The present paper reports the results obtained in examining 191 freshly isolated saccharolytic Clostridium strains for growth under anaerobic conditions in a synthetic nutrient solution with or without addition of (+) -biotin and p-aminobenzoic acid. Further, the pH of the substrates was determined electrometrically after completed fermentation. The strains were isolated from soil, sewage, green plants, silage, potato-, corn-, rye- and chestnut-mash, spoiled Emmentaler and process cheese, faeces of cow and sheep, etc. The pure cultures were obtained from the pasteurized material directly by means of smear-cultures on glucose-meat infusion-agar, glucose-yeast water-agar, etc. or after enrichment on skim milk, glucose-skim milk, corn- and pea-broth, glucose-meat-peptone-broth, Winogradsky's solution, etc. The pH measurements and analyses of the fermented substrates showed that out of the 191 strains thus isolated 88 (46 per cent) were true butyric acid bacteria producing great amounts of butyric and acetic acids, along with traces of neutral compounds, whereas 22 strains (11,5 per cent) were butanol-isopropanol or butanol-acetone bacteria capable of forming large quantities of neutral products, together with only small amounts of fatty acids. 81 strains (42,5 per cent) were found to be intermediate types. The synthetic medium used contained 20 amino acids in addition to glucose, ammonium acetate and mineral salts. (+) -biotin and p-aminobenzoic acid were supplied in amounts of 0.001 μg and 0.05 μg, respectively, per ml of substrate. In order to check the quality of the pasteurized spore suspensions used for inoculation parallel cultures were grown in a modified Karnicki-Dorner medium. It was demonstrated conclusively that 178 strains (93 per cent) were heterotrophic for (+) -biotin, whereas 6 strains (3 per cent) showed auxo-autotrophic tendencies, and 7 strains (3-4 per cent) required (+)-biotin or p-aminobenzoic acid for normal growth in the synthetic substrate mentioned. The end products of fermentation found in the synthetic medium supplied with (+) -biotin are, qualitatively and quantitatively, the same as those observed in parallel cultures on a glucose-yeast extract substrate. As far as selection and genic mutations involving changes from an autotrophic to a heterotrophic growth-factor habit do not occur in the media employed for isolating the strains and growing the subcultures used in preparing the spore suspensions, we may conclude that the natural populations of butyric acid as well as butanol-isopropanol and butanol-acetone bacteria of the genus Clostridium contain a very great number of strains requiring an exogenous supply of (+) -biotin for normal growth even in media containing most of the amino acids obtained on hydrolysis of bacterial proteins. The number of strains with auxo-autotrophic tendencies is obviously very small. In some strains a replacement of (+) -biotin by p-aminobenzoic acid seems possible. [ABSTRACT FROM AUTHOR]