Last updated on June 21st, 2021The oxidase test is provided to identify bacteria that develop cytochrome c oxidase, an enzyme of the bacterial electron transfer chain. As soon as present, the cytochrome c oxidase oxidizes the reagent (tetramethyl-p-phenylenediamine dihydrochloride) to indophenols, a purple or dark blue shade end product. Once the enzyme is no present, the reagent stays reduced and is colorless.

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All bacteria that room oxidase-positive room aerobic and can usage oxygen as a terminal electron agree in respiration. This walk NOT mean that they room strict aerobes. Bacteria that space oxidase-negative might be anaerobic, aerobic, or facultative; the oxidase an unfavorable result just way that this organisms execute not have the cytochrome c oxidase that oxidizes the check reagent. They may respire using other oxidases in electron transport.Test demands for Oxidase test Moist filter record with the substrate (1% tetramethyl-p-phenylenediamine dihydrochloride), or commercially prepared document disk, wood wire, or platinum wire.Kovács oxidase reagent (1% tetra-methyl-p-phenylenediamine dihydrochloride, in water). Keep refrigerated in a dark party for no longer than 1 week.
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Various types of oxidase check procedure
Contents1 Procedure the Oxidase test1.1 Filter record Test Method1.2 Filter file Spot Method1.3 direct Plate Method1.4 Test tube Method1.5 offers of oxidase test1.6 high quality Control1.7 Precautions and Limitations: 2 list of oxidase hopeful organisms

Procedure that Oxidase test

Oxidase test deserve to be performed in assorted ways. This include, but are not minimal to, the filter record test, filter paper spot test, direct bowl method, and test pipe method.

Filter record Test Method

Soak a little piece the filter record in 1% Kovács oxidase reagent and let dry.Use a loop and pick a well-isolated swarm from a new (18- to 24- hour culture) bacterial plate and also rub onto treated filter paperObserve for shade changes.
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Oxidase-positive Pseudomonas aeruginosa (left) andoxidase-negative Escherichia coli (right).
Results Oxidase positive: color transforms to dark purple within 5 to 10 seconds.Delayed oxidase-positive: color alters to violet within 60 come 90 seconds.Oxidase negative: shade does not adjust or the takes much longer than 2 minutes.

Filter record Spot Method

Use a loop and pick a well-isolated colony from a new bacterial plate and also rub the onto a tiny piece of filter paper.Place 1 or 2 fall of 1% Kovács oxidase reagent top top the biology smear.Observe for shade changes.
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Oxidase-positive Pseudomonas aeruginosa (left) andoxidase-negative Escherichia coli (right).
ResultsOxidase positive: color alters to dark violet within 5 to 10 seconds.Delayed oxidase-positive: color changes to violet within 60 to 90 seconds.Oxidase negative: shade does not adjust or that takes longer than 2 minutes.

Direct bowl Method

Grow a fresh society (18 to 24 hours) that bacteria ~ above nutrient agar or trypticase soy agar using the streak plate method so the well-isolated nests are present.Place 1 or 2 fall of 1% Kovács oxidase reagent top top the organisms.Do not invert or overwhelming plate.Observe for color changes.
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Oxidase-positive Vibrio cholerae mirroring purple colonies, and also oxidase-negative Escherichia coli with absence of shade change
ResultsOxidase positive: color alters to dark violet within 5 come 10 seconds.Delayed oxidase-positive: color transforms to violet within 60 come 90 seconds.Oxidase negative: shade does not readjust or that takes much longer than 2 minutes.

Test tube Method

Grow a fresh culture (18 come 24 hours) the bacteria in 4.5 ml of nutrient broth (or conventional media the does not contain a high concentration the sugar).Add 0.2 ml that 1% α-naphthol, then include 0.3 ml of 1% paminodimethylaniline oxalate (Gaby and Hadley reagents).Observe for color changes.
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Oxidase optimistic Neisseria sicca (left) and oxidase an adverse Staphylococcus aureus (right)
ResultsOxidase positive: color transforms to blue within 15 to 30 seconds.Delayed oxidase-positive: color alters to violet within 2 come 3 minutes.Oxidase negative: no readjust in color

Uses the oxidase test

Oxidase check is most useful in screening colonies suspected of being a member the the Enterobacteriaceae family; every the members that the Enterobacteriaceae family members including E. Coli are oxidase negative.To protect against misidentification, perform an oxidase test on all Gram-negative rods. Oxidase check is specifically important in separating Aeromonas native Enterobacteriaceae.Note: If you see swarming nests in a culture media, do not execute oxidase test, together its unique attributes of Proteus spp, which are oxidase negative.Oxidase check is used as a major characteristic for the to know of Gram-negative rods that are not in the Enterobacteriaceae family. Nests suspected of belonging to various other genera Aeromonas, Pseudomonas, Neisseria, Campylobacter, and Pasteurella are oxidase positive.Gram-negative diplococci give a positive reaction. All members of the genus Neisseria space oxidase positive. Moraxella spp. That space either Gram-negative diplococci or coccobacilli are also oxidase-positive.

Quality Control

Bacterial species showing hopeful and negative reactions must be operation as controls at regular intervals. The adhering to are suggested:A. Oxidase positive: Pseudomonas aeruginosaB. Oxidase negative: Escherichia coli

 Precautions and also Limitations:

Timing is an essential to precise testing.Use new reagents, no older than 1 week, enlarge reagents deserve to autooxidize thus offering erroneous results. Execute not usage if reagent or filter file is purple.Do not test organisms farming on media the contain glucose or water (e.g., MacConkey agar or EMB agar).Do not use nickel-base alloy wires include chromium and also iron (nichrome) to choose the colony and make smear together this may offer false-positive results.Bacteria get an impressive on media containing water may provide aberrant results.Older cultures are much less metabolically energetic so may give false-negative results within the pointed out observation time.

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The oxidase test should be performed native 5% sheep blood agar or an additional medium there is no fermentable sugar. Fermentation the carbohydrates results in acidification of the medium (e.g., lactose in MacConkey Agar or Sucrose in TCBS), and a false negative oxidase test may result if the neighboring pH is listed below 5.1. Subinoculation ~ above Nutrient agar is required prior to the oxidase test have the right to be performed.During the to know of suspected Vibrio cholerae isolate, that is not feasible to do an oxidase test straight from a TCBS society because the acid created by the sucrose fermenting nests will inhibit the oxidase reaction.

List of oxidase hopeful organisms

Bacterial genera characterized as oxidase-positive include Neisseria and Pseudomonas etc. Genera the the Enterobacteriaceae family members are defined as oxidase negative. Mneomoics because that Oxidase confident Organisms- PVNCH (It’s simply an acronym inspired by the famous mnemonic because that Urease optimistic organisms-PUNCH)P: Pseudomonas sppV: Vibrio choleraeN: Neisseria sppC: Campylobacter sppH: Helicobacter spp/ Haemophilus spp. Aeromonas sppAlcaligensReferences and further readingsPatricia Shields, Laura Cathcart. 2010. Oxidase test protocol.Clinical Microbiology procedures Handbook, fourth Edition. (2016). American culture of Microbiology. Https://doi.org/10.1128/9781555818814