HYGIENIC MANUFACTURE AND PRESERVATION 767 of tenfold serial dilutions by aseptically transferring 1 ml to 9 ml of Peptone Water Diluent. From each dilution remove 1 ml and add to separate sterile 15 X 00 mm Petri dishes. Plate out each dilution in triplicate. Add 15 ml of liquefied Plate Count Agar which has been cooled to 45-50 ø. Mix the agar and allow to harden. If necessary to prevent the swarming of bacteria, add a 5 ml overlay of Nutrient Agar and allow to harden (1). Invert the Petri dishes and incubate at 30 ø for 48 h. Remove plates and count colonies. Plate out as above in acidified Malt Extract Agar for the enumeration of yeasts and fungi. 3.44 Anaerobic counts. Method 1 Prepare dilutions as above but use Reinforced Clostridial Medium as a diluent. Plate out 1 ml aliquots in Reinforced Clostridial Agar and Plate Count Agar. Incubate for anaerobic culture at 30 ø and 37 ø. Count colonies after 48-72 h. On ordinary agar the colonies of most Clostridia are large and spreading, but their size may be minimised by over-drying the plates or separate colonies ensured by plating out on Charcoal Agar (2). Concentrated agar e.g. 2.5-3% may also be used to prevent the spread of colonies. 3.45 Anaerobic counts, Method 2 If isolations are not being made and only counts are required, the following method may be used. Transfer 1 ml aliquots from serial tenfold dilutions of the suspension into sterilized, plugged Miller-Prickett tubes*. Cool freshly prepared reinforced Clostridial Agar to approximately 50 ø and, without shaking, add about 25 ml to each tube. Seal immediately with melted sterile paraffin wax and allow to set in a water-bath at about 15 ø. Incubate at 37 ø and count colonies after 48-72 h. Run at least one blank to detect contaminants occurring during the procedure. This method provides a simple means of culturing anaerobic bacteria without recourse to the use of an anaerobic jar (3,4). REFERENCES (1) White, M., Bowman, F. W. and Kirshbaum, A. J. Pharm. Sci. 57 1061 (1968). (2) Alwen, J. and Smith, D. G. J. Appl. Bacteriol 30 389 (1967). (3) Miller, N.J., Garrett, C. W. and Prickerr, P.S. Food Research 4 (5) 447 (1939). (4) Oxoid Manual 3rd edn. 222 (1965). 3.5 Eye cosmetics: Solid, semi-solid and liquid preparations 3.51 Examination of samples in current production A representative number of samples should be examined for bacteria '*Miller-Prickett Anaerobic tubes may be obtained from Astell Laboratory Service Co. Ltd., London, S.E.6.

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.