The organism was cultured in deep culture fermen-tors at 24 C. and pB. 6-5-7-5 in a complete medium containing sucrose and corn steep liquor, maximum production being achieved in about 120 hr. The activity was determined by the agar cup-plate method using Staphylococcus aureus as test organism. The antibiotic was extracted from the clarified broth at pH 5 with methyl isobutyl ketone and concentrated further on extraction of the organic phase with aqueous sodium hydroxide at pH 11. From the concentrated aqueous solution thus obtained, fusidic acid was precipitated as a crystalline benzene solvate on acidification in the presence of benzene. Pure solvent-free fusidic acid (m.p. 192-93 C.^aJ^-O0 (all rotations in chloroform)) separated from, an ethereal solution of the recrystallized benzene solvate on standing. The acid is sparingly soluble in water, ether, and hexane, but soluble in alcohols, acetone, chloroform, and pyridine. The soldium salt is readily soluble in water.Fusidic acid (I) is a carboxylic acid, which contains carbon, hydrogen, and oxygen only. The elementary analysis and the equivalent weight, obtained by electrometric titration in 50 per cent (v/v) ethanol, agree well with the formula C31H48O6 (found: C, 72-03; H, 9-36 per cent; equiv. weight 5185. C31H48O6 requires: C, 72-06; H, 9-36 per cent; M = 516-7). The pK-value found by titration is 6-35, corresponding to a>K of approximately 5-35 in water. A non-acidic monomethyl ester (II), C32H50O6 (m.p. 153-54 C., [a]"J> - 14), is oUained on methyl-at'on with diazomethane. Fusidic acid contains one acetoxy group, and two hydroxy groups, one of which is readily acetylated. It can therefore be formulated:rCOOH r K j OCOCH3 28421 OH I OH Catalytical hydrogenation of (I) over palladium-on-charcoal in ethanol yielded dihydrofusidic acid (III), C31H5006, H20 (m.p. 182-83 C., [a]i 0), while hydrogenation over platinum oxide in glacial acetic acid afforded tetrahydrofusidic acid (IV), C31H52O6 (m.p. 172-73 C., [a]l -64). The difference between the ultra-violet spectra of (I) and (III) gives X(EtOH, max.) 204 m\L (e 4,200) for the chromophore first hydrogenated and shows that this is a trisubstituted isolated double bond. The second chromophore, lost in going from (III) to (IV) X(EtOH, max.) 220 mji (e 8,300)), is characteristic of an a,p-unsaturated acid having not more than one hydrogen atom at the double bond. Since (IV) gave no colour with tetra-nitromethane, (I) must contain a tetracyclic ring-system. Further structural work, the results of which will be reported elsewhere, is in progress.Table l. ANTIMICBOBIAL SPECTRUM OF FUSIDIC ACID Concentration required OrganismStaph. aureus, penicillin-sensitive (6 strains) Staph. aureus, penicillin-resistant (15 strains)Sir. pyogenes (2 strains) D. pneumoniae (6 strains) N. gonorrhoeae (3 strains) N. meningitidisC. diphtheriae (2 strains) S. subtilis Clostridium tetani E. coliK. pneumoniae Sal. typhimuriumSh. dysenteriae P vulgarisPs. aeruginosa Myco. tuberculosis v. hum.C. albicans Asp. fumigatusT. mentagrophytes for 50 per cent inhibition (Atgm./ml.)0-04-0-1 0-05-0-24-20 5-200-4-0-8 0-60-01-0-02 0-60-02 >100100 >100>100 >100>100 0-8>100 >100>100 The sodium salt of fusidic acid was tested against a number of micro organisms by a serial dilution method. The concentrations which cause 50 per cent inhibition are given in Table 1.Fusidic acid is non-toxic. The subcutaneous and oral LD50 in mice were found to be 1-2 gm. and 1-5 gm. per kgm. body-weight, respectively. The intravenous LD50 of the sodium salt was 0-2 gm. per kgm. tody-weight. Daily oral administration of fusidic acid to rats in doses of 0-4 gm. per kgm. body-weight over a period of 6 months was well tolerated. Post-mortem examination revealed no pathological changes.