In this article, we're going to delve into what the Oxidase Test is, how it works, and what it reveals about bacteria. Specifically, we'll discuss its principles, methods (including false positives), and its importance in food hygiene when identifying certain Gram-negative bacteria.
What is the Oxidase Test?
Originally developed for clinical testing, the Oxidase Test helps distinguish between different types of Gram-negative bacteria. For instance, if bacteria isolated from a patient are Gram-negative, this test quickly determines whether they are Pseudomonas (like Pseudomonas aeruginosa), which are oxidase-positive, or other bacteria from the Enterobacteriaceae family, which are oxidase-negative.
Initially, the Oxidase Test was developed by Gordon and MLend in 1928, mainly to identify gonococcus. However, the paper-based version of the Oxidase Test, which uses TMPD reagents, was improved by Kovacs in 1956. Through this test, Kovacs discovered that Pseudomonas turned purple, while other Gram-negative bacteria like E. coli did not change color, allowing for quick differentiation.
In clinical tests, especially in emergency cases where there's no time to wait for culture results, physicians often use the Oxidase Test for rapid bacterial identification. After isolating bacteria from a patient's blood sample and confirming they are Gram-negative, the Oxidase Test allows for quick determination of whether these bacteria are Pseudomonas or other types of Gram-negative bacteria, usually from the Enterobacteriaceae family.
Why is the Oxidase Test Important?
The enzyme Cytochrome c oxidase targeted by the Oxidase Test is part of the electron transport chain in aerobic respiration. Not all bacteria possess this enzyme:
- Lactic acid bacteria that don't even have a citric acid cycle.
- Some anaerobic bacteria that do have the citric acid cycle but don't use oxygen as the final electron acceptor.
From a practical perspective in food microbiology,
- The oxidase test is not used to test Gram-positive bacteria as almost all of them are negative, and therefore there is no need to use it.
- Instead, the oxidase test is used to differentiate between Gram-negative bacteria, particularly between the Enterobacteriaceae group (which are oxidase-negative) and other Gram-negative bacteria.
This is roughly how it should be understood. The oxidase test is also used to further classify bacteria that were previously identified as fermentative (F) in the OF test.
- Bacteria whose main habitat is seawater or freshwater, such as Vibrio and Aeromonas, are oxidase positive.
- Bacteria whose primary habitat is the gut of terrestrial animals, such as E. coli and Salmonella, are oxidase-negative.
How Does the Oxidase Test Work?
Cytochrome c oxidase is an essential enzyme in bacterial aerobic respiration, facilitating the transfer of hydrogen from the citric acid cycle to oxygen, the final electron acceptor. The Oxidase Test checks whether bacteria have this specific enzyme.
The cytochrome C oxidase oxidizes cytochrome C, allowing the hydrogen energy to be transferred to the final hydrogen acceptor, oxygen.
The oxidase test is used to determine if bacteria have cytochrome C oxidase in their electron transfer system.
The test uses TMPD (Tetra-methyl-p-phenylenediamine dihydrochloride) as a color indicator. When oxidized by Cytochrome c oxidase, TMPD turns purple. This color change lets us know if the bacteria are oxidase-positive or not.
In essence, TMPD serves as an artificial electron donor. When electrons are taken from TMPD and oxidized, the color of the reagent changes to purple, indicating that the bacteria tested are positive for cytochrome C oxidase.
How to Perform the Oxidase Test
For a detailed procedure, please consult your lab manual. Here’s a summary:
- Drop TMPD liquid onto filter paper or a glass slide.
- Smear the bacterial sample onto the liquid using a platinum loop.
- Wait 10 seconds. If the sample turns purple, it's oxidase-positive; otherwise, it's negative.
Caveats
- The test relies on the oxidation of TMPD, which naturally occurs over time. So, make your observations strictly within 10 seconds to avoid false positives.
- Avoid using Nichrome wire loops as they can cause false positives due to their own oxidative properties. Platinum loops are recommended..
- For beginners, using known oxidase-positive controls like Pseudomonas and oxidase-negative controls like E. coli can make your test results more reliable.
And there you have it, a thorough yet easy-to-understand guide to the Oxidase Test, an essential tool for differentiating Gram-negative bacteria.
