The oxidase test is used to determine if an organism possesses the

cytochrome c oxidase The enzyme cytochrome c oxidase or Complex IV, (was , now reclassified as a translocasEC 7.1.1.9 is a large transmembrane protein complex found in bacteria, archaea, and mitochondria of eukaryotes. It is the last enzyme in the respiratory elect ...

enzyme. The test is used as an aid for the differentiation of ''

Neisseria ''Neisseria'' is a large genus of bacteria that colonize the mucosal surfaces of many animals. Of the 11 species that colonize humans, only two are pathogens, '' N. meningitidis'' and ''N. gonorrhoeae''. ''Neisseria'' species are Gram-negativ ...

'', ''

Moraxella ''Moraxella'' is a genus of gram-negative bacteria in the family Moraxellaceae. It is named after the Swiss ophthalmologist Victor Morax. The organisms are short rods, coccobacilli, or as in the case of ''Moraxella catarrhalis'', diplococci in ...

'', '' Campylobacter'' and ''

Pasteurella __NOTOC__ ''Pasteurella'' is a genus of Gram-negative, facultatively anaerobic bacteria. ''Pasteurella'' species are nonmotile and pleomorphic, and often exhibit bipolar staining ("safety pin" appearance). Most species are catalase- and oxidase ...

'' species (oxidase positive). It is also used to differentiate pseudomonads from related species.MacFaddin JF, editor. Biochemical Tests for Identification of Medical Bacteria. 3rd ed. Philadelphia:Lippincott Williams and Wilkins; 2000. p. 363-7

Classification

Strains may be either oxidase-positive (OX+) or oxidase-negative (OX-).

OX+

OX+ normally means the bacterium contains cytochrome c oxidase (also known as Complex IV) and can therefore use

oxygen Oxygen is the chemical element with the symbol O and atomic number 8. It is a member of the chalcogen group in the periodic table, a highly reactive nonmetal, and an oxidizing agent that readily forms oxides with most elements as wel ...

for energy production by converting O₂ to H₂O₂ or H₂O with an

electron transfer chain An electron transport chain (ETC) is a series of protein complexes and other molecules that transfer electrons from electron donors to electron acceptors via redox reactions (both reduction and oxidation occurring simultaneously) and couples thi ...

. The

Pseudomonadaceae The Pseudomonadaceae are a family of bacteria which includes the genera ''Azomonas'', ''Azorhizophilus'', ''Azotobacter'', '' Mesophilobacter'', ''Pseudomonas'' (the type genus), and '' Rugamonas''. The family Azotobacteraceae was recently recl ...

are typically OX+. The

Gram-negative Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall ...

diplococci A diplococcus (plural diplococci) is a round bacterium (a coccus) that typically occurs in the form of two joined cells. Types Examples of gram-negative diplococci are '' Neisseria spp.'' and ''Moraxella catarrhalis.'' Examples of gram-positiv ...

and

are oxidase-positive. Many Gram-negative, spiral curved rods are also oxidase-positive, which includes ''

Helicobacter pylori ''Helicobacter pylori'', previously known as ''Campylobacter pylori'', is a gram-negative, microaerophilic, spiral (helical) bacterium usually found in the stomach. Its helical shape (from which the genus name, helicobacter, derives) is though ...

'', ''

Vibrio cholerae ''Vibrio cholerae'' is a species of Gram-negative, facultative anaerobe and comma-shaped bacteria. The bacteria naturally live in brackish or saltwater where they attach themselves easily to the chitin-containing shells of crabs, shrimps, and oth ...

'', and ''

Campylobacter jejuni ''Campylobacter jejuni'' () is a species of pathogenic bacteria, one of the most common causes of food poisoning in Europe and in the US. The vast majority of cases occur as isolated events, not as part of recognized outbreaks. Active surveillan ...

''.

Oxidase variable

'' Legionella pneumophila'' may be oxidase-positive.

OX−

OX− normally means the bacterium does not contain cytochrome c oxidase and, therefore, either cannot use

for energy production with an

or employs a different cytochrome for transferring electrons to oxygen. Enterobacteriaceae are typically OX−.

Mechanism

The test uses disks impregnated with a reagent such as ''N,N,N′,N′''-tetramethyl-''p''-phenylenediamine, TMPD (or ''N,N''-dimethyl-''p''-phenylenediamine, DMPD, which is also a

redox Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate (chemistry), substrate change. Oxidation is the loss of Electron, electrons or an increase in the oxidation state, while reduction ...

indicator). The reagent is a dark-blue to

maroon Maroon ( US/ UK , Australia ) is a brownish crimson color that takes its name from the French word ''marron'', or chestnut. "Marron" is also one of the French translations for "brown". According to multiple dictionaries, there are var ...

color when oxidized, and colorless when reduced. Oxidase-positive bacteria possess cytochrome oxidase or

indophenol oxidase Indophenol is an organic compound with the formula OC6H4NC6H4OH. It is deep blue dye that is the product of the Berthelot's reaction, a common test for ammonia. The indophenol group, with various substituents in place of O''H'' and various rin ...

(an iron-containing hemoprotein).Isenberg HD, editor. Clinical Microbiology Procedures Handbook. American Society for Microbiology; 2004. p. 3.3.2-3.3.2.13. These both catalyze the transport of electrons from donor compounds (

NADH Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an aden ...

) to electron acceptors (usually oxygen). The test reagent TMPD acts as an artificial electron donor for the enzyme oxidase. The oxidized reagent forms the colored compound

indophenol blue Indophenol is an organic compound with the formula OC6H4NC6H4OH. It is deep blue dye that is the product of the Berthelot's reaction, a common test for ammonia. The indophenol group, with various substituents in place of O''H'' and various rin ...

. The cytochrome system is usually only present in aerobic organisms that are capable of using oxygen as the terminal electron acceptor. The end-product of this metabolism is either water or hydrogen peroxide (broken down by catalase).

Procedures

#Wet each disk with about four inoculating loops of deionized water. #Use a loop to aseptically transfer a large mass of pure bacteria to the disk. #Observe the disk for up to three minutes. If the area of inoculation turns dark-blue to maroon to almost black, then the result is positive. If a color change does not occur within three minutes, the result is negative. In alternative manner, live bacteria cultivated on

trypticase soy agar Trypticase soy agar or tryptone soya agar (TSA) and Trypticase soy broth or tryptone soya broth (TSB) with agar are growth media for the culturing of bacteria. They are general-purpose, nonselective media providing enough nutrients to allow f ...

plates may be prepared using

sterile technique Asepsis is the state of being free from Pathogen, disease-causing micro-organisms (such as pathogenic bacteria, viruses, pathogenic fungi, and parasitism, parasites). There are two categories of asepsis: medical and surgical. The modern day notio ...

with a single-line streak inoculation. The inoculated plates are incubated at 37 °C for 24–48 hours to establish colonies. Fresh bacterial preparations should be used. After colonies have grown on the medium, 2-3 drops of the reagent DMPD are added to the surface of each organism to be tested. * A positive test (OX+) will result in a color change violet to purple, within 10–30 seconds. * A negative test (OX-) will result in a light-pink or absence of coloration.

References

* American Society for Microbiology, Oxidase Test Protocol. 2013. ASM MicrobeLibrary, 1–9. * Cheng W J, Lin C W, Wu T G, Su C S, Hsieh M S. 2013. Calibration of glucose oxidase-based test strips for capillary blood measurement with oxygen saturated venous blood samples. Clinica Chimica Acta. 415, 152–157. * Corchia L, Hubault R, Quinquenel B, N'Guyen. 2015. Rapidly Evolving Conjunctivitis Due to Pasteurella Multocida, Occurring after Direct Inoculation with Animal Droplets in an Immuno-compromised Host. BMC Ophthalmology 15.1, 21. * Floch C, Alarcon-Gutiérrez E, Criquet S. 2007. ABTS assay of phenol oxidase activity in soil. Journal of Microbiological Methods. 71, 319–324. * Gaby W L, Hadley C. 1957. Practical laboratory test for the identification of Pseudomonas aeruginosa. Journal of bacteriology. 74, 356–358. * Gilani M, Munir T, Latif M, Gilani M, Rehman S, Ansari M, Hafeez A, Najeeb S, Saad N. 2015. In Vitro Efficacy of Doripenem against Pseudomonas Aeruginosa and Acinetobacter Baumannii by E-test. Journal of the College of Physicians and Surgeons Pakistan 25, 726–729. * Kuss S, Tanner E E L, Ordovas-Montanes M, Compton R G. 2017. Electrochemical Recognition and Quantification of Cytochrome C Expression in and Aerobe/anaerobe Using, ','-tetramethyl—phenylene-diamine (TMPD). Chemical Science 8.11, 7682–7688. Web. * Ivanova N V, Zemlak T S, Hanner R H, Hebert P D N. 2007. Universal primer cocktails for fish DNA barcoding. Molecular Ecology Notes. 7, 544–54. * Prince C. 2009. Practical Manual of Medical Microbiology (Jaypee Brothers Medical Publishers (P) Ltd.) 112–112. * Shields P, Cathcart L. 2013. Oxidase Test Protocol - Library. American Society for Microbiology, ASM MicrobeLibrary, 1–5. * Steel K J. 1961. The Oxidase Reaction as a Taxonomic Tool. Journal of General Microbiology. 25, 297–306. * Zanderigo F et al. 2018. 1Carmine Binding to Brain Monoamine Oxidase A: Test-Retest Properties and Noninvasive Quantification. Molecular Imaging and Biology. 20, 667–681.

External links

* Oxidase tes
video

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