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.

764 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS N.B. TTC is photosensitive in solution and should be stored in the dark in an amber bottle. The solution can be sterilized satisfactorily by mem- brane filtration or by autoclaving at 121 o for 15 min. The solution cannot be incorporated in the medium and autoclaved. This'causes a reduction to formazan. 3.351 Test medium (TCA) The medium is made by adding 0.3% Oxoid Agar no. 1 to Oxoid Blood Agar Base no. 2 before making up according to the manufacturer's instruc- tions in 200 ml amounts in screw-capped bottles. For use, add 0.5 ml of a 1% freshly prepared sterile aqueous solution of 2:3:5-triphenyltetrazolium chloride to 200 ml of Oxoid Blood Agar Base no. 2 which has been suitably liquefied. Mix the contents and pour plates. Allocate about 20 ml of medium to a standard 100 mm Petri dish. Store the plates in the dark at 4 ø and dry off before use at 37 ø for about 1 h. If only five plates are required, add 0.25 ml of 1 •/o solution of TTC to 100 ml of medium. 3.352 Test Imprint 5-6 circles in each plate of TCA medium with a sterile, lightly flamed no. 7 cork borer (10 mm diam.). With a sterile 5 mm wire loop, transfer a sample of liquid emulsion to the centre of an imprinted circle. For the transfer of creams, take a small sample, about 0.1 g, on the end of a sterile wooden applicator* and apply to a fresh circle of agar. Incubate at 37 o. Read tests within 18 h of incuba- tion. 3.353 Results Heavily contaminated emulsions give a red spot within the imprinted circle. Increasing density of red spots indicates various intensities of bacterial contamination. Fine reddish granules or dots (stipples) can be seen in positive creams. Also the area surrounding the cream is deeply coloured. Bacteria-free emulsions give no colour reaction, but a negative result does not necessarily mean the total absence of organisms. Negative samples may be re-checked in nutrient broth and agar in the usual way. N.B. W/o emulsions tend to 'splutter' when heated in a naked flame. To prevent the spread of infected particles, wire loops charged with these materials should be sterilized in a burner which is equipped with a protective hood. A Kampff pattern micro-incinerator (Arnold R. Howell Ltd.) with a *Obtainable from Arnold R. Horwell Ltd., London, N.W.6. The sticks should be autoclaved at 121 ø for 15 min before use and afterwards destroyed by incineration.