274 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS medium and Fluid Thioglycollate (Difco) broth for the Chlorella and anaerobic bacteria, respectively, were also employed. lnocula for the aerobic bacteria consisted of one drop (0.007 mi.) of a 1-100 distilled water dilution of a 24-hour, 35 ø, Tryptic Soy Broth (Difco) culture onto the surface of the agar plates. Inoculum for Chlorel/a vulgaris consisted of one drop of a 1-100 dilution of a 5-day, 25 ø culture to 10 ml. of broth containing the test compound. Anaerobic bacterial inocula consisted of one drop of a 1-100 dilution of a 72-hour, 35 ø broth culture to 15 ml. of broth containing the test compound. Yeast inoculum was one drop per plate of a 1-100 water dilution of a heavy cell suspension prepared by the addition of 10 ml. of distilled water to a 72-hour agar slant. Mold inocula were prepared by suspending the conidia from the surface growth of a 7-day slant in 20 ml. of distilled water with no further dilution. Incubation for aerobic bacterial plates was 35 ø for forty-eight hours, at the end of which the level completely inhibiting growth was recorded. Anaerobic tubes were incubated for fourteen days at 35 ø , after which bacteriostatic levels were recorded. Yeast results were noted after four days incubation at 30 ø and mold results after fourteen days at 30 ø . In- hibition of Chlorella was recorded at the end of thirty days incubation at 25 ø over two 30-inch fluorescent tubes at a distance of 20 inches. Because of inherent error in the procedure, tests were performed in triplicate on different days for aerobic bacteria. Tests with all other organisms (cf. Table II) were performed in duplicate on different days. The average inhibitory levels, calculated as the geometric mean of the values obtained, are reported in Table III. TABLE III--ANTIMIOROBIAL SPECTRA or METHYLENEBIS(TKICHLOKOPHENOL) ISOMEKS. (MINIMAL INHIBITORY LEVEL, /.ZG./ML.) Compound (by Number) Micro6rganism 1 2 3 40 65 66 67 42 S. aureus 0.93 0.61 0.39 10 2 S. epidermidis 0.93 0.78 0.23 12.5 2 B. subtilis 0.19 0.39 0.19 3.9 1 B. ammoniagenes 0.39 0.48 0.19 15.6 2 P. vulgaris 3.9 1.9 3.9 25 E. coli 25 * 12.5 * S. typhosa 40 * 10 * Ps. aeruginosa 25 * 50 * Ps. fiuorescens 0.23 (t.61 0.19 12.5 2 Sh. sonnei 40 * 9.9 * K. pneumoniae 50 * 15.6 * T. mentagrophytes 1.74 3 12 3.12 8.9 8 T. rubrum 4.4 3.12 8.9 8.9 3 M. audouini 3.12 1.74 3.12 3.12 4 C. albicans and P. ovale * * * * C1. tetani 0.19 0.39 0.09 1.56 0 C1. perfringens 0.78 0.39 0.55 1.74 0 C1. sporogenes 0.78 0.55 0.55 1.56 0 C. vulgaris 3.12 4.4 8.9 * .5 20 1.56 25 ß 4 20 1.56 25 .9 9.9 1.56 15.6 .4 40 1.56 31.5 * 63 10 63 .4 31.5 1.56 25 .9 6.25 4.4 6.25 _1 1.56 1.74 9.9 .4 3.12 8.9 4.4 .27 1.74 0.19 4.4 .55 6.25 0.39 6 25 .55 6.25 0.55 6.25 * 17.8 4.4 17.8 * Denotes growth at 100 •g./ml.

CHEMICAL STRUCTURE AND ANTIMICROBIAL ACTIVITY OF BIS-PHENOLS 275 DISCUSSION It may be noted from Table III that each of the isomers tested was found to be biologically active and that the activity varied over a wide range in certain instances. Compound No. 42, which has the methylene bridge in the 4,4'-positions, was found to be the least active, which is in agreement with previous findings (1, 9). Of the 3,3'-methylenebis compounds, the weakest was No. 66, followed by No. 40. Why these compounds were decidedly inferior to Nos. 65 and 67 cannot be explained at this time. The most active 3,3'-isomer was No. 67 in which the three chlorine atoms are adjacent to each other. It was shown previously (1, 9) that maximum antibacterial activity of halogenated bis-phenols was observed with compounds containing the 2,2'-methylene bridge the isomers of hexa- chlorophene are no exception to this rule. Hawever, this is apparently true only for bacteria against the dermatophytes the activity of the isomers was found to be of the same magnitude. Hexachlorophene and four of its isomers were very active against the Clostridia tested less active were Nos. 42 and 66, which were also the poorest against the aerobic bacteria. A similar pattern was observed with Chlorella vulgaris. Hexa- chlorophene and all isomers consistently lacked activity against the two yeasts, Candida albicans and Pityrcsporum ovale. Major differences in bacteriostasis may be noted with the Staphylococci and other Gram-positive organisms, compound No. 3 being of outstanding activity. It might be of interest to note that compound No. 3 is the only isomer which has no chlorine substituents adjacent to the hydroxyl groups. Only hexachlorophene and one of the 2,2'-isomers (compound No. 3) demonstrated activity against the more resistant Gram-negative bacteria such as Escherichia coli, Salmonella typhosa, Pseudomonas aeruginosa, Shigella sonnei and Klebsie#a l•neumoniae. Proteus vulgaris and Pseudo- mor,'as fluorescens were generally found to be extremely susceptible to all three 2,2'-isomers. SUMMA RY The broad spectrum antimicrobial activity of hexachlorophene and of seven of its nine isomers was investigated. Maximum activity against bacteria was shown by the 2,2'-methylenebis-phenols 2,2'-methylenebis- (3,4,5-trichlorophenol) was found to be generally more potent than hexa• chlorophene and the other 2,2'-isomer. Great variations between the 3,3'-isomers were noted the 4,4'-isomer was definitely the least active of the compounds tested. Against the dermatophytes the activity of the isomers was found to be of the same magnitude. None of the bis-phenols was active against C. albicans or P. ovale at a concentration of 100 ug./ml. (Received March 25, 1963)