DETECTING MOLDS IN PERSONAL CARE PRODUCTS 41 POSITIVE MOLD DETECTION IN 18 HOURS BY USING AN ATP BIOLUMINESCENCE ASSAY A comparative study was performed that included the use of R-TAT broth, R-TATP broth (R-TAT broth with a 5 mM concentration of L-glutamic acid), and Letheen broth to evaluate the effi cacy of L-glutamic acid for detecting the presence of mold organisms by each of these enrichment broths. By using two different inoculum levels of A. brasiliensis ATCC 16404 for inoculating 100 ml aliquots each of the abovementioned enrichment broths, detection for the presence of microorganisms by using an ATP bioluminescence assay was conducted after 18 and 24 h of incubation at a temperature of 30.0°C ± 1.0°C. With a A. brasiliensis inoculum of 20 CFU and an incubation period of 18 h, the RLU ratios for R-TAT broth, R-TATP broth, and Letheen broth were found to be as follows: 2.1, 3.5, and 1.7 (Figure 3). By using an A. brasiliensis inoculum of 38 CFU, the RLU ratios for each of the abovemen- tioned enrichment broths were as follows: 2.5, 9.3, and 3.2 (Figure 3). By extending the time of the incubation period from 18–24 h, the RLU ratios had increased further with R-RATP broth showing the highest RLU ratio of 21 and 155 with an A. brasiliensis in- oculum of 20 and 38 CFU (data not shown). The effect of adding a 5 mM concentration of L-glutamic acid to R-TAT broth on the growth of another Aspergillus species, A. oryzae ATCC 10124, was also observed with RLU ratios of 6.4 at 18 h of incubation and 64 at 24 h of incubation (Table II). These test results indicated that the new enrichment broth of R-TATP broth with a 5 mM concentration of L-glutamic acid is able to promote the growth of mold, including A. brasiliensis and A. oryzae to a detectable level only after us- ing an 18 h incubation period at a temperature of 30.0°C ± 1.0°C by performing an ATP Figure 2. Eighteen-hour detection RLU ratios for Aspergillus brasiliensis ATCC 16404 growth in 100-ml aliquots of R-TAT broth, R-TATP broth, and Letheen broth.

JOURNAL OF COSMETIC SCIENCE 42 bioluminescence assay. The result of an 18 h incubation period for detecting the presence or absence of mold in a test sample is signifi cantly improved by having greater amounts of ATP being produced by mold in comparison with ATP bioluminescence assays using R-TAT broth and Letheen broth as the enrichment broth. EFFECT OF AN 18 HOUR R-TATP BROTH INCUBATION PERIOD ON THE GROWTH OF OTHER TYPES OF MICROORGANISMS Besides for the possibility that mold being present in a test sample of a nonsterile raw ingredient or personal care product formulation, other types of microorganisms may also be present. To determine whether R-TATP broth (with a 5 mM concentration of L-glutamic acid) could have an adverse effect in preventing the detection of other micro- organisms by using an ATP bioluminescence assay, a study was performed to determine whether other representative microorganisms could also be detected. The following representative test microorganisms were used: S. aureus ATCC 6538, E. coli ATCC 8739, B. subtilis ATCC 6633, and C. albicans ATCC 10231. Additional microbial inoculation studies with the abovementioned microbial species at inoculation levels of 18–38 CFU were conducted by either using 100 ml aliquots R-TATP broth alone or a 1% test suspension of a nonsterile raw ingredient or personal care product formulation in R-TATP broth. Figure 3. Background comparison of RLU ratio values for R-TAT, R-TATP, and Letheen broths. Table II RLU Ratios for Bacteria, Yeast, and Mold in Only R-TATP Broth after 18- and 24-h Incubation Periods Test microorganism S. aureus 6538 E. coli 8739 B. subtilis 6633 C. albicans 10231 A. brasiliensis 16404 A. oryzae 10124 Inoculum level CFU/100 ml 18 38 35 29 24 32 R-TATP broth Hours of incubation 18 h 1,871 5,000 5,000 21 3.7 6.4 24 h 5,000 5,000 5,000 1,229 21 4.2