INOCULUM PREPARATION IN PRESERVATION TESTING 203 of preservative efficacy testing, the rate of inactivation of test organisms is independent of the concentration of organisms introduced in the sample (up to the point at which the preservative system is overwhelmed by the inoculum). This was substantiated by demonstrating that different concentrations ofS. aureus (from 1.5 x 103 to 1.8 X 106 S. aureus/mL) gave similar rates of inactivation in preservative efficacy tests of lotion (3). Thus, use of inocula adjusted to a standard turbidity is not needed for preservative efficacy testing performed by the linear regression method. Although the studies on the effects of incubation of cultures up to 72 hr used emul- sions, it is believed that similar results would be observed with different test organisms in preservative efficacy tests of different samples. Nevertheless, the suitability of using cultures incubated for extended periods should be determined using cosmetic and/or pharmaceutical products of interest in each laboratory. The effect of extended incubation of yeasts and molds was not studied because these organisms typically have slower growth rates than the test bacteria used in this work and because preservative testing generally is performed with cultures that have been incubated for 2-7 days at 20-25øC (1). The quantitative data on the kinetics of bacterial inactivation obtained by use of the linear regression method often provide investigators with a better understanding of preservative testing than is possible by use of non-quantitative methods. This work demonstrated that culture conditions and the type of inoculum affect the kinetics of bacterial death during preservative efficacy testing. It is evident that the acceptance criteria used in determining the suitability of preservative systems are dependent on the results of test methods used (1-3,8) and that changing methods may necessitate reeval- uation of acceptance criteria. It is recommended that test methods be modified to use saline suspensions of test organisms grown on solid agar media rather than broth ino- cula. ACKNOWLEDGMENTS We thank Mr. Mark Entrup of Hill Top Bioresearch Laboratories for furnishing the culture of B. subtilis. REFERENCES (1) Anon, "Microbiological Tests, Antimicrobial Preservatives--Effectiveness," in United States Pharma- copeia XXI (The United States Pharmacopeial Convention, Rockford, MD, 1985), pp. 1151-1152. (2) Preservation Subcommittee of the CTFA Microbiological Committee, A guideline for the determina- tion of adequacy of preservation of cosmetics and toiletry formulations, TGA Cosmet. J., 2, 20-23 (1970). (3) D. S. Orth, Linear regression method for rapid determination of cosmetic preservative efficacy,J. Soc. Cosmet. Chem., 30, 320-332 (1979). (4) E. J. Scibienski, J. J. O'Neill, and C. A. Mead, An accelerated preservation test. Presentation at the Annual Scientific Meeting of the Society of Cosmetic Chemists, Dec. 11, 1981. (5) M. Chan and H. N. Bruce, A rapid screening test for ranking preservative efficacy, Drug & Cosmet. Ind., 129, 34-37, 80-81 (1981). (6) G. K. Mulberry, M. R. Entrup, and J. R. Agin, Rapid screening methods for preservative efficacy evaluations, Cosmet. Toilet., 102, 47-50, 52-54 (1987).

204 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (7) D. S. Orth and L. R. Brueggen, Preservative efficacy testing of cosmetic products. Rechallenge testing and reliability of the linear regression method, Cosmet, Toilet., 97, 61-65 (1982). (8) D. S. Orth, "Evaluation of Preservatives in Cosmetic Products," in Cosmetic and Drug Preservation. Principles and Practice, J. J. Kabara, Ed. (Marcel Dekker Inc., New York, 1984), pp. 403-421. (9) C. N. Baker, C. Thornsberry, and R. W. Hawkinson, Inoculum standardization in antimicrobial susceptibility testing: Evaluation of overnight agar cultures and the rapid inoculum standardization system, J, Clin, Microbiol,, 17, 450-457 (1983). A. W. Bauer, W. M. M. Kirby, J. C. Sherris, and M. Turck, Antibiotic susceptibility testing by a standardized single disk method, Am. J. Clin. Pathol,, 45, 493-496 (1966). R. F. D'Amato and L. Hochstein, Evaluation of a rapid inoculum preparation method for agar disk diffusion susceptibility testing, J. Clin. Microbiol,, 15, 282-285 (1982). Preservation Subcommittee of the CTFA Microbiological Committee, Preservation Testing of Aqueous Liquid and Semi-Liquid Eye Cosmetics (Cosmetic, Toiletry and Fragrance Association, Inc., Wash- ington, D.C., 1975). C. C. Garber and R. N. Carey, "Laboratory Statistics," in Clinical Chemistry. Theory, Analysis and Correlation, L. A. Kaplan and A. J. Pesce, Eds. (C.V. Mosby, St. Louis, 1984), pp. 287-300. E. Freese, C. W. Sheu, and E. Galliers, Function of lipophilic acids as antimicrobial food additives, Nature 241, 321-325 (1973). R. E. Buchanan and N. E. Gibbons (Eds.), Bergey's Manual of Determinative Bacteriology, 8th ed. (The Williams & Wilkins Company, Baltimore, 1974), p. 532. Ibid. p. 217. C. W. Moss and M. L. Speck, Identification of nutritional components in trypticase responsible for recovery of Escherichia coli injured by freezing, J. Bacteriol., 91, 1098-1104 (1966). H. S. Bean, Preservatives for pharmaceuticals, J, Soc, Cosmet. Chem,, 23, 703-720 (1970). D. S. Orth, C. M. Lutes, S. R. Milstein, and J. J. Allinger, Determination of shampoo preservative stability and apparent activation energies by the linear regression method of preservative efficacy testing, J. Soc. Cosmet. Chem,, 38, 307-319 (1987). (20) A. L. Barry, R. E. Badal, and R. W. Hawkinson, Influence of inoculum growth phase on microdi- lution susceptibility tests, J, Clin, Microbiol., 18, 645-651 (1983). (21) J. H. Wicks, R. L. Nelson, and G. E. Krejcarek, Rapid inoculum standardization system: A novel device for standardization of inocula in antimicrobial susceptibility testing, J. Clin, Microbiol., 17, 1114-1119 (1983). (22) C. G. Mayhall and E. Apollo, Effect of storage and changes in bacterial growth phase and antibiotic concentrations on antimicrobial tolerance in Staphylococcus aureus, Antimicrob. Agents Chemother., 18, 784-788 (1980).