Oxidase Test- Principle, Uses, Procedure, Types, Result Interpretation

The scientific method is packed to the brim with hundreds of thousands of different tests. With so many methods and procedures, it can start to feel like they blend after a certain point.

So many of them have seemingly identical-sounding names, 10 different ways to carry them out, and twice as many ways to make the results you find invalid!

Not only is it frustrating to carry out the entire process again, but you may also be working with a limited resource pool that you are testing, making a second try at a scientific study a logistical nightmare.

Fortunately, there are some simple and easy ways around it.

For starters, having a good understanding of the specific test you are carrying out at that moment, especially why and how you are doing it, can be a great way to focus on paying attention to the procedure.

Even if you aren’t a dedicated researcher, and you’re unlikely to see the lens of a microscope in your day-to-day life, who doesn’t love to know a little more about the scientific process?

In this particular article, we are going to be taking a closer look at the Oxidase test.

We are going to discuss what exactly its purpose is and what it hopes to find or understand, the uses that it has in the scientific method and beyond, how the procedure is carried out for testing, as well as what the results that you can expect from it are.

We’re also going to discuss some precautions that you may want to take, before or during this test, to help keep your results as valid as possible.

Whilst a mistake is always a learning experience, it’s also a frustrating lesson. One that can sometimes be avoided, with a little extra prep-work.

So, with that out of the way, let’s get started!

The Purpose Of The Oxidase Test

So, if you are carrying out, or are planning to carry out the oxidase test shortly, it probably pays to know exactly what you’re looking for when carrying out this test.

When you are running the oxidase test, you are testing for the presence of an enzyme that is found in many different types of organisms, known as Cytochrome c oxidase.

What Cytochrome C Oxidase Is, And Why It Is Important

This particular enzyme is found in the membrane of many cells and is a vital part of the transport chain of electrons used in the respiratory process of organisms.

The enzyme in question will receive the electrons from Cytochrome C molecules, which donate electrons to other respiratory sites.

This is the final hydrogen receptor used in the respiratory process, and is used at the end of a long chain of processes that releases a large amount of energy and water molecules, and is vital for aerobic respiration in organisms that use this biological process.

The Principle Of The Oxidase Test

In the Oxidase test, this process is observed by putting the bacteria you are testing in question as a type of reagent, a substance used to test the presence of other test samples in many biological, chemical and biochemical tests.

Because the Cytochrome c oxidase enzyme helps transport electrons, the regent that is used helps make their presence of them visible for researchers to observe and see for themselves by oxidizing them, hence the name of the test.

The reagent, usually a colorless solution before testing begins, will start to change if the presence of the enzyme in question is found, going from the original clear appearance, to a deep purple color.

The oxidase test can be done with several different reagents.

Whilst Kovacs oxidase reagent, a 1% tetramethyl-p-phenylenediamine dihydrochloride solution mixed with water, is the most common, there are many other reagents that this test can be carried out with.

Gordon and Mcleod’s reagent is made from 1% dimethyl-p-phenylenediamine dihydrochloride solution and is also used in oxidase tests, amongst others.

The Uses For The Oxidase Test

When doing this test, it also helps to know what the uses for this test are, as a scientist and a researcher. You don’t want to be carrying out a test that doesn’t have some useful result to measure, after all.

As we’ll see, this test is vital for research in the fields of biochemistry and medicine.

As we discussed in the purpose of this test, and as the test name implies the oxidase test is very useful for detecting the presence of the Cytochrome C oxidase enzyme in bacteria and other organisms, as well as discerning if and which bacterial organisms do not have this particular enzyme.

Whilst this may seem to be somewhat niche research, the enzyme that is being tested is a vital part of most bacterial functions, as well as for many bacteria that live inside the human body.

The consequences of defects and mutations can have disastrous effects on organisms that otherwise depend on this enzyme to carry out the last stages of the respiratory process.

Tests such as this can help establish whether these enzymes are or are not being produced in bacteria that otherwise should, which can be an indicator of metabolic or mitochondrial diseases.

The oxidase test is also an effective way to differentiate between various species of Pseudomonas, a family of antibiotic-resistant bacteria, but can also play a key role in the clearing of polluting agents in the environment, as well as preventing crop plant species from being infected with other pathogens that can ruin crop yields.

As we have shown, this test has a wide array of uses across many areas that potentially affect our everyday lives.

Methods And Procedures Used In The Oxidase Test

As you can imagine, there are a variety of different ways the procedure of this test can be carried out, depending on your method of preference, or the resources you have available.

Generally speaking, however, the most common ways that the oxidase test is carried out are the following methods:

Wet Filter Paper Method

This method involves a reagent solution, a bacteria culture that you are looking to test, a platinum loop, and a strip of filter paper.

  1. The filter paper is first dampened with the 1% solution of whatever reagent you are using and has been prepared recently.
  2. Using the platinum loop, the culture of bacteria that you are examining is rubbed onto the dampened spot of your filter paper.
  3. After a small period, usually around 5 to 10 seconds, the areas of the paper dampened with the reagent will start to react with the bacteria culture if the oxidase enzyme is present, turning from colorless to pink, before then becoming maroon, before a deep purple color.
    • The exact time will vary depending on the amount of enzyme present, although a result should take longer than 60 seconds to appear.

Dry Filter Paper Method

This method is very similar to the wet filter method. However, the different steps and equipment used to make this a very different method overall.

  1. A piece of filter paper is first soaked in the researcher’s reagent of choice, that has been recently prepared for the experiment.
  2. Once the filter is soaked, it is then allowed to drain for 30 seconds, before it is then freeze-dried, whilst being stored in a dark space. The usual vessel of choice is a bottle that has been darkened to prevent light from spoiling the reagent that is sealed airtight.
  3. A strip of the freeze-dried filter is then taken out, and moistened with distilled water.
  4. Then, using a platinum loop, as with the wet method, the colony of bacteria you are testing is transferred to the moistened spot on the filter.
  5. The results should be clear to an observer in a similar time frame to the wet filter method.

This is a great way to prepare testing samples if you have already prepared a reagent, which generally has a very short usable shelf life once ready to use.

Direct Plate Method

This method uses both the reagent that all oxidase tests require and the bacteria colony, as well as a non-selective agar plate. Aside from the equipment, this method is relatively straightforward.

  1. Place the cultures of bacteria you are testing in the agar plate.
  2. Then place 2 to 3 drops of your reagent on the colonies in the agar plate.
  3. Your results should start to be visible for observation within a few seconds.

A few other notes when carrying out this method:

  • The color that your reagent will change once the bacteria colony is added will change depending on what variant you are using.
    • Kovac’s reagent oxidase will turn into a deep purple.
    • Gordon and Mcleod’s reagent oxidase will instead turn red, before becoming a black color.
  • Regardless of which reagent you use, the change of color will signify the presence of the enzyme.

Test Tube Method

  1. You will need your bacteria, reagent, and nutrient broth to help cultivate the bacteria colony, as well as a test tube for this method.
  2. Cultivate and grow your bacteria culture in a test tube for 18 to 24 hours before the test, using the nutrient broth. You’ll need approximately 4.5 grams to get the appropriate amount.
  3. Add 0.5 ml of Gabley & Habley’s reagent (approximately 0.2 ml of 1% α-naphthol and 0.3 ml of 1% p-aminodimethylaniline oxalate, respectively) to the test tube.
  4. Once the reagent has been added, make sure to thoroughly shake and mix your test tube for 30 seconds, either through mechanical or manual means
  5. Once the shaking has stopped, observe any color changes that take place. You should start to see them within 30 to 45 seconds, although some slower reactions may take up to 3 minutes to be observable.

Swab Method

This is a very simple method and only requires a medical swab alongside the usual reagent and bacteria cultures.

  1. Add a small amount of reagent to your medical swab.
  2. Wipe a small amount on to bacteria colony that is isolated.
  3. Observe the color changes within 10-15 seconds.

The Results Of The Oxidase Test

There are a few results that you are likely to get found in this test.

  • Large amounts of Cytochrome c oxidase will create a very strong and quick positive reaction, changing colors in less than 30 seconds.
  • A weak positive reaction will cause some color change within a few minutes.
  • A negative result will not change the color of the reagent in any way, staying clear.

Advice For Carrying Out The Oxidase Test

  • The reagent needs to be prepared fresh for your test. Leaving it for more than a few hours may invalidate your test results.
  • Make sure your bacteria culture are grown in non-dyed containers, otherwise the result may yield a false positive.

Final Thoughts

So, whilst the test is a little complicated to understand at first, the result you’re looking for is pretty simple to understand. Now you’re ready to carry it out for yourself!

Jennifer Dawkins