7{32 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS be adopted for shampoo testing. The membrane filter should not, however, be used for testing shampoos containing preservatives which are not com- pletely soluble in water. For the recovery of Pseudomonads and similar micro-organisms a membrane with a pore size of 0.22 lim is recommended. A wide range of membrane filtration systems, including disposable filter units, is now available commercially. For further technical information concerning membrane filter apparatus, filtration and dilution of sample, recovery media and incubation of membranes, the following technical references should be consulted: GENERAL REFERENCES Microbiological Analysis of Toiletties and Cosmetic Products. Application Report AR-16. (Millipore U.K. Ltd., Heron House, Wembley, Middlesex). Membrane filtration with 'Oxoid' membrane filters and membrane media. Temporary leaflet (July 1967) (Oxoid Ltd., London). Burman, N. P. et al. "Membrane Filtration Techniques" in Shapton, D. A. and Gould, G. W. Isolation methods for microbiologists, Technical Series 3. {Academic Press, London). Collins, C. H. Microbiological Methods gnd Edn. 150-152 and 313-314 (Butterworths, London). Mulvany, J. G. Membrane filter techniques in microbiology, in Norris, J. R. and Ribbons, D. W. Methods in microbiology 1 (1969) (Academic Press, London). 3.3 Creams and lotions 3.31 Assessment of preservative capacity When practical, test in the final pack and use the product without preservative as a control. Samples should be inoculated in triplicate with spoilage organisms (bacteria, moulds, yeasts and fungi). Incubate one set of samples at 22 ø , one set at 32 ø and another set at 35-37 ø . When testing for bactericidal action, steps should be taken to neutralise any residual preservative (see Section 1.4, p. 750). 3.32 Inoculation with bacteria This is carded out by stirring about 0.25 ml of a bacterial suspension into each 25 g sample of the product, using a sterile spatula. The inoculum should be suitably diluted with Peptone Water Diluent to give a final con- centration of about 106 organisms g-1 of product. The test inocula should include micro-organisms recently isolated from spoilt preparations. Addi- tionally, the following strains obtainable from Type Culture Collections are recommended as test organisms: Staphylococcus aureus, Streptococcus faecalis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Escherichia coli, Klebsiella spp. and Proteus spp. Immediately after inoculating the samples and thereafter at regular intervals, estimate the number of viable organisms by plating out in Plate Count Agar. (See methods $.35 and $.$6). A drastically reduced count or the

HYGIENIC MANUFACTURE AND PRESERVATION 763 demonstration of an apparently sterile product which remains sterile on three successive samplings will suggest that the preservative is likely to give good protection against bacteria. However, it is prudent to keep the samples under test for 2-3 months or longer until it has been clearly estab- lished that any surviving bacteria do not increase in numbers with further incubation 3.33 Inoculation with fungi The following fungi may be used as test organisms: Aspergillus niger, Penicillium, Cladosporium, A lternaria, Fusarium, Mucor, Rhizopus, Phoma, Trichoderma and Verticillium species. Since the samples are to be inspected visually, the dark pigmented fungi and green moulds are the most suitable. Inoculate only the surface of the cream with mould spores. This can be done by transferring a small inoculum of dry spores from the surface of a sporu- lating culture by means of a 2 mm loop, scattering the spores over the surface of the cream. Incubate in a moist atmosphere (95-100% RH) at 22-25 ø. Examine visually at regular intervals for evidence of mould growth during a six months' test period. In the absence of visible growth, sub- cultures to Malt Extract Agar and Broth can be made to establish whether the inoculum has been inhibited or killed. 3.34 Inoculation with yeasts Suitable strains can be isolated from spoilt preparations. Candida albicans and Saccharomyces cerevisiae are also recommended. Allow the organisms to grow on slopes of malt extract agar for two days. The inocula- tion and plate count procedure is the same as for bacteria except that yeast counts are estimated in Malt Extract Agar. REFERENCES Wedderburn, D. L. Advances in pharmaceutical sciences. Preservation of emulsions against microbial attack 1 (1964) (Academic Press, London). Tenenbaum, S. Toilet Goods Assoc. Cosmet. J. 2 24 (1970). 3.35 Method for detecting the survival of bacteria in emulsion systems The simple method given below is particularly useful when testing large numbers of samples. It can be used for spot checks on many emulsion systems or for following the development of bacterial contamination by daily tests on one system. The test is based on the growth of organisms in the emulsion on an indicator medium, keeping them within a defined area and noting their effect in mass. The indicator is 2:3:5-triphenyltetrazolium chloride (TTC) which becomes reduced to insoluble red formazan.