HYGIENIC MANUFACTURE AND PRESERVATION 755 the following typical spoilage organisms obtainable from Type Culture Collections have been recommended for the testing of preservatives (Appendix E, 2.7, p. 797): Bacteria Staphylococcus aureus, Staph. epidermidis, Micrococcus spp., Streptococcus spp, Corynebacterium pseudodiphtheriticum, Coliform, Coli-Aerogenes group, Proteus, Pseudomonads including Ps. aeruginosa, Bacillus cereus, and B. Subtills. Moulds and yeasts Fungal organisms should include Penicillium, Aspergillus, Mucor, Clado- sporium, Alternaria, Trichoderma viride, Candida and Saccharomyces species. Naturally occurring bacterial contaminants will usually flourish more vigorously in a manufactured product than will the type culture strains of medical importance. It is advisable, therefore, first to inoculate the un- preserved formulation. Only those organisms showing vigorous growth and surviving for more than ten days in the product should be selected for testing preservative effectiveness. The selection of appropriate spoilage organisms and the number of strains to be used in a particular study is obviously important the final choice and method of inoculation is left to the discretion of the microbiologist performing the test. An unpreserved formulation should always be included to serve as control this should be checked for microbial content before commencing preservation studies. Inoculation with bacteria may be carried out with an actively growing broth culture by stirring 0.1 ml of culture into about 30 ml or the equivalent of product. (See also methods 2.121 and 2.122.) A viable count is performed on each suspension to estimate the number of live bacteria which has been mixed into each test sample. Inoculation with moulds and bacteria must be carried out separately. Care must be exercised in the handling of preparations which have been deliberately infected with potentially dangerous micro-organisms. Strains of Pseudomonas aeruginosa recently isolated from clinical infections can be particularly harmful to the eyes and should only be used in preservation studies with great care to avoid disseminating infection. The culture used for test purposes may be grown on solid or in liquid medium a young and actively growing culture is essential. One of the following procedures will normally be applicable.

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.