j. Soc. Cosmet. Chem., 40, 193-204 (July/August 1989) Effect of culture conditions and method of inoculum preparation on the kinetics of bacterial death during preservative efficacy testing D. S. ORTH, C. M. LUTES, and D. K. SMITH, The Andrew Jergens Company, 2535 Spring Grove Avenue, Cincinnati, OH 45214. Received September 2, 1988. Synopsis The linear regression method of preservative efficacy testing was used to evaluate the effect of culture conditions and type of inoculum on the kinetics of death of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis or B. cereus, and Escherichia coli in nonionic emulsions. The rates of death, as determined by use of D-values, were essentially unchanged when saline suspensions of bacteria grown for 24, 48, or 72 hr on agar media were used in testing. Broth inocula performed differently in preservative efficacy testing than saline inocula prepared from surface growth on agar media. The use of 0.2% broth inocula (0.1 ml/50 mL sample) or the addition of 0.2% sterile broth to test samples produced significant decreases in the rates of inactivation of the test organisms. Broth inocula changed the kinetics of bacterial death and should not be used in routine testing. These results demonstrate the need for standardized procedures for preparation of inocula. It is recommended that preservative efficacy tests be performed using saline suspensions of bacteria grown aerobically on solid agar media. INTRODUCTION The development of cosmetic products includes preservative efficacy testing to demon- strate that the products are adequately preserved. Test methods include standardized procedures: the United States Pharmacopeia (USP) method (1) and the Cosmetic, Toi- letry & Fragrance Association (CTFA) method (2) and rapid procedures: the linear regression method (3), the accelerated preservative test (4), and the presumptive chal- lenge test (PCT) (5). These methods were reviewed recently by Mulberry et al. (6). The preservative efficacy test methods have a number of similarities, including the test organisms, recovery systems, and the method of performing aerobic plate counts (APCs). The methods have several differences, including culture media and growth temperatures used in preparing the inocula and the times at which APCs are deter- mined. These differences may produce variations in test results, statistical treatment of the data, and criteria used to determine whether a given product is satisfactorily pre- served (1-8). 193

194 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Transferring test organisms to new growth media is one of the first steps in preparing for preservative efficacy testing. Antibiotic susceptibility testing is performed with broth cultures in early log phase of growth, such as 2-5 hr growth at 35øC in Tryptic Soy Broth (TSB) (9-11). Cosmetic preservative efficacy testing routinely is performed using overnight cultures of test organisms. Thus, the USP method requires growth of bacterial cultures on a solid agar medium at 30-35øC for 18-24 hr. Lower incubation temperatures and longer incubation times are recommended for yeasts and molds (1). The testing guidelines proposed by the CTFA suggest "that no less than one million cells per milliliter or gram be used as the challenge" (2). Specific instructions are not given for growth of the test organisms or the volume of inoculum to use in testing however, these guidelines state that harvested broth cultures or surface growth on a solid medium may be used for inoculating the product. The CTFA guideline for preser- vation testing of aqueous liquid and semi-liquid eye cosmetics stipulates that fresh cultures of bacteria should be used for challenging preservative systems and that broth cultures or surface growth on solid media may be used in testing (12). Different methods recommend the use of bacterial cultures grown for various combinations of 18-24 hr or 24 hr at 30-35øC, 32-35øC, 35-37øC, or 37øC (1-5,12). It is possible that different preservative efficacy test results obtained by laboratories reporting to use the same method and test organisms may, in fact, be due to differences in the manner in which test organisms were grown prior to testing or to the type of inocula used. The quantitative data from the linear regression method enabled us to determine the effect of culture conditions, including age and growth media, and the type of inoculum on preservative efficacy test results. The studies reported here demon- strate that the culture medium affects the metabolism of bacteria and that the type of inoculum affects the results obtained in preservative efficacy testing. Specifically, the use of broth inocula decreased the rates of inactivation of the test organisms, compared to the use of saline inocula prepared from surface growth on agar media. EXPERIMENTAL TEST ORGANISMS The test organisms used in this study were Staphylococcus aureus (FDA 209 strain), Pseu- domonas aeruginosa (ATCC 9027), Bacillus cereus (ATCC 11778), B. subtills ATCC 6633 (kindly furnished by Mr. Mark Entrup, Hill Top Biolabs, Inc.), Bacillus sp., and Escherichia coli (ATCC 8739). CULTURE CONDITIONS The test organisms were grown on solid agar media: Tryptic Soy Agar (TSA), TSA with 0.07% (vol/vol) lecithin and 0.5% (vol/vol) Tween 80 (TSALT), or Plate Count Agar (PCA) or in broth media: TSB, Tryptic Soy Broth w/o dextrose (glucose) [TSB(-G)], and TSB(-G) with 1-5% (wt/vol) glucose. The cultures were incubated aerobically for 24, 48, or 72 hr at 37øC. All culture media were obtained from Difco Laboratories, Detroit, MI.