352 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS TABLE 5--COMPARISON OF DIAPHENE, HEXACHLOKOPHENE• AND A MERCURIAL AGAINST PATHOGENIC FUNGI Trichophyton Microsporum Microsporum Test Material rnentagrophytes rubrum audouini Control No zone No zone No zone Diaphene 8 mm. 12 mm. 14 min. Hexachlorophene 2 mm. 4 mm. 4 ram. Phenyl mercuric lactate 1 mm. No zone No zone phene. This test was performed using 0.01 p.p.m. of diaphene in the culture media in which Micrococcus pyogenes var. aureus was being grown. Subculturing occurred at each twenty-four-hour period with equivalent concentrations of diaphene in the culture media. After six subcultures had been made the inhibiting concentration of diaphene was determined on the original micro/Srganism strain and on each subcultured growth. In each case it was found that the same concentration of diaphene in- hibited the growth of the micro/Srganism this concentration was deter- mined to be 0.1 p.p.m. in each case. Additional studies in this area have shown diaphene to be active also against various strains of antibiotic- resistant staphylococci. The inhibition concentration of salicylanilide against Trichophyton inter- digitale is 40 p.p.m., against Micrococcus pyogenes var. aureus 50 p.p.m., and against E. coli 1013 p.p.m., using a deep broth culture technique. The inhibition concentrations of diaphene against these micro/Srganisms are 2 p.p.m., 0.1 p.p.m., and 1 p.p.m., respectively. In these deep broth culture experiments the following procedure was used. A twenty-four- hour culture broth was used of which 0.1 $ mi. was diluted 1: 10 and added to the compound under investigation which had been dissolved in the culture dilution series. The incubation of these samples took place in an oven at 37øC. for twenty-four hours. Evaluation of growth was carried out on a growth-no growth basis. A question which is of considerable practical importance is the pene- trability of antimicrobial substances into the skin and their ability to attack microbes found in the deeper layers. For this effect the physical and chemical properties of the material used are important, as well as the nature of the vehicle employed. However, it is of fundamental importance to determine the penetrability of the material itself in an oleaginous and aqueous vehicle. Therefore, in the following experiment an oily ointment and an aqueous jelly base were used in order to attempt to exclude as much as possible the influence of the vehicle and to determine how far the active material itself is capable of penetrating into the lower skin levels. The principle in the following experimental conditions is to use the high ef- fectiveness of diaphene toward Micrococcus pyogenes var. aureus in order to determine its presence in skin sections cut with a microtome.

SALICYLANILIDE COMPOSITION FOR SOAPS AND COSMETICS 353 Figure 1.--Comparison of antibacterial activity of soap containing 0.3% diaphene (No. 1) with three commercial antibacterial soaps. For this purpose shaved fresh calfskin was used in the experiment. A surface area of 100 square centimeters was marked out with a marking pencil and one gram of product under investigation was applied to the area, distributed as evenly as possible, and rubbed in lightly. The skin por- tions marked out were carefully separated and one square centimeter areas were cut out and immediately sliced with a freezing microtome in a thick- ness of 35 microns parallel to the surface. Six slices with one square centi- meter surface area were then collected and boiled briefly with 1 ml. of 1 per cent sodium hydroxide in a test tube. The volume was made up to 10 ml. with standard nutrient broth and the solution adjusted to pH 7 to 8 with a few drops of dilute acetic acid solution, after it had been determined previously that Micrococcus pyrogenes var. aureus grows unhindered at this pH. Further control experiments with untreated skin pieces showed that these do not weaken the effectiveness of the substance under investigation toward Micrococcus pyrogenes var. aureus and may be therefore neglected. The broth tubes prepared as above were then further diluted in geometric series and inoculated with 0.15 ml. of a twenty-four-hour Micrococcus pyro- genes var. aureus culture. The growth of this micro/3rganism was read after twenty-four and forty-eight hours at 37øC.