INOCULUM PREPARATION IN PRESERVATION TESTING 195 TEST SAMPLES The test samples used in this study were hand and body lotions and a face cream. Lotion A contained water, glycerin, cetearyl alcohol, ceteareth-20, palm oil glyceride, glyceryl dilaurate, mineral oil, petrolatum, dimethicone, isopropyl palmitate, stearic acid, al- lantoin, and other ingredients. Lotion B contained water, sorbitol, stearic acid, glyceryl dilaurate, cetearyl alcohol, ceteareth-20, lard glyceride, stearamide MEA, hydroge- nated vegetable oil, isopropyl palmitate, glyceryl stearate, PEG-100 stearate, dimethi- cone, petrolatum, allantoin, and other ingredients. The preservative system in both lotions contained methylparaben, propylparaben and Quaternium-15. Lotion C con- tained water, mineral oil, dimethicone, palm oil glyceride, allantoin, other ingredients, and was preserved with phenoxyethanol, methylparaben, and propylparaben. Lotion D was similar to lotion C, but contained propylene glycol. Lotion E was similar to lotion B, and lotion F was similar to lotion A, except that it contained glyceryl monolaurate in place of glyceryl dilaurate. The face cream contained mineral oil, water, beeswax, mag- nesium aluminum silicate, sodium borate, fragrance, isopropyl myristate, and colors. TEST PROCEDURE For broth inocula, 0.1 ml of each broth culture was added directly to separate 50-mL test samples. Saline inocula were prepared by suspending a 1oopful of TSALT surface growth of each test organism in 5 mL of saline and adding 0.1 mL of each saline suspen- sion to separate 50-ml test samples (3). Samples were taken, APCs were performed at specified time intervals (0, 3, 5, and 24 hr) using Letheen Broth with 0.01% (vol/vol) Triton X-100 diluent and TSALT as the recovery system, and D-values were calculated as described previously (3). pH DETERMINATIONS The pH of the TSA, TSALT, and PCA in Petri dishes was determined after 24, 48, and 72 hr growth by the test organisms and in uninoculated (i.e., control) samples using a pH meter equipped with a flat-surface combination pH electrode. The electrode was moistened with a drop of demineralized water, and pH measurements were read directly after the electrode was placed on a sterile portion of the agar, approximately 1 cm from the region of growth. The pH determinations of TSB and TSB(-G) cultures were made as follows: A 3-mL sample was piperred into the barrel of a 10-mL syringe, to which was attached a sterile Sweeney-type filter with a 0.45 •m membrane. The plunger was inserted into the barrel, forcing the broth through the filter and into a small beaker. The pH measure- ments were taken on the filter-sterilized broth samples prepared in this manner. EFFECT OF CULTURE BROTH ON PRESERVATIVE EFFICACY TESTING The effect of bacteriological culture broth on preservative efficacy testing was deter- mined by adding 0.1 mL of filter-sterilized TSB to 50-mL samples of lotion A immedi- ately before inoculating with saline suspensions of the test organisms. The control samples were prepared by adding 0.1 mL of sterile demineralized water to samples of

196 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS lotion A immediately before inoculation with the test organisms. Samples were taken, APCs were determined, and D-values were calculated as described previously (3). STATISTICS -- Statistically significant differences between mean D-values (X) of duplicate experiments were determined by the t test (13). RESULTS AND DISCUSSION Organisms with different physiological capabilities may respond differently to changes in growth media and culture conditions. Thus, S. aurem, P. aeruginosa, B. subtilis, and E. coli may exhibit different growth rates and time in the maximum stationary phase of growth in any given culture medium. It would not be unreasonable to expect that the length of the maximum stationary phase may range from a few minutes to days or weeks, depending on the growth conditions (i.e., temperature, pH, rate of growth, etc.) and the ability of the organism to withstand the metabolic stresses imposed by the unfavorable environment (i.e., depletion of nutrients, accumulation of toxic products, low pH, etc.). The organic acids produced during fermentation may be bacteriocidal. This antibac- terial effect increases as the pH decreases (14). It is believed that metabolic stress may be reduced by the use of culture media that allow aerobic growth without causing the development of unfavorable conditions (i. e., acid pH). This may enable the population of organisms to remain in the maximum stationary phase for extended periods. The changes in pH during aerobic growth of the test organisms on the solid media were monitored. Although the 24-hr pH determination for S. aureus on PCA revealed that the agar was pH 6.7, all other pH determinations for S. aureus and all other test or- ganisms were alkaline at 24, 48, and 72 hr (Table I). PCA contains 0.1% glucose TSA Table I pH of PCA, TSA, and TSALT After Aerobic Growth of Test Organisms on Solid Agar Media for 24, 48, and 72 hr at 37øC Test culture Medium, time S. aureus P. aeruginosa B. subtilis E. coli PCA, 24 hr 6.7 8.1 7.9 7.8 PCA, 48 hr 8.2 8.4 8.4 8.2 PCA, 72 hr 8.3 8.4 8.4 8.3 TSA, 24 hr 7.8 8.2 8.0 8.3 TSA, 48 hr 8.3 8.4 8.4 8.4 TSA, 72 hr 8.4 8.5 8.4 8.4 TSALT, 24 hr 8.3 8.5 8.2 8.2 TSALT, 48 hr 8.2 8.5 8.2 8.3 TSALT, 72 hr 8.4 8.5 8.4 8.4 Table values are mean values of duplicate pH determinations on sterile agar, ca. 1 cm from growth. pH of uninoculated media on the day of preparation: PCA -- pH 6.8, TSA = pH 7.1, TSALT = pH 7.1.