280 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Empicol ESB 3/S (sodium lauryl ether sulphate) (27.3•o active) Lauryl isopropanolamide 1.5 Sodium chloride 0.5 Sterile distilled water to 100.0 •o w/w 36.6 (equivalent to 10•o active detergent) After preparation the model shampoo was sterilized by filtering it through a 0.2 [tm filter membrane. Sodium lauryl ether sulphate (SLES) was chosen as the detergent because products containing it are known to be amongst those most suscep- tible to contamination, possibly due to its relatively low toxicity to Gram- negative bacteria. It is also widely used as a detergent in shampoos. The bacterium selected as the experimental contaminant was a strain of Enterobacter cloacae isolated from a contaminated shampoo. We have found this organism to be particularly resistant to anionic detergents. Three 16 oz nutrient agar slopes were each inoculated with 1 ml of a 24 h nutrient broth culture of Enterobacter cloacae and incubated at 28 ø overnight. The bacteria were washed from the slopes using distilled water and washed three times with water after centrifugation. The bacteria were finally re-suspended in distilled water and stored at 4 ø for 4 days before use. Total viable counts Total viable counts on bacterial suspensions and inoculated shampoos were performed in Tryptone Soya Agar using the pour-plate method with 0.1 •o peptone water as the diluent. Total viable counts on mains water were performed in Nutrient Agar using sterile mains-water as the diluent. In each case the plates were incubated at 28 ø for 7 days before counting. Influence of inoculum size on the survival and multiplication of bacteria in a shampoo Total viable counts were performed on the stored suspension of E. cloacae. A series of decimal dilutions of the suspension were then made in distilled water so as to give the following range of concentrations of bacteria: 109, 108, 107, 10 ø, 105, 104, and 10 a ml -•. 0.2 ml of each suspension was added to duplicate 20 g amounts of sterile shampoo. The inoculated shampoo was incubated at 28 ø and total viable counts were performed at intervals up to 14 days.

WATER-BORNE BACTERIA AND SHAMPOO SPOILAGE 281 Influence of shampoo volume on the survival and multiplication of bacteria 10 g, 100 g and 1000 g aliquots of the model shampoo were each inocu- lated with either 50 or 400 bacteria contained in 0.2 ml of suspension. After inoculation the shampoo was mixed thoroughly and then incubated at 28 ø . Total viable counts were performed at intervals up to 14 days. Isolation from mains water of bacteria capable of multipIyb•g in a model shampoo To determine the minimum volume of water necessary to contaminate the model shampoo a series of 100 g quantities of the shampoo were inocu- lated with bacteria isolated from volumes of water from 1 ml to approxi- mately 101. With the exception of the 1 ml inoculum the shampoos were not inoculated with water but with bacteria-proof membrane filters through which the water had been passed. Five replicates of each volume of water were inoculated into model shampoo. The water for testing was collected from a tap fed directly from the mains. Before collection the tap was swabbed with alcohol which was then burned off. The tap was then opened fully and allowed to run for 3 min to ensure that the sample was composed of water directly from the mains and excluded water which had stagnated in the pipes. Volumes of water from 10 to 1000 ml were collected in sterile conical flasks and filtered through membranes of mean pore-size 0.22 [tm supported in Millipore Sterifil filter holders. In order to estimate the number of organisms trapped on the membrane total viable counts were performed on the water before filtration. It was not convenient to filter a 10 1. volume of water using the Sterifil filter holder so these volumes were filtered through a 0.22 !•m membrane held in a stainless steel Carlson-Cox Model 1000 filter holder. The filter holder was attached to a length of sterilized flexible plastic hose the other end of which was fitted to the tap. After the water had been passed through the membranes each membrane was transferred to 100 g of sterile shampoo in a 4 oz sterile glass bottle. To dislodge as many organisms as possible from the surface of the membrane the bottle was shaken vigorously in a Griffin flask shaker for 5 min before transferring to an incubator. Total viable counts were performed immediately and at various intervals during incubation at 28 ø .