Fatty Material in Bacteria and Fungi Revealed by Staining Dried, Fixed Slide Preparations

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Abstract
An improved technique for staining intracellular lipid in microorganisms is outlined, as follows: (1) prepare film, let it dry in air, and fix it by heat in the usual way; (2) flood the slide with Sudan Black B sola. (0.3% in 70% ethyl alcohol), let stand for 5 mins. or longer; (3) drain slide and blot dry; (4) clear with cpxylol; (5) blot dry; (6) counterstain with safranine (0.5% aqueous soln.) for 5-10 secs., or (in case of acidfast organisms) with dilute carbol fuchsin for 1-3 mins.; (7) wash in water, blot dry.. Application of this stain to the principal spp. of bacteria and fungi reveals an abundance of stainable lipid in fungi, and in Gram-positive bacteria, little or none in Gram-negative bacteria (except in a few saprophytic kinds, e.g., Azotobader, Alcaligenes, Spirillum). Acidfast bacilli often stain blue-gray throughout and may show deep-staining fat-droplets as well. The atnt., form and location of the intracellular lipid is remarkably constant in any one sp., and related spp. or genera show similar pictures. Certain spp. have distinctive appearances which are of practical differential value. Stainable lipid is present in bacteria in primary cultures and in prepns. made directly from the human or animal body, as well as in stock cultures. Some of it appears to result from cellular degeneration, rather than from a food storage process. It arises at the periphery of cells, presumably in some relation to the cytoplasmic membrane, which is itself stained by Sudan Black B in some spp. The fatty material is extracted by recognized fat solvents. It is suggested that this stain should prove helpful in the routine characterization and identification of microorganisms, and especially in studies of bacterial cytology and metabolism.