Oxidation Fermentation Test - Principle, Purpose, Biochemistry, Media, Procedure, Result, Interpretation
Introduction to Oxidation Fermentation Test
The oxidation fermentation (O/F) test is a biochemical test used to determine/differentiate the ability of microorganisms to metabolize carbohydrates and related compounds.
Principle of Oxidation Fermentation Test
Carbohydrates are large organic molecules that can be categorized into three types- monosaccharides (which contains 3-7 carbon atoms), disaccharide (which contain two monosaccharide molecules), and polysaccharide (which contains eight or more monosaccharide molecule). Each carbohydrate molecule is made up of hydrogen, oxygen, and carbon in the ratio (CH2O)n.
Many microorganisms including bacteria and fungi are able to decompose carbohydrates to release energy which is referred to as catabolism. However, each organism may utilize carbohydrates in a different way depending on the enzyme complement system which results in various different products.
This differentiation in enzyme activity and end-product release has been used to differentiate as well as identify genera, species, or groups of organisms.
The OF (Hugh and Leifson's) medium can be used to differentiate such a group of organisms on the basis of carbohydrate utilization. Two test tubes containing OF media inoculated with test organisms are used. One test tube is sealed by pouring a thick layer of paraffin oil to maintain an anaerobic environment.
If the organism utilized tryptone, the media turns alkaline hence the dark blue color. And if the organism metabolizes glucose, acid is produced turning BTB in media to yellow. The microorganisms able to produce acid in both closed and open tubes are fermentative while the organisms which produce acid in just the open tube is called oxidative.
Purpose of Oxidation Fermentation Test
The oxidation fermentation test is performed for the following reasons:
To distinguish between the aerobic and anaerobic breakdown of carbohydrates
To show the organism's ability to utilize a particular substrate
To test for specific breakdown products
To show the types and range of carbohydrates that can be attacked and spoiled
Biochemistry of Oxidation Fermentation Test
Organisms release extracellular enzymes such as amylases and intracellular enzymes such as maltase, and lactase to cleave the carbohydrate molecule to produce end products such as glucose and galactose. Microorganisms having the ability to metabolize organic carbohydrate compounds to produce energy are called chemoorganotrophs.
In a cell, glucose molecules are catabolized aerobically (where oxygen serves as the final electron acceptor) or anaerobically (where NO3- or SO42- may serve as the final electron acceptor) or both aerobic and anaerobic pathways. Some organisms, on the other hand, may even lack a pathway to metabolize glucose molecules.
In the case of carbohydrate fermentation, the end product may be organic acids (lactic acid, formic acid) and/or gas (hydrogen, carbon dioxide).
Media used for Oxidation Fermentation Test
To distinguish between aerobic and anaerobic paths used by microorganisms when metabolizing carbohydrates, a semi-solid medium containing carbohydrates and indicators such as Bromothymol blue (BTB) or Bromocresol purple (BCP) is used. One such commercially available medium is Hugh and Leifson's medium (OF medium).
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Image: O/F test - fermentative (a), oxidative (b), glucose not metabolized (c) (Source: Auburn University)
Procedure of Oxidation Fermentation Test
Take two test tubes containing O/F medium and inoculate each tube with a freshly cultured test organism.
Pour 3-4 ml of paraffin oil into one of the inoculated tubes.
Incubate each test tube at 35-37°C for 18-24 hours (for bacteria) or at 28°C for 3-4 days (for fungi).
Observe for microbial growth and color change of media
Result, Interpretation of Oxidation Fermentation Test
The change of OF media from green to yellow in both tubes indicates a fermentative organism.
If only one tube (without paraffin oil) has its color changed after incubation, the organism is an oxidative organism. The tube with paraffin oil remains green.
Fermentative organism
E. coli
Oxidative organism
Pseudomonas aeruginosa