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SUMMARY: "he polysaccharide content of Aerobucter aerogenes surface cultures grown on defined media was estimated by the anthrone method and related to the amount of growth as measured by the non-dialysable nitrogen content. Obser- vations after different periods of incubation at 35" showed that on a medium containing excess sugar, growth was complete after 24hr. and polysaccharide production was complete after 4 days. While most of the polysaccharide was pro- duced after the cessation of growth, the rate of production per cell was greatest in the logarithmic phase. The polysaccharide : nitrogen ratio was low in 4-day cultures on media wherein cessation of growth was due to exhaustion of sugar (the sole carbon and energy source) and also was low in growths limited by anaerobic con- ditions or by the development of acidity. It was increased 10-20-fold in cultures wherein cessation of growth was due to exhaustion of limited nitrogen, phosphorus or sulphur sources, so that an excess of sugar remained available for extra poly- saccharide synthesis. This great increase in the polysaccharide : nitrogen ratio of the culture was paralleled by similar increases in the degree of mucoidness as measured by the wet weight : nitrogen ratio, in the intracellular polysaccharide content as measured by periodate-Schiff staining, in capsular polysaccharide as measured by capsule diameter, and in loose slime polysaccharide as measured chemically in the supernatant after centrifugation of the culture. After 4 days at 55" the loose slime accounted for about half of the total polysaccharide in the highly mucoid cultures and about a fifth of that in the non-mucoid cultures. The polysaccharides of bacteria can occur intracellularly, and extracellularly as capsules and loose slime. Intracellular polysaccharide is demonstrable microscopically by the specific periodate-Schiff staining method of Hotchkiss (1948) and McManus (1948) (Pennington, 1949; Bunting, Robinow & Bunting, 1949; Lankford, Hoyo & Lutteringer, 1951). Capsules and slime are demon- strable microscopically by a variety of methods, most satisfactorily in wet films with India ink (Duguid, 1951). Previous investigations of the influence of cultural conditions on poly- saccharide production by bacteria mostly have been made by methods which reveal only part of the polysaccharide. Birch-Hirschfeld (1936) extracted a polysaccharide-containing material from nutrient agar cultures of Sal- monella paratyphi B, and found greater amounts after growth at 25" than at 37'. Morgan & Beckwith (1939) observed the degree of mucoidness of surface cultures as indicative of the amount of capsule and slime; they found that a low temperature of incubation (10-ZOO) and the provision of readily utilizable carbohydrate were necessary for highly mucoid growth by sal- monella and escherichia strains. Hoogerheide (1939) observed the viscosity of cultures in relation to their cell count and regarded this as a measure of capsule size. He found that the capsules of Klebsiella pneumoniae were

The Influence of Cultural Conditions on Polysaccharide Production by Aerobacter aerogenes