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.

DETECTING MOLDS IN PERSONAL CARE PRODUCTS 45 condition can be provided for detecting the presence or absence of mold contamination and allow the expression of other non-mold microorganisms that may present in an incubated test sample. By adding PDB (Difco), Becton™ Neopeptone, Sucrose to Difco™ TAT Broth Base, the purpose of these components is to provide additional microbial nutrients to support the growth of mold. By adding polysorbate 20 and sodium thiosulfate to the Difco™ TAT Broth Base, the purpose of these two components is to neutralize the anti- microbial activity of preservative systems that may be present in a test sample. Further- more, the addition of L-glutamic acid to this new enrichment broth mixture is used to accelerate mold growth by shortening the lag phase of the mold growth cycle in which mold can be detected faster by using an ATP bioluminescence assay. The effects of amino acids including glutamic acid on fungi growth had been previously studied on Saccharo- myces cerevisiae for transaminase activity (11), as a carbon source for the growth of Cryptococcus albidus (12) and on Streptomyces viridoochromogenes for spore germination (13). However, none of these articles had reported on the effects of glutamic acid in shortening the lag phase of the microbial growth cycle, and there had been no studies conducted that involved the usage of a rapid microbial detection method such as an ATP bioluminescence assay. Microbial growth or proliferation is defi ned as an orderly increase of the components of an organism that is followed by cell multiplication. The microbial growth cycle consists of three phases. The fi rst phase of the microbial growth cycle is the lag phase in which a microorganism adapts to their new environment by forming proteins and metabolites for multiplication. For mold, the lag phase involves the preparation of hyphal elongation and branching. The second phase of the mold growth cycle is the exponential phase when new cell materials are synthesized at a constant rate and the amount of cell mass increases in an exponential manner. For mold, this is the germination period. The third phase of the mold growth cycle is the stationary phase where there is an exhaustion of microbial nu- trients or the accumulation of toxic byproducts from mold metabolism that causes growth or proliferation to cease completely (10,14). The length of the lag phase of the growth cycle for a microorganism will depend on the microorganism and as to whether microbial nutrients are available for metabolism. The lag phase of the mold growth cycle such as A. brasiliensis is 0–15 h in length in comparison with bacteria which is 0–6 h in length (10,15,16). For a yeast such as C. albicans, the length of the lag phase is between 0 and 3 h (17). The length of the lag phase will vary between different types of mold species (18). Without direct biochemical evidence, we are proposing that the signifi cant increase in the RLU by the ATP production by A. brasiliensis is a result of a shorter lag phase in the fungal growth cycle allowing the advancing and enhancing of the exponential phase for mold growth. This is refl ected by the increased production of ATP from A. brasiliensis that directly corresponds to the increased concentrations of 1 and 10 mM L-glutamic acid. Furthermore, the RLU signal up at least 40-fold in R-TATP broth alone after 24 h incubation period (Figure 1). After 18 h of incubation, the RLU signal was greater than a 20-fold in the 1% test suspensions in R-TATP broth for 15 of 17 personal care product formulations and 13 of 15 raw ingredients (Tables III and IV). However, this rate of de- tection in a test sample at 18 h is not a concern because this type of situation will be de- tected during the validation testing of an ATP bioluminescence assay for a nonsterile raw ingredient or product formulation by a personal care or pharmaceutical company. If mold could not be detected after using an 18 h incubation period, it looks like that the presence of mold in a 1% test sample suspension in R-TATP broth would eventually be detected instead with a 24 h incubation period based on our test results.