756 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 2.121 Culture on solid medium Inoculate slopes from stock culture and incubate for 24 h, or longer if necessary, at the optimum temperature for the organisms. Maintain in continuous active culture by sub-culturing onto fresh solid medium at intervals of 24 h on three successive days. 2.122 Culture in liquid medium Inoculate a tube of Nutrient Broth from the stock culture and incubate at the optimum growth temperature of the organism. Maintain in continu- ous active culture by subculturing into fresh medium at intervals of 24 h on three successive days. The cells should be well washed by centrifugation to remove surplus nutrient medium. A suspension should be prepared in sterile distilled water and standardized to an opacity or optical density calculated to produce a viable count usually in the order of 107 to 109 organisms. Yeasts and fungi are grown on slopes of Malt Extract Agar or Sabouraud Dextrose Agar for three and seven days respectively at 22-25 ø. Dry spores, rather than mycelium, suspended in small volumes of water are recommended for tests with fungi. One drop or a loopful of suspension should be applied to the surface of the product (usually a cream or emulsion) to be tested. In all tests, the inoculum should be in a form which interferes as little as possible with the properties of the product. The sample should be inspected visually for mould growth. 2.13 Storage tests Long-term storage tests need to be conducted before finally approving a preservative for use in a particular product. These tests consist of exposing the finished product, processed under normal manufacturing conditions so as to include the usual environmental microflora, to a wide range of temperature and relative humidity conditions along with regular exposure to atmospheric contamination. The preparation should be inspected for visual and olfactory evidence of deterioration and samples periodically cultured for evidence of microbial contamination. Packaging matedhals may reduce the efficacy of preservatives by physico-chemical interaction. It is therefore important that the product be tested with the same type of con- tainer and closure that will actually be used. During the period of surveil- lance, which might be from six months to two years, resistant organisms may slowly develop in the product until they eventually flourish in what was previously a hostile environment. Ample time should be allowed for such adaptation. It follows that no final conclusion regarding the efficacy of a preservative in a given system can be reached for several months.

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