HYGIENIC MANUFACTURE AND PRESERVATION 769 circles 9 cm diam. will conveniently fit into the lower half of a standard size dish). Prior to incubation saturate each filter circle with about 1.5ml of sterile distilled water and moisten the surface of the preparation with 0.1-0.2 ml of sterile water. Transfer the samples, in covered Petri dishes, to a plate box or other suitable container with a tight fitting lid and incubate at 18-22 ø. Alternatively, when dealing with a number of samples, large culture dishes (30 X 30 X 2.5 mm)* suitably lined with moistened filter paper and sealed with an air-tight covering may be used. Periodically moisten the samples and inspect visually at regular intervals for evidence of fungal growth. Under favourable growth conditions, moulds may appear as discrete colonies visible to the naked eye within 10-14 days of incubation. Negative samples should continue to be incubated in a moist atmosphere for at least 6 weeks. 3.52 Assessing preservative capacity It is recommended that all products intended for use in or around the eye should contain a preservative bactericidal to Pseudomonas A eruginosa. 3.521 Preservation against bacterial contamination When practical, test in the final pack and use the product without preservative as a control. Inoculate the preparation in triplicate with appropriate strains of bacteria at a level of 106 organisms g-1. Allow the inoculum to soak into the surface of solid and semi-solid products or mix in with fluid products. The following test organisms are recommended: Pseudomonas aeruginosa, Ps. fluorescens, Staphylococcus aureus, Micro- coccus luteus, Streptococcus faecalis. In addition spoilage organisms isolated from contaminated products may be used. Incubate the inoculated samples at 30 ø and 37 o. To observe bactericidal action, carry out viable counts or do sterility checks on the test and control samples immediately after inoculation, after 1 h and 6 h, and then at daily intervals for one week, followed by counts and sterility checks at further weekly intervals. In con- ducting these procedures, it is important that effective preservative in- activation is carried out and that a sufficiently large volume of broth is used in sterility testing. 3.522 Preservation against mould growth Studies on mould contamination should be carried out separately. When possible the preparation should be tested in its final pack. The method of *Jencons (Scientific) Ltd., Mark Road, Hemel Hempstead, Herts.

770 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS inoculation and range of fungal organisms is generally the same as used in the testing of creams and lotions. Moulds isolated from spoiled products should also be used. After inoculation, incubate test and control samples at 22-25 ø in a moist atmosphere as previously described. Inspect regularly for evidence of fungal growth for a test period of not less than 3 months. 3.523 Saliva test A test which simulates the customary practice of the user in moistening such products as eyeliners and mascara with saliva, thereby contributing large numbers of micro-organisms to the preparation, may be carried out in the laboratory. For this purpose, pool together 4-6 samples of fresh saliva. The mixed sample will carry a rich microbial flora that is normally indigenous to saliva, including large numbers of Streptococci, Staphylococci, and Neisseria. Spread an inoculum of about 0.2 ml of the pooled saliva over the surface of duplicate preparations. Incubate the test samples, and a control, in a moist atmosphere at 22 ø. Periodically moisten dry samples with 0.1-0.2 ml of sterile distilled water. Observe bactericidal action by culturing the infected samples on Blood Agar at 37 ø. Sub-culture at regular intervals according to the schedule previously described for bacteria. If a complete kill has been achieved, the challenge test may suitably be repeated on the same sample with fresh saliva. 4. CHOICE OF PRESERVATIVES 4.1 Requirements Modern cosmetic preparations, unless adequately preserved, often present a favourable environment for microbial growth. Apart from the health hazard, this may cause separation of emulsions, discoloration, changes in viscosity, the formation of gas and of off-odours. The extent of microbial growth depends largely on the type of formulation, water content, phase composition of an emulsion, pH, nutritive value, amount and type of perfume, and the type of container in which the product is packed. After manufacture, most products are likely to be exposed to further con- tamination from filling machines, containers and caps. Fungal growth on the surface of cosmetic creams has often been traced to infected cap-liners. Products packed in wide-mouth jars are easily contaminated by the user's fingers. Flexible bottles which draw air back into them are liable to suffer airborne contamination during use. Products packed in collapsible tubes and bottles with small apertures are less prone to contamination in use but