oxidase Test
- Obtain an oxidase DrySlide.
- Using a toothpick, add a sample of each species into separate panels of the DrySlide.
- Start a timer for 20 seconds.
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Watch for purple color to develop on the oxidase test strip within 20 seconds.
- purple or bluish color change in less than 20 seconds is oxidase positive
- no color change within 20 seconds is oxidase negative
- Repeat for each bacterial species you are testing.
Attributions
- "Microbiology Laboratory Manual: Labs, 1.19 Cytochrome C Oxidase" by Dr. Rosanna Hartline , West Hills College Lemoore, LibreTexts: Biology is licensed under CC BY-NC 4.0
- Chapter Image: Red Mountain Microbiology by Jill Raymond Ph.D.; Graham Boorse, Ph.D.; and Anne Mason M.S. is licensed under CC BY-NC 4.0
- MB352 General Microbiology Laboratory 2021 (Lee) by Alice_Lee@ncsu.edu is licensed under CC BY-NC-SA 4.0
- Microbiology by OpenStax is licensed under CC BY 4.0
- Red Mountain Microbiology by Jill Raymond Ph.D.; Graham Boorse, Ph.D.; Anne Mason M.S. is licensed under CC BY-NC 4.0
- "Gonorrhea: About Gonorrhea" by U.S. Center for Disease Control and Prevention , CDC Use of Materials Statement
- "The Laboratory Diagnosis of Neisseria gonorrhoeae" by Lai-King Ng, Irene E Martine , Canadian Journal of Infectious Diseases and Medical Microbiology/Volume 16, Issue 1/323082 p. 15-25 , Wiley Online Library is licensed under CC BY 4.0