478 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS type procedures could be used, viz. the phenol coefficient method. Extinc- tion type tests, due to lack of sensitivity, would not readily detect differ- ences between weakly bactericidal compounds. EXPE•.IMENT^I. AND D•scuss•o• Compound numbers of the Ns-phenols employed in this investigatiom refer to the numbers in a previous publication (1) and are listed in Table 1.1 Bis-phenols not demonstrating bactericidal action of 95 per cent kill better are omitted from this report. TABLE 1--NAtaES s•o NvtaB•Rs ov BIS-PHENOLS Compound No.* 1 2 3 4 5 6 7 8 9 15 16 19 20 21 24 30 31 32 33 34 36 37 38 39 40 41 42 43 47 48 2,2'-methylenebis (3,4,6-trichlorophenol) 2,2'-methylenebis (4,5,6-trichlorophenol) 2,2'-methylenebis (3,4,5-trichlorophenol) 2,2'-methylenebis (4~bromo-3,6-dichlorophenol) 2,2'-methylenebis (4-bromo-5,6-dichlorophenol) 2,2'-methylenebis (4-chlorophenol) 2,2'-methylenebis (4-methylphenol) 2,2'-methylenebis (4,6-dichlorophenol) 2,2'-methylenebis (5,6-dichlorophenol) 2,2'-ethylidenebis (4-chlorophenol) 2,2'-ethylidenebis (6-bromo-4-chlorophenol) 2,2'-(2,2,2-trichloroethylidene)-bis (6-bromo-4-chlorophenol) 5,5'-dichloro-2,2'-stilbenediol 3,5,3',5'-tetrachloro-2,2'-stilbenediol 2,2'-benzylidenebis-(4,6-dimethylphenol) 2,2'-thiobis (4-chlorophenol) , 2,2'-thiobis (4-bromophenol) 2,2'-thiobis (4,6-dichlorophenol) 2,2'-thiobis (6-bromo-4-chlorophenol) 2,2'-thiobis (3,4,6-trichlorophenol) 2,2'-thiobis (4-chloro-6-isopropyl- 3-methylphenol) 2,2'-thiobis (4-chloro-3,5-dimethylphenol) 2-hydroxy-2-methoxy-5,5'-dichlorodiphenyl sulfide 1,1 '-methylenebis (2-naphthol) 3,3'-methylenebis (2,4,6-trichlorophenol) 3,3'-methylenehis (4-bromo-2,6-dichlorophenol) 4,4'-methylenebis (2,3,6-trichlorophenol) 2,2'-oxybis (tetrachlorophenol) 4,4',6,6'-tetrabromo-o,o'-diphenol salicil (2,2'-dihydroxybenzil) * Numbers kept unif•)rm with numbering in J. Soc. Cosmetic Chemists, 11,307 (1960) for future reference purposes. The basic test procedure for ascertaining bactericidal activity of the compounds was that of inoculating a test system with a known number ofl organisms followed by enumeration of the survivors after twelve minutes i contact time. The test system consisted of 100 ml. distilled water contain- ing 30 ug./ml. sodium dodecylbenzene sulfonate (Ultrawet©-K) (Atlantic) and the specified level of bis-phenol. Aliquots of alcoholic (S,D.A. 30) solutions of the test compounds were added to the anionic-water system,

CHEMICAL STRUCTURE AND ANTIMICROBIAL ACTIVITY 479 but in no case was the final alcohol concentration greater than one per cent. The inoculum consisted of one mi. of a distilled water dilution of a 24-hour A.O.A.C. broth culture of either Staphylococcus aureus A.T.C.C. 6538 or •Escherichia coli A.T.C.C. 11229 (11) added to 100 mi. of the test system. 3_11 solutions were equilibrated to a temperature of 20øC. before inoculum addition. One ml. sample aliquots of the inoculated test system were removed at the end of the specified time and placed directly into Astell Roll •Fubes (Consolidated) containing 4 mi. antidote agar. The roll tubes were incubated at 34øC. and the survivors counted at the end of 48-hours in- eubation. Roll tube agar consisted of Dextrose Tryptone Extract Agar [(Difco) containing one per cent Tween © 80 (Atlas) fortified with an addi- itional 0.5 per cent agar. The bis-phenols were initially screened in the surfactant system against IS. aureus and E. coli at levels of 15/•g./ml. and 100 •g./ml. respectively. IPercentage kills were calculated and those compounds demonstrating a 95 Iper cent kill or better were further evaluated at lower levels. All com- [TABLE 2--BIs-PHENOLS DEMONSTRATING BACTERICIDAL ACTIVITY AGAINST ,•. aureus AND E. coli , S. aureus-- , , E. coli , Compound 15•g 10•g 5•g l•g 100•g 50•g 15•g (level) No. 95 99 95 99 95 99 95 99 95 99 95 99 95 99 (% kill) 5 * * 6 * * * * 7 * * 15 * * * * 16 * * * * 19 * * * * 20 * * * * * * 21 * * 24 * 30 * * * * 31 * * * * 32 * * * * 33 * * * 34 * * * * 36 * * 37 * * 38 * * 39 * * * 40 * * 41 * * 42 * * * * 43 * * * * 47 * * * 48 * * Initial count/•nl, of test system 8000 6500 4000 4000 18,000 17,000 17,000