A Nile Blue Culture Medium for Lipolytic Microorganisms

Abstract

A culture medium for the enumeration of lipolytic microörganisms is presented which makes use of Nile blue sulfate as a specific indicator for detecting changes in neutral fats. It is prepared as follows:—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. amounts are distributed in flasks and sterilized in the autoclave at 15 pounds pressure for 20 minutes. 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° C. and sufficient N/10 NaOH added to bring the reaction to pH 7.2. The gelatin solution 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 thru a hand homogenizer until the oil globules are about 10μ in diameter. After sterilization in the autoclave at 15 pounds pressure for 20 minutes, 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° 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 temperature. Non-lipolytic colonies appear white or pink. The medium is equally efficient both with pour and steak plates, the former being desirable for quantitative work.