HYGIENIC MANUFACTURE AND PRESERVATION 757 3. PRODUCT TESTING 3.1 Toothpaste 3.11 Examination of samples in current production Extrude a small portion of paste from each tube and discard in order to eliminate chance contamination. Add approximately 1 g of paste to 9 ml of Peptone Water Diluent, mix thoroughly and plate out 6 aliquots each of 0.1 ml on Oxoid Nutrient Agar. Incubate the plates in triplicate at 28 ø or 37 ø and examine at 24 h intervals. Discard the plates after 7 days if no growth occurs. 3.12 Plate-count technique for estimating viable bacteria in toothpaste Measure 1 g amounts of toothpaste into sterile bottles containing 9 ml of Peptone Water Diluent and mix thoroughly. Prepare decimal dilutions of the suspension in the same diluent down to 10 -7. Transfer four aliquots, each of 1 ml from each dilution to sterile Petri dishes and mix with Nutrient Agar. Incubate the plates in duplicate at 28 ø and 37 ø followed by colony counting after 48 h. 3.13 Method for estimating survival of bacteria added to toothpaste Culture the test bacteria, preferably an isolate from a contaminated toothpaste, for 24 h at 28 ø on Nutrient Agar slopes. Wash the cells from the slopes and wash again twice before re-suspension to a final concentration of 109 ml-1 use sterile distilled water throughout this procedure. The paste for this test should have been made in the normal fashion and amounts of approximately 400 g placed in sterile glass jars (100 mm diameter) to a depth of 60 mm. Ensure that the surface is as smooth as possible. Layer 50 ml of the bacterial suspension on the surface of five such portions of toothpaste. During subsequent storage the cells become evenly distributed throughout the paste. Cover each jar to minimise evaporation but allow condensation to occur within the head space. Carry out viable counts at 28 ø (see 3.12) immediately and after 1, 2, 3 and 4 weeks' incuba- tion at 28 ø . Prior to each viable count, mix the contents of the jar thorough- ly for 15 min. If a preservative is included in the toothpaste a suitable inactivator must be included in both the diluent and the Nutrient Agar. 3.2 Shampoo 3.21 Microbial spoilage Shampoos based on detergents such as triethanolamine lauryl sulphate and sodium lauryl ether sulphate are usually able to support the growth of

758 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS bacteria if no effective preservative is present. Organisms responsible for shampoo spoilage consist largely of gram-negative, non-spore-beating rods. Moulds and yeasts are rarely encountered. When contamination occurs, a sediment or "ropiness" may become visible but not always the aroma may alter noticeably and also there may be a potential risk to the user. To avoid these undesirable consequences, it is theoretically possible either to employ sterile raw matehals and handle them aseptically so as to avoid extraneous contamination altogether, or alternatively, to operate with a reasonably high standard of cleanliness and include a bactericide. Thorough aseptic technique would be unduly expensive and elaborate for shampoo manufacture and it is therefore customary to adopt the second course. 3.22 Testing the preservative Before adding the preservative to the shampoo, set aside about 400 ml of the unpreserved product. Store at about 4 ø until ready for use as a control. After the preservative has been added to the shampoo, allow the product to stand at room temperature for at least 48 h. The length of time that should be allowed for chemical interaction between shampoo and preservative will largely depend on the stability of the preservative under test, e.g. formalin disappears very quickly from some formulations, but products containing less reactive preservatives should be retained before testing for at least 5-10 days. It is obviously best to allow a prolonged interaction time to be sure that the preservative will be stable in the shampoo formulation. 3.23 Test inoculum The test inoculum should comprise representative strains of aerobic non-sporing gram-negative bacteria initially isolated from factory plant, spoilt shampoo, and contaminated detergent preparations. The inoculum should include a selection of Pseudomonas species. The organisms should be recent isolates and capable of vigorous growth in detergent solutions. The test bacteria should be grown on slopes of Nutrient Agar containing 5% of the unpreserved shampoo, for 18-24 h (see methods oe.1oe1 and •.1oe•, p. 756). Experimental inoculation of the shampoo should be carried out in duplicate using a washed suspension of micro-organisms suitably diluted in sterile water. For test purposes a suitable inoculum of viable organisms is between 107 and 109 ml-1. To each 100 ml sample of shampoo add 1 ml of the standard bacterial suspension. Mix thoroughly but avoiding excessive froth. After inoculation, incubate the samples at 22 ø and 30 ø. Estimate bacterial counts in Tryptone