DETECTING MOLDS IN PERSONAL CARE PRODUCTS 43 Each of the inoculated R-TATP broth alone or the 1% test suspensions in R-TATP ali- quots were incubated at a temperature of 30.0°C ± 1.0°C. After an incubation period of 18 h, an ATP bioluminescence assay was conducted on each of R-TATP broth alone or the 1% test suspensions of 17 personal care product formulations and 15 raw ingredients in R-TATP. At 18 h, positive ATP detections (RLU 2) were observed for all tested aliquots of R-TATP broth by itself and the 1% test suspensions of tested raw ingredients and personal care product formulations in R-TATP broth that had been inoculated with the abovementioned selected bacterial species and C. albicans. For A. brasiliensis inoculated aliquots of R-TATP broth by itself and the 1% test suspensions of tested raw ingredients and personal care product formulations, positive ATP detections were detected for 15 of 17 personal care product formulations and 13 of 15 raw ingredients after 18 h of incuba- tion at a temperature of 30.0°C ± 1.0°C (see Tables III and IV). After 24 h of incubation, 100% of the tested raw ingredients and personal care product formulations were found to have positive ATP detections for A. brasiliensis. By having positive ATP detections after 18 and 24 h of incubation, the test results confi rm that low levels of A. brasiliensis can be detected in nonsterile raw ingredients and personal care product formulations that are susceptible to microbial contamination in which in R-TATP broth is used as an enrich- ment broth in an ATP bioluminescence assay. DISCUSSION Rapid microbial detection such as ATP bioluminescence assay for the has attracted many personal care and pharmaceutical companies for its ability to rapidly screen for the presence Table III RLU Ratios for Raw Ingredients Inoculated with Test Microorganisms Using an 18-h Incubation Period Test microorganism: S. aureus 6538 E. coli 8739 B. subtilis 6633 C. albicans 10231 A. brasiliensis 16404 Inoculum levels (CFU/100 ml of enrichment broth) 14–19 15–38 32–54 21–51 20–52 Name of raw ingredient RLU Ratios Sorbitan monooleate 5,000 5,000 5,000 63 7.8 Hydroxyethylcellulose 601 5,000 5,000 326 2.0 Sodium citrate 1.1 5,000 70 8.3 1.5a POP (2M) myristyl ether propionate 3,603 5,000 5,000 18 6.9 PPG-12-buteth-16 255 5,000 439 23 4.5 POE (20M) sorbitan monopalmitate 790 5,000 5,000 79 11 C12-15 alkyl ethyhexanoate 1,832 5,000 5,000 62 2.2 Flamenco sparkle green #820J 1,997 5,000 5,000 52 2.2 Boron nitride 808 5,000 5,000 68 5.4 Chroma-lite dark blue #CL4501 4,198 5,000 225 14 3.0 Timiron super silver 1,422 5,000 5,000 73 14 Cationic emulsion 35% APE free 5,000 5,000 5,000 35 1.7b Titanium dioxide coated grew-MP149 5,000 5,000 109 20 7.3 Diglyceryl diisosterate 5,000 5,000 5,000 40 5.7 PPG 20 methylglucose ether 5,000 5,000 54 26 4.6 a With a 24-h incubation period for sodium citrate, the RLU ratio was 2.5. b With a 24-h incubation period for cationic emulsion, the RLU ratio was 101.

JOURNAL OF COSMETIC SCIENCE 44 or absence of microbial contamination in nonsterile raw ingredients before they are used in manufacturing and personal care product formulations before they distributed for sale to consumers. However, there are four main issues in using ATP bioluminescence tech- nology. The fi rst issue is that the detection of slow-growing microorganisms such as mold might be ambiguous because of the limited amount of ATP that is initially produced by a growing mold after 24 h of incubation. The second issue is the high RLU background from nonmicrobial ATP that may be present in either a raw ingredient or a personal care product formulation. This high level of nonmicrobial ATP of a test sample could mini- mize the RLU ratio between an incubated and non-incubated test sample that could cause a false-negative test result for the absence of contaminating microorganisms. The third issue is that the presence of high levels of nonmicrobial ATP in an enrichment broth might lead to having a false-positive test reaction for the presence of microbial ATP in a test sample that is not contaminated with microorganisms. The fourth issue is the dis- satisfaction in detecting the presence or absence of mold in a test sample by using Letheen broth as the enrichment medium in which a longer incubation period has to be used in comparison for testing the presence or absence of bacteria in a test sample. All living microorganisms must rely on the uptake of microbial nutrients from the envi- ronment to sustain energy, metabolism, and growth (6,9,10). By creating a more nutritious enrichment broth that has a low level of nonmicrobial ATP, the best possible growth Table IV RLU Ratios for Personal Care Product Formulations Inoculated with Test Microorganisms Using an 18-h Incubation Period Test microorganism S. aureus 6538 E. coli 8739 B. subtilis 6633 C. albicans 10231 A. brasiliensis 16404 Inoculum levels (CFU/100 of enrichment broth) 14–19 15–38 32–54 21–51 20–52 RLU ratios R-TATP broth (positive control) 55–1,871 434 to 5,000 46 to 5,000 21–138 3.7–11 Type of personal care product formulation Body cream 1,474 5,000 5,000 15 3.7 Body lotion 2,341 5,000 5,000 17 1.8a Body lotion 1,286 5,000 5,000 19 3.1 Skin toner 3,923 5,000 511 75 5.0 Skin toner 5,314 5,000 3,294 49 2.6 Body lotion 5,000 5,000 3,398 31 2.2 Body cream 3,739 5,000 2,353 48 2.6 Mascara 727 5,000 263 14 7.3 Night cream 5,000 5,000 5,000 29 15 Mascara 543 5,000 3.2 31 4.3 Eyeshadow 745 5,000 867 22 6.2 Shampoo 168 5,000 106 3.4 2.3 Foundation 1,941 434 232 7.7 3.6 Cleanser 1,479 5,000 949 21 2.6 Hand cream 190 5,000 646 32 4.2 Shampoo 15.1 5,000 5,000 20 6.5 Skin lotion 40.4 5,000 5,000 49 1.7b a With a 24-h incubation period, the RLU ratio was 28. b With a 24-h incubation period, the RLU ratio was 9.3.