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Metachromatically staining granules of volutin, first observed in C(oryrebacterium xerosis and Corynebacterium diphtheriae, have since been demonstrated in a wide variety of bacteria, yeasts, fungi, and algae. In the species normally forming volutin, the amount produced is influenced greatly by the conditions of culture. Moreover, there are some species which form the granules only in exceptional circumstances. Thus, Aerobacter aerogenes forms no volutin when grown on ordinary nutrient media, but much when grown under acid conditions (pH 4.0 to 5.0) on a poorly buffered medium containing sugar and a small amount of phosphate (Duguid et al., 1954). Volutin production is induced also in A. aerogenes by certain conditions of nutrient imbalance not involving development of acidity; this paper describes an investigation of such conditions. There is evidence that volutin accumulates when cell growth is limited. Pesch (1924) found that volutin production in C. diphth.eriae on glucose blood agar was increased when the amount of growth was decreased by reduction of the blood content. Winkler (1953) noted that the granules of corynebacteria and mycobacteria were small in the early stages of growth and enlarged greatly in the later stages. Stich (1953) observed that Acetabularia mediteranea formed large cytoplasmic granules of volutin ("metaphosphate") when its growth was terminated naturally or artificially. Similarly, in the present study, A. aerogenes was found to form volutin when its growth was halted by exhaustion of the nitrogen or sulfur source. The restoration of phosphate supply following phosphate starvation constitutes another form of nutrient imbalance inducing production of volutin. Jeener and Brachet (1943) found that yeast cells grown on a phosphate deficient medium lost most of their normal basophilia; when transferred to a fresh medium containing phosphate, such cells synthesized much basophilic substance within a short time. Wiame (1946a) showed that the basophilic substance formed in the subcultures of phosphate starved yeast, unlike that in normal yeast, gave the metachromatic reaction with toluidine blue which is characteristic of volutin. Duguid (1948) found that volutin formation was induced siilarly in A. aerogenes, granules appearing abundantly in the first hour after transfer of the phosphate starved bacilli onto nutrient agar. The present paper reports a further study of this mode of volutin formation. Wiame (1946a, b; 1947a, b) obtained convincing evidence that the volutin ("metachromatic substance") of yeasts is a polymenized inorganic metaphosphate. Duguid et al. (1954) obtained similar findings for the volutin of A. aerogenes grown in acid conditions. Synthesis of volutin requires adequate provision of certain nutrient factors, including favorable sources of energy and phosphorus (Elser and Huntoon, 1909; Zikes, 1922; Duguid et al., 1954). The requirement of potassium ions for uptake of phosphate prior to metaphosphate formation by bakers' yeast was demonstrated by Schmidt et al. (1949).

VOLUTIN PRODUCTION IN AEROBACTER AEROGENES DUE TO NUTRIENT IMBALANCE