Oxidase Test Procedure for Bacteria

In this oxidase test procedure for bacteria post we have briefly explained oxidase test principle, objectives, requirements, oxidase test in microbiology procedure, uses and limitations.

Oxidase Test in Microbiology

The electron transport chain is a collection of membrane-encased components that make up the final stage of bacterial respiration. The utilisation of the enzyme cytochrome oxidase, which catalyses the oxidation of cytochrome c while reducing oxygen to generate water, could be the final step in the chain.

As an artificial electron source for cytochrome c, the oxidase test in microbiology frequently employs the reagent tetra-methyl-p-phenylenediamine dihydrochloride. When cytochrome c oxidises the reagent, it transforms into indophenol blue, a dark blue or purple compound.

One electron from each of four cytochrome c molecules is momentarily transferred to the enzyme in bacteria that contain the enzyme cytochrome oxidase. This creates four electron-poor cytochrome c molecules and an electron-rich cytochrome oxidase enzyme.

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.

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.

Principle

Oxidase test in microbiology 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.

Requirements

  1. Kovács oxidase reagent
  2. Gordon and McLeod reagent
  3. Gaby and Hadley oxidase test
  4. Inoculating loop
  5. Incubators
  6. Supplemental media
  7. Whatman (No.1) filter paper
  8. Wooden applicator stick

Oxidase Test Procedure for Bacteria

Filter Paper Method

1. Allow to dry a small piece of filter paper that has been soaked in 1 percent Kovács oxidase reagent. Pick a well-isolated colony from a fresh (18 to 24-hour culture) bacterial plate with a loop and rub it onto treated filter paper.

2. Keep an eye out for hue shifts. When the colour shifts from light purple to dark purple in 5 to 10 seconds, microorganisms are oxidase positive.

3. When the colour turns to purple within 60 to 90 seconds, microorganisms are delayed oxidase positive. If the colour does not change or takes longer than 2 minutes, the microorganism is oxidase negative.

Oxidase Test

Oxidase Test in Microbiology: Filter Paper Method

Filter Paper Spot Method

1. Pick a well-isolated colony from a fresh (18–24 hour culture) bacterial plate with a loop and rub it onto a small piece of filter paper.

2. On the organism smear, put 1 or 2 drops of 1 percent Kovács oxidase reagent. Keep an eye out for hue shifts.

3. When the colour shifts from light purple to dark purple in 5 to 10 seconds, microorganisms are oxidase positive. When the colour turns to purple within 60 to 90 seconds, microorganisms are delayed oxidase positive.

4. If the colour does not change or takes longer than 2 minutes, the microorganism is oxidase negative.

Oxidase Test

Oxidase Test in Microbiology: Filter Paper Spot Method

Direct Plate Method

1. Use the streak plate method to grow a fresh culture of bacteria (18 to 24 hours) on nutrient agar such that well-isolated colonies are present.

2. On the organisms, drop 1 or 2 drops of 1 percent Kovács oxidase reagent or 1 percent Gordon and McLeod reagent. Inverting or flooding the plate is not recommended.

3. Keep an eye out for hue shifts. Microorganisms are oxidase positive when the colour changes to dark purple within 5 to 10 seconds while employing Kovács oxidase reagent.

4. When the colour turns to purple within 60 to 90 seconds, microorganisms are delayed oxidase positive. If the colour does not change or takes longer than 2 minutes, the microorganism is oxidase negative.

5. Microorganisms are oxidase positive when the colour changes to red within 10 to 30 minutes or to black within 60 minutes when using Gordon and McLeod reagent. If the colour of the microorganism does not change, it is oxidase negative.

Oxidase Test

Oxidase Test in Microbiology: Direct Plate Method 

Test Tube Method

1. In 4.5 mL of nutrient broth, grow a fresh culture of bacteria (18 to 24 hours) (or standard media that does not contain a high concentration of sugar.

2. Add 0.2 mL of 1% -naphthol, followed by 0.3 mL of 1% paminodimethylaniline oxalate. Keep an eye out for hue shifts.

3. When the colour changes to blue within 15 to 30 seconds, microorganisms are oxidase positive.

4. When the colour changes to purple within 2 to 3 minutes, microorganisms are delayed oxidase positive. If the colour of the microorganism does not change, it is oxidase negative.

Oxidase Test in Microbiology: Test Tube Method

Results

Positive Result

Microorganisms are oxidase positive when the colour changes to dark purple within 5 to 10 seconds while employing Kovács oxidase reagent. When the colour turns to purple within 60 to 90 seconds, microorganisms are delayed oxidase positive.

Negative Result

Colour does not change or takes longer than 2 minutes; the microorganism is considered oxidase negative

Limitations

1. Because oxidase reagent has auto-oxidizing properties, it should always be used fresh. Oxidase reagents should not be kept for more than one week.

2. Because both bacteria and yeast on culture media containing high concentrations of glucose, such as Nutrient agar, inhibit oxidase activity, it is critical to test colonies grown on culture media without excess sugar. A great media to use is tryptic soy agar.

3. Bacteria that grow on dye-containing media can produce unexpected results. Because the reagents will kill the organisms, it will be necessary to subculture the microorganisms before adding any additional reagent to an active culture plate.

4. The oxidase test in microbiology can be performed to determine whether or not Neisseria is present, as well as to differentiate and identify Gram-negative bacteria. Microorganisms that test positive for oxidase must be stained with gram stain to see or check their morphology and gram reaction.

5. Wire loop made of nichrome or other iron-containing materials can give false positive results. The best and most recommended wire loops are platinum wire loops.

6. To ensure accurate results, gram-negative bacilli should have their oxidase reactions measured on non-selective and non-differential media.

7. Colonies obtained from culture conditions containing a high glucose concentration may produce misleading negative results in oxidase test in microbiology.

8. It’s crucial to use colonies that are 18 to 24 hours old for oxidase test in microbiology, as younger colonies may have weaker reactions.

Further Readings

Reference