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

232 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS test as described by Hsu (1). This test depends on the enzymatic breakdown of a chloroform-soluble complex of detergent and methylene blue. On hydrolysis of this complex by the enzyme, water-soluble methylene blue is released which is estimated spectrophotometrically. The organisms were grown for 24 h at 28 ø on various media including nutrient broth (Oxoid CM1) (NB), NB containing 0.1}/o detergent (as above) and a sulphate-free salts solution (3) containing 0.1•o detergent (as above) as the sole source of carbon and sulphur. The cells were harvested, washed with sterile distilled water and resuspended in the dye-detergent complex solution which contained 33 nmol each of SLS and methylene blue ml -• of 0.14 M sodium chloride and 0.05 M tris-HC1 buffer at pH 7.2. The mixture was incubated at 28 ø. Aliquots were removed at intervals of 10 min and the free methylene blue estimated following centrifugation. The specificities of the enzyme inducers were examined by growing the bacteria in SLS, MLS and SLES and testing for SLS degradation. Slime production by test organisms Slime production was promoted in the test organisms by growing them without shaking in salts solutions containing increasing concentrations of detergent. SLES was increased from 0.1,% to 1, 5, 10 and 13,5/o by sub- culture. A new challenge test for shampoos Bacteria isolated from tap water or contaminated shampoos were grown in NB, sulphate-free salts solution containing either 0.1 •o SLES (for induction of sulphatase) or 13}/o SLES (for production of slime). The cells were harvested, washed with and resuspended in water at a concentration of about 109 ml-L 0.1 ml of the suspension was inoculated into 20 g unpre- served SLES-based shampoo and mixed well. 1 ml aliquots were removed for viable count on TSA immediately and at intervals during several weeks' incubation at 28 ø . Adaptation of a bacterial strain to a particular detergent was tested by using bacteria grown in 13,5/o MLS to challenge a shampoo containing a different detergent, e.g. SLES. In addition, these same bacteria were tested for survival in shampoo after growth in NB.