744 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS diluent or medium those organisms which may be embedded or trapped within the bulk of the material. Standardization of the preliminary pro- cedure is important. Excessive speed of the cutting blades of a blender or unduly prolonged use of a mixer may cause injury to the microbial cells either mechanically or by heat generated. Care must be taken to minimize risk from aerosols created by a mechanical blender when pathogens are present or suspected. 1.11 Preparation of sample for testing Mix the sample and transfer approximately 1 g or 1 ml to 9 ml of sterile Nutrient Broth to give an approximately 10% solution or suspension. Other dilutions may be prepared as necessary. The transfer and dilution technique must be carried out in such a manner as to avoid the introduction of extraneous contaminants. If the sample is not miscible with water, it should be dispersed with the aid of an emulsifying agent. Lubrol W, Tween 80 and Triton X100 are useful for this purpose. The following method may be adopted: Prepare a supply of Universal bottles containing 4 ml aliquots of 4% Lubrol W. Sterilize by autoclaving. Add approximately 1 g or 1 ml of the test sample to 4 ml of 4% Lubrol W. Place in a 44 o water bath for 10 min with intermittent shaking. At the end of the time add 5 ml of Nutrient Broth, previously warmed to 44 o, to make a final volume of 10 ml. 1.12 Inoculum Select a suitable range of agar and broth media and with a sterile Pasteur pipette inoculate each plate with 2 to 3 drops of suspension spread with a sterile wire loop and add 0.5 to 1 ml of suspension to each bottle of liquid medium. Additionally, streak out on Nutrient Agar or other suitable medium a loopful of the undiluted product (or heavy suspension). Streak-plate cultures of the preparation in its original form should be made routinely, since spoilage organisms tend to become dependent on the product for their nutritional requirements and may fail to grow or show only sparse growth when first isolated from this environment. When spoilage organisms fail to grow satisfactorily on standard culture media, it may be advantageous to employ diluted, e.g. half-strength, media this provides conditions more suitable for organisms adapted to an environ- ment of poor nutritive status. On incubation, blood agar is lysed by prolonged contact with Lubrol W broth. If contamination with haemolytic

HYGIENIC MANUFACTURE AND PRESERVATION 745 micro-organisms is suspected, an inoculum from a plain broth suspension should be used. 1.2 Quantitative examination The problem of representative sampling has been discussed in 2.1 above and the value of quantitative studies will obviously depend to a consider- able extent on the validity of the samples examined. Quantitative test procedures generally involve the replication of experiments on several aliquots derived from a single initial sample but it should be stressed that the validity of a test procedure is greatly strengthened by ensuring that replicate samples are included as well as replicate aliquots from a single sample. 1.21 Plate count procedure All glassware and equipment must be chemically clean and sterile. When preparing serial dilutions use a fresh sterile pipette for each trans- ference. A considerable saving in 1 ml pipettes can be achieved by using an automatic syringe connected to a sterile Pasteur pipette. The special syringe* is pre-set to deliver 1 ml. A fresh sterile disposable Pasteur pipette is used for each dilution but the same syringe can be used for any number of operations since the diluent is not sucked into the barrel. 1.211 Diluent Use sterile 0.1 w/v peptone water at pH 7.0. Inoculation of media should be carried out within 30 min of the preparation of the dilution. When examining a sample for anaerobic bacteria use freshly prepared Reinforced Clostridial Broth as the diluent. 1.212 Liquid samples Pipette aseptically 10 ml of the thoroughly-mixed sample into a sterile glass bottle fitted with a ground glass stopper and add 90 ml of diluent to give a 1:10 dilution v/v. Alternatively, weigh with aseptic precautions 10 g of the thoroughly-mixed sample into the bottle and add 90 ml of diluent to give 1:10 dilution w/v. Prepare further serial decimal dilutions in 0.1% sterile peptone water as necessary. *R. B. Turner & Co. Ltd., Church Lane, London, N.2.