Oxidative/Fermentative Test (O/F test)


Oxidative/Fermentative (O/F) test is done to distinguish between aerobes and anaerobes through their ability to breakdown carbohydrates. In this test, a semi solid medium containing a carbohydrate (usually glucose) and a pH indicator is used.  If acid is produced only at the surface of the medium, where conditions are aerobic, the attack on sugar is oxidative. If acid is found through out the tube, including the lower layers where conditions are anaerobic, the breakdown is fermentative. Media Preparation (Hugh and Leifson)

Peptone                                                      2 g

Sodium chloride                                       5 g

Dipotassium hydrogen phosphate        o.3 g

Bromothymol blue (1% aq. solution)   3 ml

Agar                                                             3g

Water                                                          1 litre

Note: The pH of the medium should be adjusted to 7.1 before adding bromothymol blue and the medium should be autoclaved in a flask at 121oC for 15 minutes. Carbohydrate to be added is sterilized separately and added to give final concentration of 1%. The medium is then poured on sterile tubes to a depth of 4 cm. This medium can also be used for recording gas production and motility.


  • Inoculate the organism using single colony in two tubes of medium.
  • Immediately cover one tube with a layer of sterile melted petroleum jelly to a depth of 5-10 mm.
  • Incubate both tubes at 35°C for 48 hours for gram negatives.Slow growers may take 3-4 days before the result can be observed.


Resultsimages (3)


  1. Fermenting organisms- produce an acidic reaction throughout the medium in the covered (anaerobic) as well as the open (aerobic) tube. e.g. Enterobacteriaceae, Aeromonas, Vibrio
  2. Oxidative organism- produce an acidic reaction only in the open tube (this begins at the surface and gradually extends downwards) e.g. Pseudomonas
  3. Organisms that do not break the carbohydrates aerobically or anaerobically produce an alkaline reaction in the open tube and no change in the covered tube. e.g. Alcaligenes faecalis

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