A Nile Blue Culture Medium for Lipolytic Microorganisms

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Abstract
A culture medium for the enumeration of lipolytic microorganisms is presented which makes use of Nile blue sulfate as a specific indicator for detecting changes in neutral fats. It is prepared as follows:[long dash]20 g. agar (Bacto) is dissolved in 1000 cc. beef infusion broth prepared in the usual way; the whole filtered and the reaction adjusted to pH 7.4; 100 cc. amts. are distributed in flasks and sterilized in the autoclave at 15 lbs. pressure for 20 min. A dye-fat emulsion is then prepared by first dissolving 4 g. ossein gelatin (200 Bloom, isoelectric point at pH 5.6) in 100 cc. distilled water at 60[degree] C and sufficient iV/10 NaOH added to bring the reaction to pH 7.2. The gelatin soln. is first shaken vigorously in a stoppered bottle which contains 100 cc. U.S.P. cotton-seed oil to which has been added 0.1 g. Nile blue sulfate (C.I. No. 913, dye content 90%, solubility 0,15 g. in 100 cc. water) and then put through a hand homogenizer until the oil globules are about 10m in diam. After sterilization in the autoclave at 15 lbs. pressure for 20 min., the emulsion which was originally blue, turns deep pink. When plates are to be poured, 100 cc. of the sterile infusion agar base are liquified and while still hot, 10 cc. of the sterile dye-fat emulsion are added aseptically. The whole is well mixed and allowed to cool to 45[degree] C before being poured into plates. Before inoculation the medium has a rich salmon-pink color. Colonies of organisms which decompose fats appear as light blue colonies surrounded by a narrow zone of decolorized, transparent medium when incubated for 3-4 days at room temp. Non-lipolytic colonies appear white or pink. The medium is equally efficient both with pour and streak plates, the former being desirable for quantitative work.

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