OXIDASE TEST: RATIONALE, PROCEDURE, AND USES - BIOLOGY - 2024

The oxidase test is a diagnostic method that shows the presence of the enzyme complex called cytochrome oxidase c. This system induces the transformation of reduced cytochrome to oxidized, since it captures oxygen and this in turn acts as the last electron acceptor (H ⁺) in the respiratory chain.

The term oxidase is a shorthand way of referring to the enzyme cytochrome oxidase, also known as indophenol oxidase. In ancient times it was believed that the enzymes cytochrome oxidase and indophenol oxidase were two different enzymes, but today they are known to be the same.

Positive and Negative oxidase test. Source: No machine-readable author provided. Alpha.prim ~ commonswiki assumed (based on copyright claims).

For their part, cytochromes are hemoproteins that contain iron and complete the cytochrome oxidase system. Cytochromes can vary from species to species.

There are different varieties of cytochromes (cytochromes a1, a2, a3 and 0). Some bacteria can produce only one, but others up to two or three at a time. In this sense, the presence of cytochrome a and a3 is known as cytochrome oxidase c. This is the type of cytochrome that the oxidase test detects.

The genera Neisseria and Pseudomonas contain cytochrome oxidase c. These genera give the positive oxidase test, helping to differentiate them from the Acinetobacter and Stenotrophomonas genera respectively.

There are also other genera that are oxidase positive.

Basis

Characteristics of the cytochrome oxidase c system

The cytochrome oxidase c system works in the following way: oxidase-positive microorganisms use oxygen to generate energy through aerobic respiration. This system works thanks to the transport of electrons from donor substances such as NADH ⁺ to receptor substances, in this case oxygen.

This results in the production of energy (ATP) and water or hydrogen peroxide, depending on the cytochrome oxidase system that the microorganism possesses.

That is why most of the oxidase positive bacteria are also catalase positive, a necessary condition to eliminate the hydrogen peroxide produced, since this substance is toxic to bacteria.

The cytochrome oxidase c system is present in some aerobic bacteria, some facultative anaerobes, few microaerophilic ones, and no strict anaerobes. The latter is understandable, since strict anaerobes cannot live in the presence of oxygen, therefore they lack the cytochrome oxidase system.

Test principle

In this test it uses substances that act as artificial electron acceptors, substituting natural ones within the electron transport chain.

Mainly, dyes such as paraphenylenediamine and indophenol are used, which act as receptor substrates and artificial electron donors.

Paraphenylenediamine is oxidized by the cytochrome oxidase c system. The dye in its reduced form is colorless, but in its oxidized form it is colored.

This is how the presence of the cytochrome oxidase c system is evidenced; since a positive reaction will generate a lavender or blue-purple color depending on the reagent used.

On the other hand, if the last electron accepting substance in the respiratory chain is different from oxygen, the oxidase test will be negative (there is no color production); this is the case with anaerobic microorganisms.

Likewise, if the cytochrome used by the microorganism is different from cytochrome oxidase c, it will also give the negative test.

Process

There are various reagents and protocols for the oxidase test, all for the same purpose.

Reagents

Kovacs reagent, Gordon and McLeod reagent, Nadi reagent, Carpenter, Suhrland and Morrison reagent, and use of oxidase discs.

-Kovacs oxidase reagent

It is made up of 1% tetramethyl-p-phenylenediamine dihydrochloride.

Kovacs's reagent is prepared by dissolving 1 g of the above-mentioned substance in 50 ml of distilled water. It is heated subtly until completely dissolved. Transfer to an amber bottle of sufficient capacity and make up the volume to 100 ml with distilled water. Wait at least 15 minutes before using. Store in a refrigerator protected from light.

It is labeled Kovacs oxidase reagent, to differentiate it from the Kovacs reagent used to reveal the indole test. This reagent is the most sensitive, less toxic but more expensive than the rest of the reagents.

A positive reaction will be evidenced with this reagent with the colony color change to lavender, which rapidly turns purple almost black. A negative reaction is evident because there is no color change in the colony or it takes on a slight pinkish color. The medium can also darken, but that does not mean a positive reaction.

With this reagent, the reaction time is crucial, a color change that occurs between 5 to 15 seconds is considered a positive reaction.

-Gordon and McLeod reagent

It is composed of dimethyl-p-phenylenediamine dihydrochloride, also known as N-dimethyl-p-phenylenediamine or p-aminodimethylaniline monohydrochloride. It is prepared as described for the Kovacs oxidase reagent, substituting for the substance involved.

This reagent is slightly more stable than Kovacs oxidase reagent, although all reagents containing p-phenylenediamine are unstable.

This reaction is later, it is interpreted as positive with the appearance of a blue-purple color within 10 to 30 minutes.

-Nadi reagent

It is composed of 1% α-naphthol in ethyl alcohol (95% ethanol) and 1% aminodimethylaniline. The mixture is prepared in equal parts and using absolute ethyl alcohol as a diluent, until making up a sufficient quantity for 100 ml.

-Carpenter, Suhrland and Morrison reagent

It is composed of 1% p-aminodimethylalanine oxalate. Prepare in the same way as described for Kovacs oxidase reagent, changing for the corresponding substance.

With the solution ready, test strips are prepared as follows: 6-8 cm Whatman No. 1 filter paper strips are impregnated with 1% dimethyl-p-phenylenediamine oxalate reagent.

They are allowed to dry without contact with metal, store in screw-capped jars with desiccant and store in the refrigerator. These strips are stable for up to 6 months.

It is the most stable reagent of all those mentioned, and can last up to 6 months in solution. Another plus point is that it does not color the medium around the colony, if it is used directly on the plate.

The appearance of a red color is interpreted as a positive test.

-Oxidase discs

They are commercial discs that are impregnated with reagent for the oxidase test. There are several commercial brands on the market.

Its use is quite practical, since it is not necessary to prepare fresh reagents, which facilitates the work. The results obtained are reliable as long as the discs are properly preserved.

Protocols

Direct plate method, indirect method on paper and use of discs impregnated with oxidase reagents.

-Direct board method

2 or 3 drops of any of the aforementioned reagents are added for this purpose directly on the colony (s) contained in a plate of culture medium that does not contain glucose.

The change or not of color of the colonies is interpreted, not of the medium. The valid reaction time depends on the reagent used.

-Indirect method on paper

Cut a piece of filter paper (Whatman No. 1) to a size of 6 cm ² and place it inside an empty Petri dish.

Add 2 or 3 drops of the Kovacs oxidase reagent to the paper, take part of the colony to be studied with a platinum handle or wooden toothpick and spread it in a straight line on the reagent impregnated paper. Interpret within 5 to 10 seconds.

With the strips prepared with Carpenter, Suhrland and Morrison reagent, a colony is spread on the dry strip. A single strip is used to test several strains. Interpret in 10 sec.

-Disks (m

Subtly moisten the commercial discs with sterile distilled water and superimpose on the colony to be studied. It is recommended to use the plates at 35 ° C, if plates at room temperature or refrigerated plates are used the reaction is a little slower. Interpret the color change between 10 to 20 sec.

Colonies contained on blood or chocolate agar can be used.

-Disks (indirect method)

Dampen the disc as previously described. Place it in an empty Petri dish. Take a sufficient amount of the colony to study with a platinum handle or wooden toothpick and place on the disk. Interpret the color change between 10 to 20 sec.

Use

The genus Neisseria and Acinetobacter are sometimes very similar morphologically because even though the genus Acinetobacter is a Gram-negative rod, it can sometimes take a coccoid form and be distributed in pairs, simulating the genus Neisseria.

In this case the oxidase test is really useful. The Neisseria genus is positive and Acinetobacter negative.

However, the genus Moraxella is very similar to the genus Neisseria and both give a positive reaction; This is why carbohydrate fermentation tests must always be carried out for definitive identification.

On the other hand, the oxidase test is useful to differentiate a bacterium belonging to the Enterobacteriaceae family (all oxidase negative) from other fermenters, such as the genus Pasteurella, Aeromonas, Plesiomonas (oxidase positive).

The genus Vibrio and Helicobacter are also oxidase positive.

QA

Use known strains of Escherichia coli as a negative control and strains of Pseudomonas aeruginosa as a positive control.

Limitations

-The reagents must be used freshly prepared, their shelf life in solution at room temperature is short because they are very unstable. Refrigerated they can last between 5 days to 2 weeks.

-The reagents are colorless, if they change color they must be discarded. Damaged discs show up because they darken over time.

-A positive reaction with the Kovacs oxidase reagent between 15-60 sec is considered a delayed reaction and after 60 seconds it should be considered negative.

-Haemophylus influenzae gives a negative oxidase reaction if any reagent with dimethyl-p-phenylenediamine is used, but positive if the Kovacs oxidase reagent (tetramethyl-p-phenylenediamine) is used.

-Mediums containing glucose interfere with the test, giving false negatives.

-Bordetella pertussis strains can give a false positive reaction if they come from highly concentrated blood agar plates.

-The use of metal (iron) handles gives a false positive reaction.

recommendations

-Because the reagents are very unstable and tend to self-oxidize, it is recommended to freeze aliquots of 1 to 2 ml and remove as needed.

-Another way to delay the auto-oxidation of the reagent is to add 0.1% ascorbic acid when preparing the reagents.

-As the reagents are unstable, a weekly quality control is recommended.

-Reagents that do not pass the quality control test should not be used.

References

1. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.
2. Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.
3. "Oxidase Test." Wikipedia, The Free Encyclopedia. Jan 15 2018, 10:32 UTC. 3 Apr 2019, 14:03
4. World Health Organization. Laboratory Manual for the Identification and Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public Health Importance in the Developing World. 2004. Available at: who.int/drugresistance/infosharing
5. Reagent strips for the diagnosis of oxidase activity in bacteria. Rev Cubana Med Trop. 2000; 52 (2): 150-151.