HYGIENIC MANUFACTURE AND PRESERVATION 759 Glucose Yeast Extract Agar by plating out on the day of inoculation and after 1, 2, 3 and 4 weeks' incubation. To obtain more detailed information on bactericidal activity, viable counts can be performed more frequently. The following schedule may be used: viable counts are made on control and test samples immediately after inoculation, after 1 h, 6 h, and 24 h, then at daily intervals up to one week. If at any time there is an immediate decrease to a zero count which is maintained for two consecutive weeks, a second challenging inoculum should be added with further periodic culturing. This provides information on the sustained ability of the preservative to protect the product. The following controls should be included: 3.231 Test shampoo manufactured without preservative. This sample is not inoculated. 3.232 Shampoo manufactured as above, heated in a water-bath for 1 h at 65 ø, allowed to cool and inoculated with 1 ml of standard suspension. {The heat treatment is usually sufficient to destroy extraneous contaminants but before experimental inoculation the sample should be checked for viable bacteria). 3.233 Another shampoo formulation, known to be definitely self-sterilizing. Inoculated with 1 ml of standard suspension. Findings on these controls should be interpreted thus: $.251 Since this sample was not inoculated, the recovery of viable organ- isms will confirm the need for an antibacterial preservative. $.252 The inoculated sample without preservative should normally demon- strate a colony count of at least 106 organisms ml-1 throughout the test period and thus serves as a check on the viability of the test inoculum. $.255 Following experimental inoculation, the shampoo should normally demonstrate a self-sterilizing action within 7 days. 3.24 Acceptance of shampoo preservative systems Incubation of the shampoo should proceed at 22 ø and 30 ø for at least one month. Plate count and sterility tests should be carried out during this time to ensure that the product remains sterile. An effective shampoo preservative should be bactericidal in action so that, following experimental inoculation, the product becomes virtually sterile within 1-7 days. Although rapid bactericidal action is highly desir- able, a slow-acting but stable preservative is, however, equally acceptable it is most important that the preservative should not only yield a sterile

760 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS product but should continue to retain its activity within the product in order to cope with any subsequent contamination. 3.25 Bacteriological examination of shampoo production (bulk samples.) Transfer a requisite amount of shampoo to a sterile bottle suitably calibrated, e.g. 30 g of shampoo made up to 300 ml with sterile diluent will give an initial dilution of 1:10. If the shampoo is highly contaminated it may be necessary to prepare a number of 10-fold serial dilutions by aseptic- ally transferring 1 ml to 9 ml of Peptone Water Diluent. From each dilution remove 1 ml and add to separate sterile 20 X 100 mm Petri dishes. Add 15 ml of sterile liquefied Plate Count Agar which has been cooled to 45 ø- 47 ø. Swirl the agar and allow to harden. Invert the Petri dishes and place in a 30ø incubator for 48 h. Remove and count colonies. In favourable circumstances, bulk shampoo will prove to be sterile. However, since the action of the preservative is not instantaneous, a finite time will be required for sterilization of shampoo initially bearing a reason- able level of contamination. The product will only fail to become sterile within 48 h if the initial contamination is exceptionally heavy or if resistant strains are encountered. 3.26 Bacteriological tests on shampoo packs The schedule of testing given below should be regarded as a minimum requirement. The conduct of further studies from time to time will help to ensure that standards of hygiene are not falling below safe levels for example, it is useful to keep a proportion of packed stock up to one month before testing to make certain that growth does not occur. In conducting tests for sterility, aseptic precautions should be taken throughout all procedures. Testing should not be conducted under direct exposure to ultraviolet light or in areas under disinfectant aerosol treatment. Suitable environmental control tests, including plate counts, should be performed at regular intervals. Test each lot of culture medium for sterility and for its growth-promoting properties. The medium must be appro- priately modified or precautions taken to neutralize the activity of any antimicrobial in the preparation tested (see 1.41). Wide-bore pipettes are suitable for the transference of viscous liquids, e.g.H.J. Elliott Ltd. E-mil 1 ml disposable serological pipettes (Cat. No. D.P. 1010, in sterile packs). 3.261 Broth sterility test 3.2611 Take two bottles or sachets for each half-day's filling from each filling line. Reserve one of these samples as a duplicate for examination only