SYNERGISM IN VITRO OF CERTAIN ANTIMICROBIAL AGENTS 541 It will be clear from Tables V11I and IX that one is able to go some way in approaching an all-purpose preservative by using combinations of materials. We have now obtained a product with a considerably enhanced anti- microbial system and in considering further possibilities of synergism this combination rather than a single ester should be used. In our work, there- fore, in considering possible bacteriological experiments, an ester combin- ation has been used as the one component of further mixtures, as already indicated in Tables VIJI and IX. I wish to emphasise that in our bacteriological tests we have laid con- siderable stress on determining the effectiveness of combinations on the organism Pseudomonas pyocyanea and other gram-negative organisms. The reasons for this have been our observations of the notable increase of this organism in cosmetic preparations. One suggestion put forward for the ever increasing incidence of Pseudomonas pyocyanea is the widespread use of hexachlorophane. Continued use of this material considerably reduces the degree of gram-positive skin bacteria, but clears the way for infection by gram-negative organisms such as Pseudomonas pyocyanea, Proteus, Coli, etc., against which this product is ineffective. This finding, amongst others, has led to the present bacteriological examination of combinations of the above type. A report (13) refers to a possible connection between the increase in pyocyaneus infections and the widespread use of disinfectants containing hexachlorophane which are ineffective against gram-negative organisms. Recently, Savin (14) in an investigation of Pseudomonas pyocyanea infections in a hospital skin ward, found a diluted steroid cream to contain large numbers of these organisms. It is resistant to most antibiotics and able to fill the biological vacuum left when these are used. Since the intro- duction of antibiotics, Pseudomonas pyocyanea has played an increasing role in human infection. Reference (15) was made early last year to several instances of in- fection by Pseudomonas which developed despite the fact that treatment had been carried out with creams containing antibiotics, such as hydro- cortisone, amphomycin and neomycin to which this organism is usually sensitive. After these cases had been investigated further, it was found that opened tubes of the used ointment were themselves infected with Pseudo- monas pyocyanea and that several other gram-negative bacteria are able to survive in what might. be regarded as an unfavourable environment, pro- viding a trace of moisture is present. Last year Burdon and Whitby (16)

542 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS reported the contamination by Pseudomonas species of two commercially available preparations, one containing chlorhexidine and the other a combination of chlorhexidine and cetrimide, these preparations being recommended as the disinfectants of choice against Pseudomonas. In all these investigations, Pseudomonas is not the only offending micro-organism. Others which have been found include Staphylococcus pyogenes, Escherichia coli and different Salmonella strains. In further consideration of all these facts, it is necessary not only to determine the actual sterility of a final preparation, but also to determine the resistance of a preparation when inoculated with high concentrations of micro-organisms of the above type. We have carried out much work on the determination of observing the percentage reduction of a given inoculant concentration over short intervals. This work has involved emulsions containing various concentrations of hexachlorophane, tribromosalicylanilides, alone and in combination with Phenonip*, a liquid broad spectrum antimicrobial system. Our investi- gations have been concerned with short time kills, i.e. less than 10 min and the results are shown in Fig. 2. A combination of a 3% hexachlorophane emulsion with 1.0ø/0 Phenonip thus provides the desired kill rate more closely than 3% hexachlorophane emulsion by itself, against both gram-positive and gram-negative bacteria. Phenonip itself is a synergistic combination of certain antimicrobial agents. It inhibits the growth of spoilage organisms at concentrations well within its maximal water miscibility of 0.50/0 . Lethal concentrations are higher but killing times are impressive at maximum solubility. Nonionic surfactants can be used to solubilise Phenonip providing sufficient con- centrations which are highly effective. Tests with anionic surfactants had a negligible interference effect on the product, in contrast to quaternaries and compounds such as chlorohexidene, which are incompatible with anionics. Generally speaking the product shows inherent efficiency against a broad- spectrum of spoilage micro-organisms. The lethal effects are impressive and inhibiting activity is maintained at low concentrations. Recent work by Tracy, Glass, Nicholson and Pivnick (17) on pre- servatives for poliomyelitis vaccine, has demonstrated the effectiveness of' using combinations of antibiotics with formaldehyde, and the esters of p-hydroxybenzoic acid and the phenoxetols. These workers found, for example, that the failure of low concentrations *Phenonip: based on p-hydroxybenzoates