MODEL SYSTEM FOR DANDRUFF INVESTIGATION 191 of sloughing being recorded 5 days after application. The sloughing re- action was graded subjectively on a 0-4 scale after the treated areas were reclipped, with the most severe reactions being given the highest score. Photographs showing' the severity of sloughing relative to scores are shown in Fig. 1. Oleic acid (reagent grade) was applied to the test sites at the indicated concentrations in a propylene glycol vehicle. Control sites indicated that the vehicle was not irritating. Organisms Representative strains of the major constituents of the scalp microbial population were utilized in this study. These strains were isolated from scalp washings obtained by placing a glass collar (25-mm diameter) firmly in place on the scalp and scrubbing with 5 ml of 0.05M phosphate buffer (pH 7.0) for 30 sec. Fresh cell preparations harvested from broth media were routinely used. The P. ovale strain was isolated using the medium described in Table II. Table II Formula of Medium for Growth of P. o•ale and Scalp Diphtheroid g/1. Tryp ticase soy broth (BBL) 30.0 Tween 40 1.0 Oleic acid 0.1 1N Lactic acid 20.0 Streptomycin sulfate • 0. 005 Penicillin G a 0. 045 Cycloheximide • 0. 025 pH 6.0 Omitted from diphtheroid medium. For growth and maintenance of pure cultures, the antibiotics in the above formulation were omitted. Incubation was normally for approxi- mately 14 days at 37øC at which time the cells were harvested by centrif- ugation or filtration. Diphtheroids were cultured on the above medium minus antibiotics. In every instance, these organisms were grown anaerobically at $7øC and harvested by centrifugation after 5 days of incubation. These cultures have been identified as Corynebacterium aches.

192 .JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Cocci were classified in accordance with the Baird-Parker (11) system of classification. The organisms conformed to the characteristics of Staphylococcus subgroup II. Growth was in Brain Heart Infusion (Difco) medium at g7øC with the cells harvested by centrifugation after 48 hours of incubation. l Inless otherwise specified, organisms were added to artificial sebum at a concentration of 100 mg/g. The microbial concentrate was ob- tained by centrifuging growth medium at 10,000 rpm in a refrigerated centrifuge for 20 min. The spent medium was decanted and the cen- trifuged cells were then weighed and added directly and with vigorous stirring to the artificial sebum. This mixture was then applied to the delineated areas on the dorsal surface of the guinea pig. Fatty A cid Analyses Extractions of guinea pig skin were carried out by placing a glass collar 25 mm in diameter firmly on the test site. A 5-ml volume of 4:1 diethyl ether:methanol was placed within the collar and the area was scrubbed for 30 sec with a glass rod flattened at the tip. The extract was then withdrawn and filtered and the constitttent free fatty acids were converted to the corresponding methyl esters by the methods of Metcalf et al. (12) Gas chromatography was carried out with an FgcM model 720 dual column gas chromatograph* utilizing a thermal conductivity de- tector. A 10-foot stainless steel column of l/4-in. diameter, packed with a 1:9 mixture of diethylene glycol succinate* and 60/80 mesh Gas Chrom P,* and operating at a temperature of 185 ø or 190øC, was used in these studies. RESULTS Early experiments had shown that the addition of buffered suspen- sions of scalp microorganisms directly to guinea pig skin produced no visible irritation or other reaction. In view of the high concentrations of fatty materials on the human scalp and the relatively low concentra- tions of lipid on guinea pig skin, an attempt was made to approximate more closely the conditions existing on the httman scalp by supplement- ing the guinea pig skin lipids with the artificial sebum for•nulation. The various strains of scalp organisms were then admixed with the at- * F & M Scientific Corp., Avondale, Pa. ? Analytical Engineering Laboratories, Hamden, Co•m. • Applied Sciences I•aboratories, State College, Pa.