Oxidase Test Definition

The oxidase test is a biochemical reaction that assays for the presence of cytochrome oxidase, an enzyme sometimes called indophenol oxidase. In the presence of an organism that contains the cytochrome oxidase enzyme, the reduced colorless reagent becomes an oxidized colored product .

• In 1928, Gordon and McLeod (5) introduced the use of a dimethyl-pphenylenediamine dihydrochloride solution to test for the presence of oxidase systems.
• Mainly they used this test to differentiate Neisseria gonorrhoeae(oxidase positive) from Staphylococcus spp. and Streptococcus spp.(oxidase negative).
• The sensitivity of this test was expanded when Kovács discovered that a tetra-methyl-p-phenylenediamine dihydrochloride solution provided a quicker reaction.
• Gaby and Hadley developed a modified oxidase test using p-aminodimethylaniline oxalate with α-naphthol to detect oxidase in test tube cultures.
• This test helps to detect the appearance of a cytochrome oxidase system that will catalyze the transport of electrons between electron donors in the bacteria and a redox dye- tetramethyl-p-phenylene-diamine. 
• Different types of reagents can be used for this test such as; Kovacs Oxidase Reagent (1% tetra-methyl-p-phenylenediamine dihydrochloride, in water), Gordon and McLeod’s Reagent (1% dimethyl-p-phenylenediamine dihydrochloride, in water), Gaby and Hadley (indophenol oxidase) Reagent (1% α-naphthol in 95% ethanol, 1% p-aminodimethylaniline HCL)
• The dye will be reduced and formed deep purple color. 

Purpose of Oxidase Test

• To differentiate pseudomonads from related species.
• To determine if an organism possesses the cytochrome oxidase enzyme.
• To differentiate Neisseria, Moraxella, Campylobacter and Pasteurella species (oxidase positive).

Principle of Oxidase Test

The final stage of bacterial respiration involves a series of membraneembedded components collectively known as the electron transport chain. The final step in the chain may involve the use of the enzyme cytochrome oxidase, which catalyzes the oxidation of cytochrome c while reducing oxygen to form water. 

The oxidase test often uses a reagent, tetra-methyl-p-phenylenediamine dihydrochloride, as an artificial electron donor for cytochrome c. When the reagent is oxidized by cytochrome c, it changes from colorless to a dark blue or purple compound, indophenol blue. Figure 1 contains a diagram of this reaction.

Tetra-methyl-p-phenylenediamine dihydrochloride (TMPD), the oxidase reagent, is electron rich (reduced) and has no color (a). In bacteria that contain the enzyme cytochrome oxidase, one electron from each of four cytochrome c molecules will be temporarily transferred to the enzyme (b). 

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

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 (e).

In addition to a positive oxidase and negative oxidase reaction, some organisms are classified as variable oxidase or delayed oxidase-positive. Variability in the oxidase reaction has been attributed to differences in cytochrome ccomposition, variability in cytochrome oxidases, and overall transport chain composition variability.

Requirement

• Kovács oxidase reagent: 1% tetra-methyl-p-phenylenediamine dihydrochloride, in water Store refrigerated in a dark bottle no longer than 1 week.
• Gordon and McLeod reagent: 1% dimethyl-p-phenylenediamine dihydrochloride, in water Store refrigerated in a dark bottle no longer than 1 week.
• Gaby and Hadley oxidase test: 1% α-naphthol in 95% ethanol, 1% p-aminodimethylaniline oxalate, Store refrigerated in dark bottles no longer than 1 week.

Procedure of Oxidase Test

There are present different methods of oxidase test these are includes;

• The filter paper test
• Filter paper spot test
• Direct plate method
• Test tube method. 
• Swab Method

Filter Paper Test Method/Dry Filter Paper Method

1. Soak a small piece of filter paper in 1% Kovács oxidase reagent and let dry.
2. Use a loop and pick a well-isolated colony from a fresh (18- to 24-hour culture) bacterial plate and rub onto treated filter paper (please see Comments and Tips section for notes on recommended media and loops).
3. Observe for color changes.

Filter Paper Spot Method/Wet Filter Paper Method

1. Use a loop and pick a well-isolated colony from a fresh (18- to 24-hour culture) bacterial plate and rub onto a small piece of filter paper (please see Comments and Tips section for notes on recommended media and loops).
2. Place 1 or 2 drops of 1% Kovács oxidase reagent on the organism smear.
3. Observe for color changes.

Direct Plate Method

1. Grow a fresh culture (18 to 24 hours) of bacteria on nutrient agar using the streak plate method so that well-isolated colonies are present (please see Comments and Tips section for notes on recommended media).
2. Place 1 or 2 drops of 1% Kovács oxidase reagent or 1% Gordon and McLeod reagent on the organisms. Do not invert or flood plate.
3. Observe for color changes.

Test tube method.

1. Grow a fresh culture (18 to 24 hours) of bacteria in 4.5 ml of nutrient broth (or standard media that does not contain a high concentration of sugar, please see Comments and Tips section for notes on recommended media).
2. Add 0.2 ml of 1% α-naphthol, then add 0.3 ml of 1% paminodimethylaniline oxalate (Gaby and Hadley reagents).
3. Observe for color changes.

Swab Method

1. Dip swab into reagent and then touch an isolated suspect colony
2. Observe for colour change within 10 seconds.

Result of Oxidase Test

Filter Paper Test Method: 

• Oxidase positive: Microorganisms are oxidase-positive when the color changes to dark purple within 5 to 10 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 60 to 90 seconds.
• Oxidase Negative: Microorganisms are oxidase negative if the color does not change or it takes longer than 2 minutes.

Filter Paper Spot Method

• Oxidase positive: Microorganisms are oxidase positive when the color changes to dark purple within 5 to 10 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 60 to 90 seconds.
• Oxidase Negative: Microorganisms are oxidase negative if the color does not change or it takes longer than 2 minutes.

Direct Plate Method

• Oxidase positive: When using Kovács oxidase reagent, microorganisms are oxidase positive when the color changes to dark purple within 5 to 10 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 60 to 90 seconds. 
• Oxidase Negative: Microorganisms are oxidase negative if the color does not change or it takes longer than 2 minutes.

Note: When using Gordon and McLeod reagent, microorganisms are oxidase positive when the color changes to red within 10 to 30 minutes or to black within 60 minutes. Microorganisms are oxidase negative if the color does not change.

Test Tube Method

• Oxidase positive: Microorganisms are oxidase positive when the color changes to blue within 15 to 30 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 2 to 3 minutes. 
• Oxidase Negative: Microorganisms are oxidase negative if the color does not change.

Swab Method

• Oxidase positive: Color change to purple
• Oxidase Negative: No color change.

Example of Oxidase Positive Organisms: Moraxella, Pseudomonas, Helicobacter pylori,  Neisseria, Campylobacter, Alcaligens, Aeromonas, Vibrio, Legionella pneumophila, Brucella, Pasteurella, etc.

Oxidase Negative Organisms: Enterobacteriaceae (e.g. E. coli)

Quality Control for Oxidase Test

• Positive Control: Pseudomonas aeruginosa ATCC 27853
• Negative Control: Escherichia coli ATCC 25922

Application of Oxidase Test

• Used for the identification of Pseudomonas, Neisseria, Alcaligens, Aeromonas, Campylobacter, Vibrio, Brucella and Pasteurella, all of which produce the enzyme cytochrome oxidase.
• Used to detects the presence of a cytochrome oxidase system that will catalyse the transport of electrons between electron donors in the bacteria and a redox dye- tetramethyl-p-phenylene-diamine. 
• Used to differentiate Neisseria gonorrhoeae(oxidase positive) from Staphylococcus spp. and Streptococcus spp.(oxidase negative).
• This test is used as a major characteristic for the identification of Gram-negative rods that are not in the Enterobacteriaceae family. Colonies suspected of belonging to other genera Aeromonas, Pseudomonas, Neisseria, Campylobacter, and Pasteurella are oxidase positive.

Limitations of Oxidase Test

• The reagent of oxidase test can be auto-oxidized, so it is important to always use fresh reagents, no older than 1 week.
• Timing is critical to accurate testing.
• Avoid using media containing excess sugar, such as nutrient agar. It is recommended to use Tryptic soy agar to grow colonies. Because both bacteria and yeast grown on media containing high concentrations of glucose can show inhibited oxidase activity.
• Bacteria that thrive on media-containing dyes may produce aberrant results.
• The microorganisms can be killed after adding the test reagents, so it is important to perform sub-culturing before adding any reagent to an active culture.
• Avoid using nichrome or other iron-containing loops, it may lead to false-positive reactions. It is recommended to use Platinum loops.
• Most of the Haemophilus are shows oxidase-positive, Hence, strips or reagents with less sensitivity can lead to false-negative results.
• The non-selective and non-differential media is used to ensure the valid Oxidase reactions of gram-negative bacilli. Also, if the colonies are grown on a media containing a high level of glucose may give false-negative reactions.
• Always use colonies that are 18-24 hours old. Older colonies will produce weaker reactions.
• Any color changes appearing after 20 seconds should be disregarded.
• This test can be used in the presumptive identification of Neisseria and in the differentiation and identification of gram-negative bacilli. Oxidase-positive organisms should be examined by gram stain to determine morphology and gram reaction. Additional biochemical tests are recommended for complete identification.

References

• https://asm.org/getattachment/00ce8639-8e76-4acb-8591-0f7b22a347c6/oxidase-test-protocol-3229.pdf
• https://microbiologyinfo.com/oxidase-test-principle-uses-procedure-types-result-interpretation-examples-and-limitations/
• https://microbeonline.com/oxidase-test-principle-procedure-and-oxidase-positive-organisms/
• https://learn.chm.msu.edu/vibl/content/oxidase.html