A Simple Test for Carotenoid Pigments in Yeasts.

The imperfect yeasts are divided by Lodder (1934) into two families, Rhodotorulaceae and Torulopsidaceae, distinguished by the occurrence of carotenoid pigments in the former. Lodder employed a form of the Molisch test to demonstrate the carotenoid nature of the pigments in Rhodotaceae. This involved covering a large mass of cells with an alcoholic potassium hydroxide solution and storing in the dark for several days or even weeks. After such treatment, Lodder found well-defined carotenoid crystals under the microscope, which turned blue when treated with concentrated sulfuric acid. Over a number of years, working with numerous cultures of several species of Rhodotorula, the writers have had uniformly negative results with the foregoing procedure. Mackinney (1940) observed that several species of Rhodotorula and Sporobolomyces resist prolonged boiling in alcoholic potassium hydroxide, without liberation of pigment and without apparent change in the cells under the microscope. The pigments may be extracted, however, after careful hydrolysis of the cell mass with hydrochloric acid, with acetone, and are then readily transferred to petroleum ether. Bonner, Sandoval, Tang, and Zechmeister (1946) extracted the pigment from Rhodotorula mutants with benzene by shaking the cell suspension in a benzenealcoholic potassium hydroxide mixture for 1 to 1.5 hours. This procedure is readily applicable in most cases, but with some species producing a heavy mucilaginous slime there is scarcely perceptible color in the benzene layer, and the treatment (involving vigorous mechanical shaking) has had no visible effect on the microscopic appearance of the cells. The purpose of this note is to provide the yeast taxonomist with a simple, quick procedure for determining the presence of carotenoid pigments in yeasts. When the pigments have been brought into solution, there are obviously many tests required for rigorous proof of identity. However, in the case of all Rhodotorula and Sporobolomyces species examined, solubility of the pigments in petroleum ether is virtually an adequate criterion by itself, since a large number of confirmatory spectroscopic and chromatographic analyses have been made. The conclusion can be reinforced, if the petroleum ether solution is not too dilute, by the test with concentrated sulfuric acid, and, of course, by such other methods and equipment as may be available. The details of the simple test, which we found very useful for laboratory classes, are as follows: Yeast growth on a heavily inoculated plate is removed with the end of a glass slide. Care should be exercised to avoid removing any of the agar substrate. The yeast on the slide is transferred with the aid of a spatula to a small beaker, or test tube, and suspended evenly in 8 to 10 ml of 1:1 hydrochloric