OF Test- Objective, Principle, Procedure, Results, Limitations

Objective of Oxidation-Fermentation (OF) Test

To differentiate microorganisms based on the ability to oxidize or ferment specific carbohydrates.

Principle of Oxidation-Fermentation (OF) Test

Carbohydrates are organic molecules that contain carbon, hydrogen, and oxygen in the ratio (CH2O)n. Organisms use carbohydrates differently depending upon their enzyme complement. The pattern of fermentation is characteristics of certain species, genera, or groups of organisms and for this reason, this property has been extensively used as a method for biochemical differentiation of microbes. Glucose after entering a cell can be catabolized either aerobically in which molecular oxygen can serve as the final electron acceptor indicating oxidative metabolism or anaerobically in which inorganic ions other than oxygen, e.g. NO3¯ or SO4¯ ¯ can serve as the final electron acceptor indicating fermentative metabolism or both aerobic and anaerobic pathways and some organism lack the ability to oxidize glucose by either. The metabolic end products of carbohydrate fermentation can be either organic acids (lactic acid, formic acid, or acetic acid) or organic acid and gas (hydrogen or carbondioxide).

An oxidation fermentation test is used to determine whether an organism uses carbohydrate substrates to produce acid byproducts. Non-fermentative bacteria are routinely tested for their ability to produce acid from six carbohydrates (glucose, xylose, mannitol, lactose, sucrose, and maltose). Whether an organism is oxidative or fermentative can be determined by using Hugh and Leifson’s medium, commonly called as OF medium which contains tryptone and bromothymol blue (an indicator). Two tubes are required for interpretation of the OF test. Both are inoculated, and one tube is overlaid with mineral oil, producing an anaerobic environment. The growth of microorganisms in this medium is either by utilizing the tryptone which results in an alkaline reaction (dark blue color) or by utilizing glucose, which results in the production of acid (turning bromothymol blue to yellow). Production of acid in the overlaid tube and open tube results in a color change and is an indication of fermentation. Acid production in the open tube and color change is the result of oxidation.

Media Used in OF Test

Hugh and Leifson’s medium: Peptone 2.0gm/L,  Sodium chloride 5.0gm/L,  Dipotassium phosphate 0.30gm/L,  Glucose (Dextrose) 10.0gm/L,  Bromothymol blue 0.030gm/L, Agar 3.0gm/L,  Final pH ( at 25°C) 7.1±0.2

Procedure of Oxidation-Fermentation (OF) Test

  1. Inoculate two tubes of OF medium with organism by stabbing with a straight wire.
  2. Pour liquid paraffin over the medium to form a layer about one cm in depth into one of the tubes.
  3. Incubate the tubes at 35-37°C for 24-48 hours.
  4. Examine both open and closed tubes for the color change.

Result Interpretation of Oxidation-Fermentation (OF) Test

Result Interpretation of OF Test

Oxidative: yellow coloration in the open tube only.

Fermentative: yellow coloration on both open and closed tubes.

Limitations of Oxidation-Fermentation (OF) Test

  • OF medium is general purpose medium and may not support the growth of fastidious organisms.
  • Slow-growing organisms may not produce results for several days.

Quality Control

OrganismNature of organismAerobic fermentationAnaerobic fermentation
Escherichia coli ATCC 25922FermentativeAcid production (Yellow), positive reactionAcid production (Yellow ), positive reaction
Pseudomonas aeruginosa ATCC 27853OxidativeAcid production (Yellow ), positive reaction unchanged (green) or alkaline (blue), negative reaction


  1. Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology, Thirteen edition, Mosby, Inc., an affiliate of Elsevier Inc., 3251 Riverport Lane, St. Louis, Missouri 63043
  2. Aneja K.R. 2003. Experiments in Microbiology, Plant Pathology and Biotechnology, fourth revised edition, New Age International (P) limited, Ansari road, Daryaganj, New Delhi-110002.

About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He attended St. Xavier’s College, Maitighar, Kathmandu, Nepal, to complete his Master of Science in Microbiology. He worked as a Lecturer at St. Xavier’s College, Maitighar, Kathmandu, Nepal, from Feb 2015 to June 2019. After teaching microbiology for more than four years, he joined the Central Department of Microbiology, Tribhuvan University, to pursue his Ph.D. in collaboration with Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. He is interested in research on actinobacteria, myxobacteria, and natural products. He has published more than 15 research articles and book chapters in international journals and well-renowned publishers.

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