2S8 JOURNAL OF THE SOCIETY quent washings with an anionic soap released large numbers of live bac- teria, thus suggesting the presence of an imperceptible film of the cat- ionic substance. Alcohol, •lum and mercuric salts had also been ob- served to produce protective films masking viable organisms. We reasoned that if a film formed by a cationic soap is broken by the use of an anionic soap, the opposite should also take place. Therefore, if anionic soap containing Com- pound G-11 would form a film on the skin, then this film should be re- movable by subsequent use of cationic soap, and this should result in a flora of normal numbers. We set up an experiment on this basis. Six subjects washed through the standard 10-basin washing pro- cedure, using control soap (anionic). Three of the subjects substituted Onyxsan S, 1 per cent (a surface ac- tive agent of the alkyl amidoalkyl amidoazoline type), and the remain- ing three subjects Zephiran chloride 1 per cent in Basin 5 for the control soap. Onyxsan S and Zephiran chloride are cationic detergents the former is non-bactericidal, while the latter is a potent germicide. In the second experiment six subjects washed through the stand- ard 10-basin washing procedure, using control (anionic) soap and each subject was given a bar of 2 per cent G-11 (anionic) soap with instructions to use only this soap for all washings of the skin for the fol- lowing week and then to return for the final washings. The final washings were don- OF COSMETIC CHEMISTS ducted as follows: Three subjects washed for a two-minute period, using a 1 per cent aqueous solution of Onyxsan S, and rinsed in Basin 1. Washing with control (anionic) soap then followed in the other nine basins. The remaining three sub- jects went through this same routine except that 1 per cent Zephiran chloride was used instead of Onyx- •san S. It would appear that Zephiran chloride killed those bacteria which were removed in Basin 5, but, as was shown, the count returned to a high level as soon as the cationic film was removed by anionic soap in Basins 6 to 10. Thus Zephiran chloride apparently had only a superficial action and simply covered the bacteria temporarily with a re•' movable film. These results agreed with th4 findings of Miller 'and his associates. Onyxsan S, which is non-germicidal, had an action simi- lar to that of the ofdinary anionic soaps. After G-11 (anionic) soap was used for one week, it did not matter whether Zephiran chloride or Onyxsan S was used before the final washing. The counts were equally low and not higher than in the pre- vious experiments, in which no cationic detergent was applied prior to the washings with control soap. Obviously, if our premise was correct, G-11 did not exert its ap- parent action in reducing the resi- dent bacterial flora of the skin by obscuring the bacteria beneath a film. This substantiated the opinion that G-11 acted by actually killing the bacteria on and in the skin in

METHODS OF TESTING A GERMICIDE 259' other words, 2 per cent G-11 soap did not produce a protectant film on the skin, a condition which would give rise to a false sense of security. The Role of Bacteriostasis. We felt certain that any trace of G~11 soap left on the skin following its routine use over one week and pos- sibly carried over in the rinsings ob- tained from control soap washings would be so negligible that it could exert no bacteriostatic effect in the still more dilute cultures made from these rinsings. However, in order to prove this point conclusively, cultures were made of the rinsings obtained in the control soap washings after the routine use of 2 per cent G-11 soap for one week, by plating 1 cc. and 0.1 cc. samples from each basin. If bacteriostasis is not a factor, then the ratio of the number of colonies obtained in these two plates should be of the order of 10:1. If G-11 carried over does cause bacterio~ stasis in a certain dilution, then the bacteriostasis should be lessened by increasing the dilution of GAl. Therefore if we can obtain counts of the same ratio, regardless of the dilution, bacteriostasis is not in- volved. It was readily seen in tabulation that the ratio obtained in the two dilutions after the routine use of 2 per cent G-11 soap was approxi- mately the same as the ratio ob- tained in the two dilutions prior to the use of the 2 per cent G-11 soap. The remote possibility that the extremely small amount of Com- pound G~11, which could be carried over from the skin to the basins and then to the cultures, exerted a bac- teriostatic action was entirely ex- cluded by these findings hence the low counts obtained in control-soap washings following the routine use of 2 per cent G~11 soap were not due to bacteriostasis. Effect on Pathogens. Until now no' attempt had been made to dif- ferentiate between pathogens and saprophytes. In order to determine whether the 2% G-11 soap killed pathogens, the skin on the dorsal surface of the hands on 207 indus- trial employees was swabbed to de- termine the presence of Staphylo- coccus aureus. S. aureus was selected as the example of a pathogenic flora because it was thought to be present on approximately !0 per cent of human skins, because it was an' ac- cepted organism in evaluating the potency of germicides, and because of the relative ease with•which it is identified culturally. Eight persons were found to be carriers ofS. aureus. This was determined by doing fre- quent swabbings and repeatedly finding the organism to be present. This excluded the possibility that the organism was only incidentally present on one occasion. The eight subjects were given the use of G-11 soap for a ten-day period and again swabbed and cul- tured. One subject ran out of the soap three days before the swabbing was done and this subject was the only one on whom the organism was found. The results showed that seven out of eight cases were free and warranted the assumption that