SALICYLANILIDE COMPOSITION FOR SOAPS AND COSMETICS 349 show a decrease in antibacterial activity. Special "use" tests with soap have demonstrated that the antiseptic does not lose its potency as the soap tablet is gradually consumed. Since diaphene is composed of two phenolic compounds, it reacts with ferric chloride test solution to give a violet color, but larger amounts of iron are tolerated than with many other phenolic antiseptics. The analysis of diaphene in soap using ferric chloride reagent is carried out by the following technique: Preparation of Calibralion Curve for Fisher Electrophotomeler for Ferric Chloride Mezhod. Weigh 5 grams of shredded untreated soap into each of seven 100-ml. volumetric flasks containing a few boiling chips. Into separate flasks pipette 0, 5, 10, 15, 20 and 25 ml. of diaphene stock solution (0.1 gm. diaphene dissolved in 100 ml. of isopropyl alcohol). These flasks then contain respectively 0, 0.1, 0.2, 0.3, 0.4 and 0.5 per cent dia- phene per 5-gm. sample of soap. Make up the volume to about 50 ml. with isopropyl alcohol and boil the solution until the soap dissolves or goes into fine suspension. While the solution is still boiling add 30 ml. of methanolic barium bromide solution. This will precipitate the soap as a barium salt. Cool to 25øC. and dilute to 100 ml. volumes with methyl alcohol. Mix thoroughly and filter each solution rapidly into separate dry 500 ml. filter flasks using a Buchner funnel containing a Whatman No. 1 filter paper. The flitrate may then be stored for analysis. Pipette a 2-ml. aliquot of the tiltrate into a 50-ml. volumetric flask. Pipette 20 ml. of ethyl alcohol into the flask, and place the flask and con- tents in a constant temperature bath for five minutes. When all the solutions have been prepared and are in the bath add 1 ml. ferric chloride solution (aqueous 2.5 per cent) to the first flask. Three minutes later add 1 ml. of ferric chloride solution to the second flask. Remove the first flask from the bath, pour the solution into a 23-ml. cell of the Fisher Electro- photometer, and note the A scale reading exactly four minutes after the addition of the ferric chloride solution. (A color filter is not used this time in the electrophotometer.) Add 1 ml. of ferric chloride solution to each remaining flask in order at three-minute intervals and read the color of the previously prepared solution exactly four minutes after the addition of ferric chloride solution. Prepare a calibration curve of per cent diaphene versus A scale readings. In those instruments where a color filter must be used, a green filter (550 mu.) is recommended. Atnalysis of Diaphene in Soap. Weigh 5 grams of shredded treated soap into a 100-ml. volumetric flask. Add 50 ml. of isopropyl alcohol and a few boiling chips. Boil the solution until the soap i's dissolved or goes into suspension. Add 30 ml. of methanolic barium bromide solution to the boiling contents of the flask to precipitate the soap as the barium salt. Cool the flask and contents to 25øC. and dilute the solution to 100 ml.

350 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS with methyl alcohol. Mix thoroughly and filter the solution rapidly through a Buchner funnel containing a Whatman No. 1 filter paper into a 500-ml. filter flask. Retain the flitrate for analysis. Pipette a 2-ml. aliquot of the flitrate into a 50-ml. volumetric flask. Pipette 20 ml. of ethyl alcohol into the flask and place the flask and con- tents in the constant temperature bath for five minutes. Add 1 ml. ferric chloride solution and swirl the flask to mix the contents thoroughly. Three minutes later remove the flask from the bath, pour the solution into a 23 ml. cell of the Fisher Electrophotometer, and note the A scale reading exactly four minutes after the addition of the ferric chloride solution. From the calibration curve read the percentage of diaphene in the sample. A separate calibration curve will be required on each grade of base soap. ANTIMICROBIAL ACTIVITY The in vitro activity of diaphene has been determined against a wide spectrum of microbes using standard toxic dilution test techniques. Table 2 summarizes the data collected. TABLE 2--INHIBITING CONCENTRATION OF DIAPHENE AGAINST VARIOUS MICRO•SRGANISMS Inhibiting Inhibiting Concentration, Concentration, Micro6rganism p.p.m. Micro6rganism p.p.m. Bacillus subtilis O. 5 Rhizoctonia solani 1 Micrococcus pyogenes var. Sclerotinia fructicola 1 * aureus O. 5 Sclerotinia rolfsii 1 * Micrococcus albus O. 5 Candida albicans 1 Sarcina lutea 1 Trychophyton mentagrophytes 1 Streptococcus f aecalis 5 Microsporum rubrum 1 Proteus ammoniae 10 Microsporum audouini 1 P seudomonas aeru ginosa 10 Salmonella typhosa 5 •lerobacter aerogenes O . 5 Salmonella choleraesuis 5 Bacillus ammoniagenes O. 5 Salmonella schottmulleri 5 ,'Ispergillus niger 10 Escherichia coli 10 Bot. cinerea 1 Sclerotinia sclerotinium 1' ,'llternaria solani 1 Fusarium oxysporum 1 Penicillium italicum 10 Stemphylium sp. 1 Rhizoctonia nigricans 1 Lactobacillus casei 2 * Spore germination test. It can be seen that the antiseptic composition is effective against a wide variety of gram-positive bacteria, as well as certain gram-negative bac- teria and fungi. The inhibitory concentration is frequently less than that reported for many other soap bacteriostats and cutaneous antimicrobials. The good activity against Micrococcus pyogenes var. aureus is particularly noteworthy, for this micro 3rganism is most abundant in the resident skin flora. The effective inhibition of Pseudomonas aeruginosa, Escherichia coli and Trychophyton mentagrophytes at relatively low concentrations is most interesting.