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Oxidase Test Protocol

  • Authors: Patricia Shields 1, Laura Cathcart 1
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742
  • Citation: Patricia Shields, Laura Cathcart. 2010. Oxidase test protocol.
  • Publication Date : November 2010
  • Category: Protocol
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Figures

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Figure 1

Diagram of the oxidase test reaction.

a. Tetra-methyl-p-phenylenediamine dihydrochloride (TMPD), the oxidase reagent, is electron rich (reduced) and has no color.

b. In bacteria that contain the enzyme cytochrome oxidase, one electron from each of four cytochrome c molecules will be temporarily transferred to the enzyme.

c. This creates four electron-poor cytochrome c molecules and an electron-rich cytochrome oxidase enzyme.

d. As the final step in respiration, the cytochrome oxidase enzyme transfers four electrons to molecular oxygen and along with four protons, forms two molecules of water, returning the cytochrome oxidase enzyme to its original state.

e. Instead of acquiring an electron from another component in the electron transport chain, an electron-rich TMPD molecule passes an electron to the electron-poor cytochrome c. Cytochrome c returns to its original state and the resulting electron-poor (oxidized) TMPD radical has a dark blue color.

(Bryanna Clover, University of Maryland, College Park, MD)

Source: November 2010
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Figure 2

Oxidase test on filter paper. A positve oxidase result given by Pseudomonas aeruginosa (left) is indicated by a purple color. A negative oxidase result given by Escherichia coli (right) is indicated by the lack of color change. Both organisms were rubbed on a filter that was dipped in Kovacs' oxidase reagent and allowed to dry. (Laura Cathcart, University of Maryland, College Park, MD; Sabrina Kramer, University of Maryland, College Park, MD; Patricia Shields, University of Maryland, College Park, MD)

Source: November 2010
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Figure 3

Oxidase test on filter paper. A positive oxidase result given by Pseudomonas aeruginosa (left) is indicated by a purple color. A negative oxidase result given by Escherichia coli (right) is indicated by the lack of color change. Both organisms were rubbed on a dry filter that was then treated with one drop of Kovács oxidase reagent. (Laura Cathcart, University of Maryland, College Park, MD; Sabrina Kramer, University of Maryland, College Park, MD; Patricia Shields, University of Maryland, College Park, MD)

Source: November 2010
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Figure 4

Oxidase test using direct method. This mixed culture of oxidase-positive Vibrio cholerae, indicated by purple colonies, and oxidase-negative Escherichia coli, indicated by lack of color change, demonstrate how the direct oxidase test differentiates between the two. Kovács oxidase reagent was added directly to the colonies which were grown on trypticase soy agar at 37°C for 24 hours. (Laura Cathcart, University of Maryland, College Park, MD; Sabrina Kramer, University of Maryland, College Park, MD; Patricia Shields, University of Maryland, College Park, MD)

Source: November 2010
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Figure 5

Tube oxidase test. A positive oxidase result given by Neisseria sicca (left) is indicated by a purple color. A negative oxidase result given by Staphylococcus aureus (right) is indicated by the lack of color change. Both cultures were grown in a shaker incubator at 37°C for 24 hours in gonococcus peptone broth supplemented with Kellogg's solution and 1% sodium bicarbonate. Gaby and Hadley reagents were added to each tube. (Laura Cathcart, University of Maryland, College Park, MD; Sabrina Kramer, University of Maryland, College Park, MD; Patricia Shields, University of Maryland, College Park, MD)

Source: November 2010
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