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.

ASSESSMENT OF THE PRESERVATIVE CAPACITY OF SHAMPOOS 233 RESULTS (1) The growth of water-borne bacteria in detergents is shown in Table I. Table I Growth of water-borne bacteria on 0.1% anionic detergents in tap water Source of water Bacteria Bacteria ml -x after incubation at 28 ø for 7 days ml -• initially SLES MLS SLS ALS SDBS Ashford Common 23 107 106 20 0 10 Hampton 6 105 105 105 10 0 Isleworth 16 109 -- 109 I • Kempton Park 12 0 10 20 0 10 Surbiton 13 0 10 10 1 10 Walton 16 106 106 107 107 0 Coppermills 8 -- 0 0 -- -- Hornsey 14 i ] 07 10 • i 1 Lee Bridge 19 -- 106 107 -- -- Stoke Newington 20 -- 106 107 -- -- --= Not tested. The initial number of bacteria present in water was consistently between 6 and 25 ml -•. Six or seven different colonial types of bacteria were easily distinguishable. On one occasion the viable count of a water sample increased to 104 ml 4 simply by incubation at 28 ø, no doubt as a result of the presence of nutrient material. All the detergents tested, with the exception of SDBS, were able to support bacterial growth. Not all the water sources, however, were con- taminated with detergent-utilizing bacteria. When growth did occur the number of organisms present increased to between 105 and 109ml 4, with most of the cultures containing 106-107 organisms ml -•. Without exception there was a vast predominance of one type of bacteria in each system after incubation. Most of the bacteria isolated appeared to be either Pseudomonas spp. or Klebsiella spp. Of the Pseudomonas one proved to be Pseudomonas aeruginosa. Should it become necessary to maintain the test organisms for any length of time a suitable medium is provided by 2•o Ionagar containing 0.1 • detergent. (2) The ability of bacteria to degrade detergents by hydrolysis of the sulphate group is shown in Fig. 1. Bacteria grown on nutrient broth alone did not possess sulphatase activity, but with 0.1 • SLS added to the growth