Oxidation fermentation test (Hugh and Leifson test ) principle, the procedure for P.Aeruginosa

The oxidation fermentation test was first discovered by the two scientists Hugh and Leifson in 1953. Therefore oxidation fermentation test was nominated as Hugh and Leifson test.

The purpose of the Hugh and Leifson test is to differentiate between those organisms that oxidize carbohydrates ( aerobic organisms) and between those organisms that don’t oxidize carbohydrates (anaerobic organisms).

Pseudomonas Aeruginosa is an organism that ferments carbohydrates such as any member of the Enterobacteriaceae family..

Principle of an Oxidation Fermentation test

The test organism is inoculated in two tubes of peptone agar medium that contain sugar and the indicator Bromothymol Blue. One tube is sealed with paraffin wax to exclude the oxygen after inoculation.

Fermentative organisms utilize the sugar (carbohydrate) in both tubes (open tube, sealed tube) resulting in a change in the color of media from GREEN to YELLOW.

NOTE: Oxidative organisms only utilize sugar in the presence of air and change its color to yellow. While the sealed tube remains unchanged (Green in color).

Reagents for Oxidation Fermentation test

There are six kinds of reagents we need for the Oxidation test.

• N2cl: 5 g
• Peptone: 2 g
• Dipotassium Hydrogen Phosphate (K2HPO4) : 0.3 g
• Bromothymol Blue (1% aqueous solution) : 3 ml
• Agar : 3 g
• Water: 1 liter

NOTE: PH of the medium is adjusted to 7.1 before adding the Bromothymol Blue. After this, the medium is autoclaved in a flask at 121 C for 15 minutes.

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

Oxidation fermentation test procedure

• First of taking two tubes. Tube A and Tube B. Both tubes are inoculated by stabbing.
• Tube A is covered with melted petroleum jelly or paraffin wax to a depth of 5 to 10 cm (after inoculation).
• Now place both tubes A and B in the incubator for 30 days.
  • Fermenting organisms (Enterobacteriaceae Aeromonas, Vibo) produce acid reactions throughout tube A.
  • The acid reaction begins at the surface and gradually extends downward.
  • With each tube, positive control of Pseudomonas Aeruginosa is run, as well as positive fermentative control of E.coli.
  • An inoculated tube is used as a negative control.