230 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS mine the degree of specificity of preservation of the product being tested. The chances of contamination occurring will be lowest when the number of different organisms capable of growth in the product is small and when these organisms are rare or absent in the manufacturing and use environ- ments. Currently available tests for preservative capacity Tests for preservative capacity currently in use can be divided into three categories: (i) Inoculation of the finished product followed by incubation and examination at intervals for signs of spoilage. (ii) An inoculation, incubation, sampling cycle which is repeated until samples show evidence of viable organisms. (iii) A single inoculation followed by incubation and periodic sampling for viable organisms. Tests in the first category require a long time period for completion and only show whether a product is spoiled rather than whether it is con- taminated. It is in fact quite possible to find products containing many millions of organisms per gram but with unaltered appearance. The second type of test attempts to reproduce in-use conditions. How- ever, because of the large number of organisms introduced with successive inoculations, and the progressive dilution of the product, it is biased against preservatives readily inactivated by organic matter and those having high concentration exponents, i.e. those in which a small change in concentration is accompanied by a large change in activity. The test in the third category not only detects the growth of organisms in the product but also the survival of non-multiplying organisms, which may be an indication of possible eventual multiplication should the pre- servative system be unstable. Laboratory challenge tests have been known to indicate that a product is sufficiently well preserved even when this same product is later shown to be contaminated. The most obvious reason is that the wrong test organism was used in the challenge test. However, cases have also arisen in which organisms isolated from a contaminated product and cultured in laboratory media did not survive when re-applied to the original product, giving the impression that the formulation could not support bacterial growth. Apparently the organisms adapt to their new surroundings and the adaptive mechanisms are lost during sub-culture in laboratory media so

ASSESSMENT OF THE PRESERVATIVE CAPACITY OF SHAMPOOS 231 that death occurs upon re-entry into the product. The type of adaptation which occurs could be (a) biochemical, e.g. induction of enzymes may occur to allow the organisms to utilize constituents of the product formulation for growth (b) morphological, e.g. the bacterial cell wall may change as a protection against the high ionic strength of the product (c) genetic, e.g. mutations may produce permanent changes in the bacteria. This paper describes our attempts to test some of these possibilities in the hope of establishing a reliable challenge test for shampoos. EXPERIMENTAL Utilization of detergents by water-borne bacteria Samples of domestic tap water from 11 different sources were trans- ported to the laboratory on ice and examined within 3 h of collection. The excess chlorine was neutralized by adding a crystal of sodium thio sulphate to 100 mi. 1 ml of 10•o detergent solution was added to bring the final concentration of detergent to approximately 0.1 •o. The detergents tested were sodium lauryl sulphate (SLS), sodium lauryl ether sulphate (SLES), mono-ethanolamine lauryl sulphate (MLS), ammonium lauryl sulphate (ALS) and sodium dodecyl benzene sulphonate (SDBS) (commercial grades supplied by Marchon Division of Albright and Wilson Ltd). The solutions were then incubated with shaking at 28 ø for 7 days. The number of viable bacteria present in each sample both before and after incubation was established by plating on tryptone-soya agar (TSA) and incubating at 28 ø for 48 h. 100 ml samples of water were also mixed with 100 ml molten 4•o Ionagar (Oxoid Ltd) at 48 ø containing 0.2•o detergent. Plates were poured and incubated at 28 ø for up to 3 weeks. Bacterial metabolism of detergents Bacterial strains isolated from shampoo or water were examined for their ability to hydrolyse the sulphate group of some detergents commonly used in shampoos. Sulphatase activity was assayed using the methylene blue