Oxidation-Fermentation (OF) Test

Objective of OF Test

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

Principle of OF Test

Carbohydrates are organic molecules that contain carbon, hydrogen and oxygen in the ratio (CH2O)n. Organisms use carbohydrate 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 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).

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 contain 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. Growth of microorganisms in this medium is either by utilizing the tryptone which results in an alkaline reaction (dark blue colour) 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 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 OF Test

Result Interpretation of OF Test

Oxidative: yellow colouration in open tube only

Fermentative: yellow colouration on both open and closed tubes

Limitations of 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 of OF Test

Organism Nature of organism Aerobic fermentation Anaerobic fermentatoin
Escherichia coli ATCC 25922 Fermentative Acid production (Yellow), positive reaction Acid production (Yellow ), positive reaction
Pseudomonas aeruginosa ATCC 27853 Oxidative Acid 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.

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