SYNERGISM IN VITRO OF CERTAIN ANTIMICROBIAL AGENTS 535 and the phenoxetols. For example, Kaiser (4) has recognised the synergistic activity between 0.1% tyrothricin and 0.5% phenoxetol in the control of bacterial infection in medicinal products. Similar results were apparent when using 2, 7 diamino acridine and quaternary ammonium compounds. The need therefore to consider an antimicrobial system with activity against both gram-positive and gram-negative organisms is clearly essential. In addition, the time necessary for the complete destruction of these organisms is of utmost importance in dealing with cream products of the above type. It is therefore necessary to consider the potential combinations of antimicrobial agents for this purpose, which based on a knowledge of their inherent activity, should, when combined, provide an antimicrobial system active against both gram-positive and gram-negative organisms. It is not possible however, purely on the basis of theoretical considerations, to say "x" is active against gram-positive organisms and "y" is active against gram-negative organisms, and therefore a combination of "x" and "y" will show activity against both, as well as a synergistic effect. Initially, this has to be determined experimentally by bacteriological methods. We carried out tests, where known numbers of organisms likely to be encountered during use, were innoculated into given quantities, under sterile conditions. Samples were then removed at known time intervals on which plate counts were carried out using nutrient agar containing 3% Tween 80 as a quenching agent. From the results obtained each anti- microbial combination was evaluated. One has, of course, to distinguish between a purely additive and syner- gistic effect. Synergism is only apparent if, when usedin combination to pro- vide a more effective bactericidal or fungicidal effect, a lower concentration of each constituent is necessary than when any of the constituents are used independently. For example, a 3% hexachlorophane emulsion (o/w type containing alkyl aryl polyether sulphonate, lanolin cholesterols, and petrolatum) when used alone was ineffective in destroying Pseudomonas pyocyanea within 30 min. However, when this emulsion was combined with 1% Phenoxetol*, the Pseudomonas pyocyanea was destroyed within 3 min. Phenoxetol when used alone at 1ø/0 required 30 min to destroy the same organism. (Table I). Tribromosalicylanilide, another antimicrobial agent, when used as a 1% emulsion was only effective in destroying Pseudomonas pyocyanea in 30 min, whereas a combination of this product together with 10/0 Phenoxetol *Phenoxetol: [i-phenoxyethanol

536 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS destroyed the same organism within 5 min. When using 0.3% p-chloro- phenoxetol together with 1% tribromosalicylanilide this organism was destroyed within 2 min (Table II). Table I Pseudomonas pyocyanea 3 % Ilexachlorophane emulsion alone 3 % Ilexachlorophane emulsion plus 1% phenoxetol 2 •o Phenoxetol alone 1% Phenoxetol alone Control q- strong growth (+) slight growth -- no growth Time in minutes 1 3 5 + + + __ __ (++) 10 15 30 + + + -- -- __ -- -- __ + Table II Pseudomonas pyocyanea Time in minutes 1 2 3 5 10 15 30 1% emulsion of tribromo- salicylanilide q- q- q- q- q- q- -- 1% emulsion of tribromo- salicylanilide + 1 • Phenoxetol + + (+) .... 1% emulsion of tribromo salicylanilide +0.3 % p-chloro-phenoxetol q- ...... 1% Phenoxetol + + + + + q- -- 0.3 % p-chloro-phenoxetol q- q- q- .... Control + + + + + + + q- strong growth (+) slight growth -- no growth It should be noted that both the 3% hexachlorophane and 1% tri- bromosalicylanilide emulsions, when used alone, were very effective in destroying Staphylococcus, kills being obtained within 60 s. In comparison 2% Phenoxetol does not destroy these organisms in 60 min. Against Escherichia coli a combination of tribromosalicylanilide and Phenoxetol or p-chlorophenoxetol exhibited similar results (Table III).