DETECTING MOLDS IN PERSONAL CARE PRODUCTS 39 including ATP. An additional 100 μl aliquot of antifoam is also added to these aliquots to prevent foaming during shaking. Each of these aliquots is then shaken side to side in a linear shaker for 30 min at an RPM of 200–300. After linear shaking, 2 × 50 μl aliquots are aseptically removed and transfer to two separate 12 × 55 mm cuvettes. For those en- richment broth containers that had been inoculated with bacteria only, 2 × 50 μl aliquots are aseptically removed and transferred to two separate 12 × 55 mm cuvettes. DETECTION OF ATP An ATP bioluminescence assay was conducted by using the Celsis AKuScreen test kit (Catalog number AS1310 Charles River Laboratories, Inc.) to determine the RLU from the ATP in each of the incubated test sample suspensions. To perform the assay, a rack of 12 × 55 mm cuvettes is prepared. In four for the 12 × 55 mm cuvettes, four separate 50 μl aliquots of R-TATP broth is pipetted separately into each cuvette. Two of the 50 μl aliquots of R-TATP broth will serve as a negative control. In the two remaining 50 μl aliquots of R-TATP broth cuvettes, 10 μl of ATP is aseptically added to each cuvette as a positive test control. After incubation, aseptically transfer 2 × 50 μl aliquots of the incu- bated 1% test sample suspension in enrichment broth into two separate 12 × 55 mm cuvettes. For the non-incubated 1% test sample suspension in enrichment broth, 2 × 50 μl aliquots is transfer into two separate 12 × 55 mm cuvettes. All of the cuvettes are placed into a Celsis Advance luminometer and the unit is started to process the cuvettes. The bioluminescence reagents are added to each cuvette by the luminometer and the amount of RLU detected in each cuvette is recorded by the luminometer. For each two cuvettes of a test sample, an average of the RLU is calculated and then the RLU ratio was calculated based on the RLU from an inoculated enrichment broth sample versus an uninoculated enrichment broth sample. Positive ATP bioluminescence test result for each of the test microorganisms is reported with an RLU ratio of greater than two. RESULTS AND INTERPRETATION NONMICROBIAL ATP CONTENT OF R-TATP BROTH One of the key factors for using an enrichment broth in an ATP bioluminescence assay is that it must have a low nonmicrobial ATP background in order not to provide false- positive test reactions for the presence of microbial contamination in a test sample. In comparing the RLU values for the presence of nonmicrobial ATP in uninoculated 100 ml aliquots of R-TAT broth, R-TATP broth, and Letheen broth, R-TATP broth had the low- est mean RLU value of 78 with a standard deviation value of 15 (Figure 1). The mean RLU values and standard deviations for nonmicrobial ATP in R-TAT broth were found to be 91 ± 14. The mean RLU values and standard deviations for nonmicrobial ATP in Letheen broth were found to be 105 ± 28. These RLU test results confi rm that R-TATP broth is an appropriate enrichment broth that can be used in an ATP bioluminescence assay for detecting the presence or absence of microbial contamination in nonsterile raw ingredients and personal care product formulations because of the fact that it contains a low level of nonmicrobial ATP.

JOURNAL OF COSMETIC SCIENCE 40 GROWTH PROMOTION OF L-GLUTAMIC ACID IN R-TATP ENRICHMENT BROTH FOR DETECTING THE GROWTH OF ASPERGILLUS BRASILIENSIS By using R-TAT broth and the Celsis AKusScreen test kit, low levels (10–100 CFU/g/ 100 ml) of bacteria and yeast can be easily detected after using a 24 h incubation period by performing an ATP bioluminescence assay for most test samples. The detection of A. brasilinesis by using TAT broth varied with an RLU ratio of 2–5 which is comparable with that of Letheen broth which is recommended for use as an enrichment broth in an ATP bioluminescence assay by Charles River Laboratories, Inc. R-TAT broth containing L-glutamic acid concentrations of 1, 5, 10, 20, and 30 mM that had been inoculated with an A. brasiliensis inoculums of 20 and 38 CFUs that were incu- bated at a temperature of 30.0°C ± 1.0°C for 24 h and then evaluated for the presence of ATP by using an ATP bioluminescence assay. The RLU ratio results of these samples demonstrated a clear dose–response relationship between the various concentrations of L-glutamic acid and the amount of ATP detected from the growth of A. brasiliensis in this enrichment broth. In the absence of L-glutamic acid in the enrichment broth, the RLU ratio for R-TAT broth was around 4.2 and 13 with A. brasiliensis inoculums of 14 and 28 CFU. By including L-glutamic acid in the R-TAT broth, the amount of ATP was signifi cantly increased by having an RLU ratio of 40 and 106 with a 5 mM concentration, and there was a maximized RLU ratio of 100 and 140 when a 10 mM concentration of L-glutamic acid was used. By using 20 and 30 mM concentrations of L-glutamic acid in R-TAT broth, the RLU ratio started to decline (Figure 2). The test results indicated that the maximum enhancement for the production of ATP from the growth of mold is at a 10 mM concentration of L-glutamic acid. However, we chose to use a 5 mM concentra- tion of L-glutamic acid in R-TAT broth to minimize in having a possible adverse effect of a higher concentration of L-glutamic acid on the growth of other microorganisms that may be present in a test sample. Figure 1. Effect of various concentrations of L-glutamic acid in R-TATP broth on the RLU ratio from Aspergillus brasiliensis growth after 24 h of incubation.