Oxidation-Fermentation (OF) Test: Introduction, Composition of the medium, Test Requirements, Principle, Procedure, Result -Interpretation, and Limitations

Oxidation-Fermentation (OF) Test Introduction

Hugh and Leifson developed an Oxidation-Fermentation (OF) basal medium in 1953. It is a recommended medium for the detection of oxidation or fermentation of carbohydrates by bacteria. This basal medium is useful to aid in the identification of gram-negative bacteria on the basis of their ability to oxidize or ferment a specific carbohydrate. As compared to other OF Media, Hugh and Leifson’s formula employs a low peptone/carbohydrate ratio and a minimal amount of agar i.e. 2% from 3%. The decreased (2% from 11%) amount of peptone reduces the formation of alkaline amines which can ultimately mask the small quantities of acid. The acid may produce from oxidative metabolism.

The increased carbohydrate (1% from 0.5%) results in an increase in the amount of acid that may form. The bromthymol blue indicator can detect it. The small amount of agar added to the medium provides a semi-solid structure that concentrates the acid at the point of reaction, thereby facilitating the visual interpretation of the pH shift.

Proper performance of the Oxidation-Fermentation (OF) Test requires an organism to be inoculated into two tubes of each OF medium. Once inoculated, one tube is overlaid with mineral oil or melted paraffin. The other tube is left open to the air. Oxidative utilization of the carbohydrate will result in acid production (yellow) in the open tube only. Fermentative utilization of the carbohydrate will result in acid production (yellow) in both the open and closed tubes. Acidic changes in the overlaid tubes are due to a result of true fermentation, while acidic development in the open tubes is due to oxidative utilization of the carbohydrate present. Asaccharolytic (non-fermenter and non-oxidizer) organisms will not produce acid in either tube.

Principle of Oxidation-Fermentation (OF) Test

The oxidation-Fermentation (OF) Test determines if certain gram-negative bacilli metabolize glucose by fermentation or aerobic respiration (oxidatively). During the anaerobic process of fermentation, pyruvate converts to a variety of mixed acids depending on the type of fermentation. The high concentration of acid produced during fermentation will turn the indicator, bromthymol blue present in OF  basal medium from green to yellow in the presence(oxidatively) or absence of oxygen (fermentatively). Some nonfermenting gram-negative bacteria metabolize glucose using aerobic respiration and therefore only produce a small number of weak acids during glycolysis and the Krebs cycle. The decreased amount of peptone and increased amount of glucose helps the detection of weak acids so produced. The dipotassium phosphate buffer present in the medium is useful to further promote acid detection. It is also useful to test the non-saccharolytic (have no ability to use the carbohydrate in the media) activities of organisms.

Composition of the OF Basal Medium

Constituents of Hugh and Leifson’s OF basal medium are as follows:

Ingredients Gram weight
Sodium chloride0.5
Pancreatic digest of Casei0.2
Di-potassium phosphate0.03
Bromothymol blue0.008
Distilled water (D/W)100 ml
Final pH 6.8+/- 0.2 at 25ºC
Table: Composition of OF Basal Medium

After autoclaving at 121°C for 15 minutes, add OF media with carbohydrates containing 10.0gm/L of specific carbohydrates i.e. dextrose, maltose, lactose, sucrose, etc.

Difco OF Basal Medium
Fig. Difco OF Basal Medium

Requirements for Oxidation-Fermentation (OF) Test

  1. Test organisms ( Gram-negative bacilli) pure well-isolated colonies from an 18-24-hour culture
  2. OF basal media
  3. Testing Carbohydrates (glucose, sucrose, maltose, etc.)
  4. Test tubes
  5. Sterile mineral oil or liquid paraffin  or sterile melted petrolatum
  6. Bunsen burner
  7. Inoculating wire
  8. Incubator
D-Glucose for Oxidation-Fermentation Test
Fig. D-Glucose for Oxidation-Fermentation Test
Liquid Paraffin for Aanaerobiotic Environment Creation in Fementation Test of Bacteria
Fig. Liquid Paraffin for Aanaerobiotic Environment Creation in Fermentation Test of Bacteria

Quality control strains

Procedure for OF Test

  1.  Bring the basal media to equilibrate to room temperature.
  2. Take pure well-isolated colonies from an 18-24 hour culture.
  3. For each test organism, inoculate two basal media tubes and inoculate by stabbing the agar to approximately 1/4 inch from the bottom.
  4. Apply with a 1 cm layer of either sterile mineral oil or liquid paraffin or sterile melted petrolatum to one of each two tubes. Tighten the cap of the overlaid tube, and loosen the cap of the non-overlaid tube.
  5. Incubate both tubes aerobically at 35ºC for 48 hours (Slow-growing bacteria may take 3 to 4 days before results can be observed).
  6. Examine tubes daily.
  7. A control tube of OF Base Medium should be inoculated and incubated in parallel with the OF tests.


Notice acid production in the medium by the appearance of a yellow color. In the case of oxidative organisms; color production may be first noted near the surface of the medium.

Interpretation of Result for OF(Oxidation-Fermentation)Test

A positive carbohydrate utilization test indicates the development of yellow color in the medium.

A negative carbohydrate utilization test indicates the absence of a yellow color (media remains green or turns blue).

The method of metabolism is determined as follows:

Fermenter: Acid production on both i.e. open and overlaid tubes. The acid produced changes the pH indicator, bromothymol blue, from green to yellow

Oxidizer: Acid production in the open tube (aerobic) and not in the oil-covered tube (anaerobic) indicates an oxidizer.

Non-saccharolytic or non-utilizer or Negative oxidative and fermentative organisms: The negative result indicates no color change in the oil-covered tube and in some cases an increase in pH (pH 7.6) changing the bromothymol blue from green to blue in the top of the open tube. The increase in pH is due to amine production by bacteria that break down the peptone (protein) in the medium.

Result of quality control strains

  1. Escherichia coli: Glucose Fermenter
  2. Pseudomonas aeruginosa: Glucose oxidizer
  3. Alcaligenes faecalis: Nonsaccharolytic
Oxidation Test of Bacteria Positive and Negative Results
Fig. Oxidation Test of Bacteria Positive and Negative Results
Fermentation Test Positive and Negative Results of Bacteria
Fig. Fermentation Test Positive and Negative Results of Bacteria

Limitations Oxidation-Fermentation (OF) Test

  1. It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
  2. Bacteria that only oxidize dextrose will not ferment any other carbohydrate. Other carbohydrates will only be oxidized. The covered tube, therefore, may be omitted when determining other carbohydrate utilization of such organisms.
  3. Some organisms do not grow in this OF the Basal Medium. It may be necessary to use another basal medium containing dextrose to confirm the negative reaction.
  4. Some mineral oils are acidic in nature and therefore may cause erroneous results.


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