464 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS A suitable medium for C. herbarum was found to be 3% Sabourauds medium in a 1% agar in water gel. The medium was prepared by dissolving the ingredients in an appropriate quantity of water and sterilising by heating in an autoclave for 30 min at a pressure of 1041 kN m -2. After cooling, but prior to gelation, an 0.1% w/w suspension of the steroid was prepared in the medium. A number of agar plates were then poured and seeded with C. herbarum, unseeded plates were also retained as controls. The test plates and controls were incubated at 25øC and examined at weeldy intervals. When plates were examined, the steroid and possible metabolites were extracted using a 90% chloroform/ 10% methanol mixture as solvent following evaporation to dryness with acetone. The extracts were then examined by means of thin layer chromatography and the Rf values compared with reference samples of the parent substance and theoretical metabolites, which had been obtained in pure form from commercial sources. Thin layer chromatograms of the extracts were prepared using 0.3mm thick layers of Kieselgel G on glass plates, activated by heating at 110øC for 60 min. The chloroform/methanol extract, together with similarly prepared extracts of Cladosporium herbarum and medium, and reference steroids, were placed on the plates and developed using (a) cyclohexane 25: ethyl acetate 75, and (b) dichloroethane 100: methyl acetate 50: purified water (saturated). Development of the chromatogram was carried out in Table I Rf values of hydrocortisone metabolites, reference substances and inoculum extracts. Extractive Prepared from ½ladosporium herbarum extract Hydrocortisone metabolite extract Rf in solvent A 0.045 0.116 0.152 0.197 0.424' 0.525* 0.076* 0.278* 0.422 0.522 Rf in solvent B 0.030 0.117 0.175 0.300 0.542* 0.640' 0.181' 0.390* 0.540 0.640 Androst-4-ene-1 l[I-ol- 0.279 0.390 3:17-dione Hydrocortisone 0.077* 0.179* indicates main spot.

THE METABOLISM OF STEROIDS BY' CLADOSPORIUM HERB.4RUM 465 lined tanks, the solvent being allowed to run for a minimum of 170mm of the 200mm plate. After removal from the developing tank, the plate was dried at 50øC for 15 min. It was then sprayed with a 50% solution of sulphuric acid in water, and heated at 160øC for 3 min. The sprayed chromatograms were examined under a tungsten lamp and then by uv light. Photographic records and tracings were made and the Rf values calculated (Tables I and II). Table II Rf values of progesterone and desoxycorticosterone metabolites, reference substances and inoculum extract. Extractive Cladosporium herbarurn extract Progesterone metabolite extract Desoxycorticosterone metabolite extract I(f in solvent A 0.045 0.110 0.151 0.198 0.420* 0.514' 0.048 O. 109 0.151 0.200 0.420* 0.515' 0.550' 0.572' 0.226* 0.342* 0.418' 0.490 0.512' 0.560* 0.580* Rf in solvent B 0.500* 0.030 0.118 0.180 0.305 0.530* 0.636* 0.460 0.534* 0.566* 0.632* 0.650* 0.350* 0.460* 0.502* 0.531' 0.620* 0.640 Progesterone 0.550* 0.565* Desoxycorticosterone 0.224' 0.350' Testosterone 0.345* 0.460* Testosterone acetate 0.560* 0.640* Androst-4-ene-3:17-dione 0.513' indicates main spot. RESULTS Progesterone The presence of testosterone and testosterone acetate was demon- strated. Unfortunately, we were unable to check for testololactone due to