Oxidative Fermentative Test


  • This test determines whether an organism can respire or ferment glucose can be tested with glucose OF medium.
  • A small amount of acid production can be associated with glucose respiration.
  • The original paper which describes the medium gives the example of Entner Douderoff pathway that is utilized by variety of generally Gram negative bacteria (including Pseudomonas) to convert glucose to pyruvate- an alternative method of pyruvate formation to that of Embden-Meyerhoff pathway. Pyruvate is further oxidized to CO2 in the aerobic respiration process. Among the intermediates in the Entner-Douderoff pathway are forms of gluconic acid.
  • If the strict aerobic organism producing this acid is growing, at the top of the medium where oxygen is available – net acidic reaction will be seen.
  • However this acidic reaction would be rendered indistinguishable if the organism were a facultative anaerobe – in which case the large amount of acid (produced by fermentation in anaerobic environment of the tube) would be diffusing throughout the entire medium.
  • Duplicate tubes are inoculated for each organism, and the medium in one of the tubes is overlayed with mineral oil.
  • Mineral oil does not in itself cause anaerobic conditions but rather prevents oxygen from continuing to diffuse into the medium.
  • After incubation, one looks for the presence and location of the growth and acid.
  • As glucose OF medium contains relatively less peptone and more glucose than the fermentation broth, the small amount of acid associated with respiration can be detected in the aerobic part of the non-overlayed tube.
  • If it is not made indistinguishable by acid production from fermentation which turns both tubes yellow throughout.
  • This medium was formulated to assist in the identification of certain groups of Gram negative rods. In fact, the medium is inhibitory to many Gram positive bacteria, most likely due to the bromothymol blue pH indicator.

Principle of Oxidative-Fermentative test

O-F medium is a semisolid medium incorporating a pH indicator, 1% glucose and peptones. The type of metabolism a bacterium has are; aerobic use of sugar vs anaerobic use of a sugar.. The overlay of sterile paraffin oil or a visper wax in one tube prevents oxygen from getting into the medium of one tube, the other tube has air inside.

  • Aerobic use of sugar is oxidative metabolism, anaerobic use of sugar is fermentative metabolism. Bacteria that oxidize carbohydrates are usually facultative anaerobes.
  • The fermentation will produce far more acid than the aerobic oxidation, but the OF glucose test is very sensitive to the small amounts of acid produced via aerobic means.
  • As the organism grows and uses the sugar, the resulting acid will change the pH and the pH indicator will change colours.
  • The indicator is bromothymol blue, starting as a forest green colour at the neutral pH and ending as yellow with low pH and blue with high pH.
  • Using 2 tubes allows the identification of whether the organism is oxidative (only open tube acidic) or fermentative (both tubes acidic). If the organism uses the peptones rather than the glucose, the pH will become basic.
  • This method (to distinguish between aerobic and anaerobic breakdown of carbohydrates) first reported by Hugh and Leifson in 1953, depends upon the use of a solid tube medium containing the carbohydrate together with pH indicator.
  • If acid is produced only at the surface of the medium, where conditions are aerobic, the attack on the sugar is oxidative. If acid is found throughout the tube, including the lower layers where conditions are anaerobic, the breakdown in fermentative.
  • Fermenting organisms (Organisms belonging to Enterobacteriaceae family, Aeromonas, Vibrio) produce an acid reaction throughout the medium in the covered (anaerobic) as well as the open (aerobic) tube.
  • Oxidizing organisms (e.g. Pseudomonas) produce an acid reaction only in the open tube; this begins at the surface and gradually extends downwards and may appear only after an alkaline reaction has been present for several days.
  • Organisms that cannot breakdown the carbohydrate (e.g. Alcaligenes faecalis) produce an alkaline reaction either in the open tube or closed tube depending upon whether it is an aerobic organism or an anaerobic organism.

Mode of action

A carbohydrate is added to the culture medium, degradation of the carbohydrate to acid is indicated by the pH indicator bromothymol blue which changes its color to yellow. The degradation is allowed to take place while the medium is exposed to air (degradation may be oxidative or fermentative) or under exclusion of air (degradation y fermentation only),

Requirements for Oxidative-Fermentative test

  1. Sterile Hugh-Leifson [Peptone (from casein) 2g Yeast extract]
  2. Test culture suspension
  3. Sodium chloride
  4. Dipostassium hydrogen phosphate – 580.2g
  5. 3ml Bromothymol blue (1% aqueous solution)
  6. 1 Litre Distilled water

The pH is adjusted to 7.1 before adding the bromothymol blue and the medium is autoclaved at 121°C for 15 minutes.

The carbohydrate to be added is sterilized separately and given a final concentration of 1%. The medium is then put into tubes to a depth of about 4 cm.

Procedure for Oxidative Fermentative test

  1. Two tubes of solidified medium are inoculated by stabbing.
  2. One tube is promptly covered with a layer of sterile paraffin oil to the depth of 5-10 mm.
  3. Both the tubes are then incubated and the results observed upto extended periods of the time (even upto 30 days in some cases).

Observations for F/O Test

A small amount of acid production can be associated with glucose respiration. Acid Production is determined by colour change of the OF Medium.

References And Sources

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