Oxidation Fermentation (OF) test: Principle, Procedure and Result Interpretation


Oxidation Fermentation (OF) test is used to differentiate those organisms that utilize carbohydrates aerobically (Oxidation) such as Pseudomonas aeruginosa, from those that utilize carbohydrates anaerobically (Fermentation) such as members of the Enterobacteriaaceae and those that do not utilize carbohydrates at all (Non fermenters) such as Alcaligenes faecalis  . This test was developed by Hugh and Leifson so the OF medium is known as Hugh Leifson medium.


The test organism is inoculated into two tubes of a tryptone or peptone agar medium containing glucose (or other carbohydrate) and the indicator bromothymol blue. The inoculated medium in one tube is sealed with a layer of liquid paraffin to prevent diffusion of oxygen.

If the inoculated organisms utilize the carbohydrate in both the open and sealed tubes which is indicated by change in the color of the medium from green to yellow, then the organism is fermentative. To explain this observation, in the anaerobic process of fermentation, pyruvate is converted to a variety of mixed acids depending on the type of fermentation. The high concentration of acid produced during fermentation will turn the bromthymol blue indicator in OF media from green to yellow in the presence or absence of oxygen.

If the inoculated organisms utilize carbohydrate only in the open tube without utilizing carbohydrate in the sealed tube (medium remains green), the organism is identified to be oxidative. To explain this observation, these bacteria metabolize glucose using aerobic respiration and produce a small amount of weak acids during the Krebs cycle and Glycolysis. The increased concentration of glucose in the medium enhances the production of these weak acids to a level that can be detected by bromothymol blue indicator. To further enhance detection of these weak acids, this medium contains a reduced concentration of peptones. This reduces the production of amines from the metabolism of amino acids, therefore reducing the neutralizing effect of these products. Dipotassium phosphate buffer is added to further promote acid detection.

If the inoculated organism do not utilize carbohydrates, then there is no change in color of the medium in the oil-covered tube and in some cases, some organism may utilize peptone resulting in amine production and cause an increase in pH of the media changing the bromothymol blue from green to blue in the top of the open tube.

Although most genera of aerobic bacteria are either carbohydrate oxidizers or fermenters, the production of acid may be slow and therefore cultures are usually incubated for 7-14 days.


Materials Required

  • Test organism
  • Hugh Leifson medium (Glucose, maltose, and sucrose are the most commonly used carbohydrates.)
  •  Sterile paraffin oil (liquid paraffin)



  • Using a sterile straight wire, inoculate the test organism using a heavy inoculum to the bottom of two tubes (or more if testing several carbohydrates) of sterile Hugh Leifson medium.
  • Cover the inoculated medium in one of the tubes (or one from each carbohydrate pair) with a 10 mm deep layer of sterile paraffin oil.
  • Incubate the tubes at 35-37oC for up to 14 days.
  • Examine daily for carbohydrate utilization.

Interpretation of Results


Bacteria that can ferment glucose give a fermentative result as indicated by acid production in both the open (aerobic) and sealed (anaerobic) tube.  The acid produced changes the pH indicator, bromthymol blue, from green to yellow. The semisolid consistency of the medium also allows for detection of motility as a hazy growth away from the stab line can be visualized.


Non fermenting bacteria metabolize glucose via oxidative metabolism give an oxidative result as indicated by a small amount of acid production in the open tube. The acid produced changes the pH indicator, bromthymol blue, from green to yellow. After 24-hours incubation, a change in pH is observed at the surface of the open tube where growth in the presence of oxygen is observed. With prolonged incubation (more than 48 hours), the reduced concentration of agar in the medium allows for the eventual diffusion of the weak acid throughout the whole tube.

No color change or reaction occurs in the oil-covered tube.

Negative result

Nonsaccharolytic bacteria give a negative OF result as indicated by no color change in the oil-covered tube and in some cases an increase in pH changing the bromthymol 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 . Other bacteria give a negative result indicated by no growth or color change in the medium.



Oxidative control: Pseudomonas aeruginosa.

Fermentative control: Escherichia coli.


The glucose can be replaced by maltose, lactose, mannitol, or sucrose in the medium and only one tube per carbohydrate is inoculated. A heavy inoculum should be used, as many of these non fermenters are slow growing. A positive result is indicated by an acid production and a change in pH in the top of the tube after 24 hours. Some slow growing nonfermenters may take several days to produce enough acid to be detected by the bromthymol blue.


Hugh and Leifson’s OF basal medium 

Peptone (tryptone)                                          2.0 g

Sodium chloride                                              5.0 g

Glucose (or other carbohydrate)                     10.0 g

Bromthymol blue                                            0.03 g

Agar                                                                3.0 g

Dipotassium phosphate                                   0.30 g

Preparation of Hugh and Leifson’s OF medium 

Weigh the desired amount of the commercially available Hugh Leifson media in a sterile flask . (Follow the manufacturer’s instruction). Add 1 liter ofdistilled water. The pH should be adjusted to 7.1 prior to autoclaving.
After the medium is autoclaved at 121°C for 15 minutes, a filter sterilized solution of 10% solution of carbohydrate is aseptically added to the medium to a final concentration of 1%.  The sterile medium containing the carbohydrate is aliquoted aseptically into sterile test tubes and cooled unslanted as stabs.

Alternatively, 10 g/liter of carbohydrate can be added to the medium prior to sterilization. The medium is then dissolved by heating to a boil on a hot plate or by steaming for 20 minutes prior to aliquoting into test tubes. The tubed medium is then steamed for 20 minutes in place of autoclaving to prevent breaking down of the carbohydrates.

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