OF Basal HiCynth™ Medium

Principle And Interpretation

Hugh and Leifson developed OF Medium to study oxidative and fermentative metabolism of carbohydrates by gram-negative bacteria. This criterion is used during taxonomic studies of Enterobacteriaceae (1). OF Basal HiCynth™ Medium is prepared by replacing animal and vegetable peptones with chemically defined peptones to avoid BSE/TSE risks associated with animal peptones. HiCynth™ Peptone No.4 in the medium provides the necessary carbon and nitrogen, vitamins etc. required for bacterial growth. Phosphate buffers the medium and the low agar concentration determines motility and dispersion of the acid produced on the surface (2). Bromothymol blue acts as the pH indicator. A carbohydrate whose fermentation reaction is to be studied is added separately.

Motility is observed as diffused zone of flaring out from the line of inoculation. Non-motile organisms grow along the line of inoculation. Dextrose (Glucose) is the most important carbohydrate for use in OF Basal HiCynth™ Medium. However, certain organisms may metabolize other carbohydrates even if they are unable to utilize dextrose. Degradation of the carbohydrate to acid is indicated by the pH indicator bromothymol blue which changes its colour to yellow. Oxidative utilization takes place when the medium is exposed to air while fermentative utilization occurs under exclusion of air. The authors Hugh and Leifson showed that when a gram-negative organism is inoculated in this medium containing a carbohydrate in duplicate, of which one tube is covered with mineral oil to exclude oxygen and the second tube is uncovered; reactions of differential value can be observed. Fermentative organisms will produce an acid reaction in both the covered and uncovered medium. Oxidative organisms will produce an acid reaction in the uncovered medium and give slight growth without change in the covered medium. Organisms which are not classified either as oxidative or fermentative show no change in the covered medium and an alkaline reaction in the uncovered medium (2). Prepare the medium with 1% dextrose and without 1% dextrose. Two tubes of each carbohydrate are used per organism and inoculated by stabbing. One of the inoculated tubes of each carbohydrate medium is covered with 2 ml of sterile mineral oil and the other is left uncovered. The tubes are incubated at 35-37°C for 18-48 hours or longer. The results are read after 48 hours. The acidic reaction of oxidative organisms is more apparent at the surface of the medium that gradually spreads throughout the medium. If the oxidation reaction is weak or slow, an initial alkaline reaction at the surface of the uncovered tube may persist for several days and eventually convert to an acid reaction.

OF Basal HiCynth™ Medium can be supplemented with 2% serum or yeast extract (0.1%) to make the medium more nutritious for the growth of bacteria (3).