DETECTING MOLDS IN PERSONAL CARE PRODUCTS 37 (Catalog number 0483C), B. subtilis ATCC 6633 (Catalog number 0486C), and C. albicans ATCC 10231 (Catalog number 0443C) had been purchased from Microbiologic, Inc. (St Cloud, MN) Spore suspensions of A. brasiliensis ATCC 16404 and Aspergillus oryzae ATCC 10124 were prepared in-house by harvesting spores from potato dextrose agar slants. ENRICHMENT BROTH COMPONENTS AND CHEMICALS The following microbial growth media and enzymatic digest of protein were purchased from Becton Dickinson Company (Franklin Lakes, NJ): Difco™ TAT Broth Base, potato dextrose broth (PDB) (Difco), and Letheen broth (Difco), and Bacto™ Neopeptone. Sucrose, sodium thiosulfate, polysorbate 20, and sodium thiosulfate 1 N solution were purchased from Thermo Fisher Scientifi c. L-glutamic acid and antifoam (Sigman A 5757) were purchased from Sigma-Aldrich Products. ATP BIOLUMINESCENCE REAGENTS AND GLASS BEADS Sterile 0.5 millimeter (mm) glass beads (Catalog number 11079105 Biospec Products Inc., Bartlesville, OK) and Celsis® AKuScreen test kit (Celsis Catalog number AS1310 containin the Celsis LuminAMP and LuminEX reagents) were purchased from Charles River Laboratories, Inc. TEST MICROORGANISM PREPARATION For each of the EZ-CFU product microorganism, one pellet of a lyophilized bacteria and C. albicans culture was aseptically transferred to a separate 1.0 milliliter (ml) aliquot of pre-warmed 10 millimolar (mM) phosphate buffer pH 7.2 solution at 35.0°C ± 2.0°C. Each culture aliquot was then immediately incubated at 35.0°C ± 2.0°C for 30 min for com- plete hydration in which there is a 103 CFU/ml suspension. An in-house method was used to prepare spore suspensions of A. brasiliensis ATCC 16404 and A. oryzae ATCC 10124 in which each of the Aspergillus cultures were grown on several on potato dextrose agar slants until sporulation had occurred at a temperature of 20.0°–25.0°C for a minimum of 7 d. After sporulation, the spores for each culture were harvested from the potato dextrose agar slants by using sterile 0.85% saline solution (0.85% NaCl) with 0.05% polysorbate 80 to a level of 107 CFU/ml that was then diluted to a spore suspension level of 103 CFU/ml by using sterile 0.85% saline solution with 0.05% polysorbate 80. PREPARATION OF ENRICHMENT R-TAT BROTH AND R-TATP BROTH R-TAT broth was prepared in two parts. Part A starts by warming up 2 liters (L) of deion- ized water to 50.0°C. After warming to 50.0°C, 50.0 g of PDB is added to the deionized water and stirred until dissolved, and then heated to boiling for 1 min to completely dis- solve the dehydrated PDB powder. Part B starts by warming 7.6 L of deionized water to a temperature of 50.0°C. After reaching a temperature of 50.0°C, the following ingredients

JOURNAL OF COSMETIC SCIENCE 38 are separately added to the deionized water and dissolved by stirring: 250.0 g of Difco™ TAT Broth Base, 100.0 g of Bacto™ Neopeptone, 25.0 g of sodium thiosulfate, 50.0 g of sucrose, and 400 ml of polysorbate 20. After adding all of the ingredients, the mixture is stirred for at least 30 min or until all of the ingredients have been thoroughly dissolved. After dissolving, part B is added to part A and stirred for an additional 5 min. R-TATP broth was prepared the same way as R-TAT broth with the exception of 7.35 g of L-glutamic acid is added to part B (see Table I). After mixing part A and part B together, 100 ml aliquots of the R-TATP broth and/or R-TAT broth is dispensed into individual widemouthed square plastic bottles and sterilized at a temperature of 121.0°C ± 1.0°C at 15 pounds for 40 min. After sterilization, the caps on each container are tightened after cooling down to ambient room temperature. The fi nal pH of R-TATP broth should be 6.5–6.6. Preparation of Letheen broth is performed by following the instructions from the manufacturer. TEST SAMPLE PREPARATION AND INCUBATION A 1.0 g or ml aliquot of a raw ingredient or personal care product formulation is asepti- cally transferred to 100 ml aliquots of R-TATP broth to make a 1% test sample suspen- sion. For each test microorganism, a 10 microliter (μl) aliquot of the 103 CFU/ml suspension (in test microorganism preparation) containing approximately 10 CFU is aseptically inoculated to a container with/without a 1% test sample suspension and incu- bated at a temperature of 30.0°C ± 1.0°C with agitation of 150 revolutions per minute (RPM). One uninoculated aliquot of R-TATP broth and one uninoculated aliquot of a 1% test sample suspension are also incubated as negative test controls. R-TAT broth con- taining L-glutamic acid concentrations of 1.0, 5, 10, 20, and 30 mM were prepared. A. brasiliensis ATCC 16404 inoculums containing 20 and 30 CFUs were aseptically added to each of the 100 ml aliquots of the enrichment broth. ATP BIOLUMINESCENCE ASSAY Incubated test samples are removed from the incubator shaker after 18 or 24 h of incuba- tion at 30.0°C ± 1.0°C. For those aliquots of R-TATP broth or 1% test sample suspen- sion that have been inoculated with A. brasiliensis and C. albicans, 15.0 g of sterile 0.5 mm glass beads are added to break up the mycelium and release the intracellular constituents Table I Ingredients in R-TAT Broth and R-TATP Broth Ingredient R-TAT broth (g/L) R-TATP broth (g/L) Difco™ TAT broth base 25.0 25.0 Potato dextrose broth 5.0 5.0 Difco™ neopeptone 10.0 10.0 Sodium thiosulfate 2.5 2.5 L-glutamic acid — 735 mg (5 mM) Sucrose 5.0 5.0 Polysorbate 20 4.0 ml 40.0 ml