768 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS and moulds after a suitable period of storage in a moist atmosphere, e.g. after 5 days and again after one month. 3.511 Detection of bacteria Add 0.1-0.2 ml of sterile distilled water to moisten dry preparations and incubate the samples in their containers at 22 ø. Culture for bacteria after 5 days' incubation, maintain in a moist atmosphere and re-check a proportion of stock after 1 month. 3.512 Swabbing technique (Qualitative examination) When dealing with solid preparations it is sometimes convenient to culture from swab samples. Swabs are preferably prepared from calcium alginate (Calgitex) wool. The swabs are made by wrapping about 30 mg of calcium alginate (Calgitex) wool around the final 2 cm of a wooden applicator stick. The swabs are sterilized by autoclaving in glass tubes for 15 min at 122 ø. Calgitex swabs are also obtainable from a commercial source*. These are supplied ready for use in sterile packs. The preparation is sampled in its container. Moisten the swab in Peptone Water Diluent or nutrient broth before collecting a sample from a dry surface. The swab should be firmly applied and slowly rotated, thoroughly covering the exposed surface. Culture to Nutrient Broth and Agar, streaking out the inoculum on the solid medium with a wire loop to obtain separate colonies. Examine cultures after 48 h incubation at 30 ø. 3.513 Viable count (Bacteria) Transfer 1 g of material aseptically to a universal bottle containing 8 glass balls, 8 ml of Peptone Water Diluent, and 1 ml of sterile 1 ø//o Tween 80. Mix on a Whirlimixer for 10 s. Prepare tenfold dilutions in peptone/Tween diluent from the 1:10 suspension. Plate out in duplicate and spread 0.1 ml aliquots of each dilution on the surface of dried Nutrient Agar plates. Allow the inoculum to dry, invert the Petri dishes and place in a 30 øincubator for 48 h. Remove the plates from the incubator and count the colonies. 3.514 Detection oftmoulds The following method may be used for the detection of mould con- taminants in solid preparations, pressed powder eyeliners, mascara, etc.: Transfer replicate samples with their coverings removed to glass Petri dishes which have been lined with filter paper (Whatman No. 1 filter *Arnold R. Horwell Ltd., 2, Grangeway, Kilburn High Road, London N.W.6.

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.