CHEMICAL STRUCTURE AND ANTIMICROBIAL ACTIVITY OF BIS-PHENOLS 723 Inocula for the bacteria were prepared by diluting a 24 hour Tryptic Soy Broth (Difco) culture 1:100 in 0.1% peptone water in the case of Bac- terium ammoniagenes a 48 hour culture was used. Inocula for imperfect fungi and dermatophytes were obtained by harvesting conidia from 10 and 15 day slants, respectively, in 50 ml. of 0.1% peptone water. As with the bacteria, one drop of the conidial suspension was added to the agar plates. Yeast inocula were prepared from 24 hour Sabouraud Dextrose Broth (Difco) cultures diluted 1-100 in 0.1% peptone water. Temperature of incubation of the cultures and of the test plates was 30 ø . Bacterial and yeast data were recorded at the end of five days imperfect fungi and dermatophytes at the end of 10 days Chlorella at the end of 30 days over continuous illumination. Because of the magnitude of activity exhibited by some of the compounds against several gram negative organisms, it was considered of interest to ascertain levels of activity in the presence of soap. For this purpose, a 1 ml. aliquot of the alcoholic stock solution of each compound was added to 100 ml. 2.5% white soap stock (Lever Brothers Co. Lux base). Aqueous serial dilutions were made, agar added to the serial dilution tubes, plates poured and the hardened plates inoculated as previously stated. Incuba- tion of plates was 72 hours at 30 ø . Soap controls were conducted to differentiate inhibition by the soap from the bacteriostatic activity of the compounds. The ratio of soap to test compound was held constant at 100-1 for gram-positive bacteria and at 50-1 for gram-negative bacteria. All tests were conducted in triplicate and the bacteriostatic level of the compound calculated as the geometric mean of the three observations in ug/ml., using a graphic technique for ease of calculation. Log•0 concen- trations of compound plotted against dilution tube number served as a rapid convenient plot for calculating geometric means. The highest level tested was 100 ug./ml., and no attempt was made to standardize inocula in any of the tests. The minimal inhibitory concentrations are reported in Tables V and VI. DISCUSSION Each of the compounds tested was found to be biologically active however, as may be noted from Table V, the isomers with hydrogens in the 6- and 6'-positions (II, III and VI) showed a broader spectrum of activity than the ones where these positions are occupied by chlorine (I, IV and V). A similar pattern had been observed previously (1) in the series of hexachlorophene and its isomers 2,2'-methylenebis (3,4,5-trichloro- phenol) was superior to hexachlorophene and the 4,5,6-trichloro isomer. In the present series, 2,2'-methylenebis (4,6-dichlorophenol), also known as G-5 ©, was found to exhibit the narrowest spectrum of activity gram

724 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS TABLE V--ANTIMICROBIAL SPECTRA OF 2,2•-METHYLENE (DICHLOROPHENOL) ISOMERS (Minimal Inhibitory Concentration, ug./ml.) Compound (by Number) Microbrganism I II 11I IV V VI S. aureus 6538* 0.48 0.50 0.50 0.78 0.48 0.48 S. epidermidis 155 0.63 0.31 0.06 1.56 0.78 0.10 B. subtilis 9372 0.25 0.20 0.05 0.98 0.63 0.08 B. ammoniagenes 6871 0.62 0.15 0.04 0.98 1.56 0.05 E. coli 11229 X t 9.80 7.80 100.00 X 7.80 S. choleraesuis 10708 X 12.50 9.80 100.00 62.00 7.80 S. typhosa 6539 X 20.00 12.50 X X 7.80 P. vulgaris 9920 3.12 3.12 1.28 5.20 0.78 1.95 Ps. aeruginosa X 25.00 20.00 X X 16.00 C. albicans 10231 X 6.25 6.25 31.50 16.00 6.25 S. cerevisiae X 6.25 6.25 7.80 5.00 4.00 P. funiculosum 11797 X 12.50 4.00 31.20 6.25 6.25 P. piscarium 12109 X 3.12 1.95 62.50 25.00 2.46 At. niger 9692 X 4.90 1.95 16.00 9.80 6.25 At./qavus 96•3 X 9.80 7.80 62.50 16.00 16.00 T. mentagrophytes 9129 0.96 0.96 0.39 0.78 0.62 0.78 T. rubrum 10218 0.50 1.00 0.78 0.62 0.30 1.00 C. vulgaris 9765 1.56 0.39 0.48 3.12 1.95 0.39 * American Type Culture Collection. t X denotes growth at 100 ug./ml. TABLE VI--BAcTERIOSTATIC LEVELS OF 2,2'-METHYLENEBIS (DICHI,OROPHENOL) ISOMERS IN THE PRESENCE OF SOAP* -Compounds (by Number) Organism I II III IV V VI S. aureus 0.98 S. epidermidis 1.25 B. ammoniagenes 1.55 E. coli • S. typhosa }-growth at 20/•g./ml. P. vulgaris• 1.25 0.63 1.25 0.98 0.98 0.63 0.16 1.95 1.25 0.20 0.31 0.16 1.55 1.95 0.16 * Soap/compound ratio: 100/1 for gram-positive bacteria. 50/1 for gram-negative bacteria. negative bacteria, with the exception of Proteus vulgaris, yeasts and molds grew at a level of 100 ug./ml. of this chemical. The most potent com- pound appears to be the 3,5-dichloro isomer (III), followed by the asym- metrical (VI) and 3,4-dichloro (II) isomers. These three compounds show superior activity against gram negative bacteria, yeasts and molds. All isomers are alike in their behavior against S. aureus, the dermatophytes and Chlorella vulgaris. In the presence of soap, no difference between the isomers could be ob- served all are active against gram positive bacteria and inactive at a level of 20 ug./ml. against gram-negative ones.