Application Note. Screening Products for Microbial Contamination Using the BD FACSMicroCount System. Summary. Authors Affiliation

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1 September 0 Screening Products for Microbial Contamination Using the BD FACSMicroCount System Authors Affiliation Contents Summary Objective Materials and Methods Acceptance Criteria 4 Results 5 Discussion 5 Conclusion Summary In keeping with United States Pharmacopeia (USP) <6> sample preparation, BD has developed a procedure to rapidly screen products for the presence of microbial contamination. Various emulsions, excipients, personal care, OTC, household, beverage, and cosmetic products were tested with the product screening procedure using the BD FACSMicroCount system and BD FACSMicroCount media kit. Fifty-five different product samples were inoculated with <0 cfu of bacteria, yeast, and mold. All inoculated samples were positive for microbial contamination within 4 to 48 hours. Of these, 87% were positive for microbial contamination within 4 hours, 9% were positive within 0 hours, and 0% were positive within 48 hours. Additionally, the BD FACSMicroCount software allowed differentiation between bacteria, yeast, and mold contamination in a single sample.

2 September 0 Objective The objective of this study was to demonstrate the ability of a product screening procedure using the BD FACSMicroCount system and BD FACSMicroCount media kit to detect low levels of microorganisms in various emulsions, excipients, personal care, OTC, household, beverage, and cosmetic products. Materials and Methods Various emulsions, excipients, personal care, OTC, household, beverage, and cosmetic products were tested with the product screening procedure using the BD FACSMicroCount system and BD FACSMicroCount media kit. Products were diluted : in Phosphate Buffer (PB) or Growth Enhancement Media (GEM) and mixed to achieve a homogeneous suspension. One milliliter was then transferred to 9 ml of GEM + substrate (Tube A in the BD FACSMicroCount media kit) and neutralized for 0 minutes. Pseudomonas aeruginosa ATCC 907, Escherichia coli ATCC 59 or 879, Staphylococcus aureus ATCC 658, Candida albicans ATCC, and Aspergillus brasiliensis ATCC 6404 were inoculated individually into Tube A for each product (<0 cfu/volume). Bacillus subtilis subsp. spizizinii ATCC 66 was substituted for E. coli in the excipient samples. Tube A GEM microbes Transfer swab from Tube A to B Tube B PB Tube C PB 5-µm filter P/A detection Figure. Illustration of the BD FACSMicroCount media kit product screening procedure. Inocula were spread-plated on Tryptic Soy Agar (TSA) for confirmation of inoculum levels. Negative control (non-spiked) product samples were analyzed using the same protocol to determine product baselines. Samples were enriched at 0 C with shaking for a minimum of 4 hours. After enrichment, the substrate was transferred to ml of PB (Tube B in the BD FACSMicroCount media kit) and vortexed. One hundred microliters was then added to.9 ml of PB through a filter cap (Tube C in the BD FACSMicroCount media kit), and the samples were analyzed on the BD FACSMicroCount system. (See Figure for an illustration of the BD FACSMicroCount media kit product screening procedure). Samples with BD FACSMicroCount outputs >X the baseline failed, indicating that the sample was positive for microbial contamination. Acceptance Criteria Internal validation studies have been conducted to determine pass/fail criteria for the BD FACSMicroCount system using product baselines. The baseline is calculated by taking the average non-spiked product results plus the standard deviation. A sample with a result X the baseline is considered a fail, and indicates that the sample is positive for microbial contamination. A sample with <X the baseline is considered a pass, and indicates that the sample does not contain microbial contamination. Note: Due to the nature of several of the products tested, it was necessary to modify the procedure described. One milliliter of each excipient was added directly to Tube A without a : dilution. This was possible because these samples lack preservatives and had low backgrounds. The fiber capsules and fiber powder required :0 and :0 pre-enrichment dilutions, respectively. This was due to the absorbent nature of the fiber, and a : product dilution was not possible for these samples. The eye shadow and mascara required an additional : dilution post-enrichment to reduce product background. One baby wipe/ disinfecting wipe was stomached in 90 ml of PB or GEM for 0 seconds, and the liquid was decanted. One milliliter was then added to Tube A.

3 September 0 Page Table. Product screening results showing detection of low levels of microorganisms in various emulsions, excipients, personal care, OTC, household, beverage, and cosmetic products using the BD FACSMicroCount system. Emulsions Inoculum Range (cfu) Time to Result* Silicone Emulsions ( and ) h Silicone Emulsion () h Excipients Inoculum Range (cfu) Time to Result* Clear Solutions (n=4) h Personal Care Products Inoculum Range (cfu) Time to Result* Baby Wipes h Body Wash h Conditioner 57 0 h Face Scrub h Hair Gel 78 4 h Hand Lotion h Hand Soap Antibacterial h Mouthwash 5 4 h Shampoo h Shave Gel 4 4 h Sunscreen h Toothpaste h Over the Counter Products Inoculum Range (cfu) Time to Result* Acetaminophen Children's Liquid 8 4 h Allergy Children s Liquid 78 4 h Antacid Tablets h Anti Itch 78 4 h Cold/Flu Liquid Night Time h Cough Syrup h Fiber Capsules 4 h Fiber Powder h Ibuprofen Children's Liquid 8 4 h Laxative, Saline h Nasal Spray h Sleep Aid, Liquid Caps h Vitamins Multi, Children s Chewable h Vitamins Multi, Adult h Household Products Inoculum Range (cfu) Time to Result* Dish Soap 58 4 h Dishwasher Rinse Agent h Disinfecting Wipes h Fabric Refresher 6 48 h Household Cleaner 56 4 h Laundry Detergent 6 4 h Beverage Products Inoculum Range (cfu) Time to Result* Antioxidant Water 8 4 h Cranberry Juice h Lemonade h Vegetable Juice h Cosmetic Products Inoculum Range (cfu) Time to Result* Eye Shadow 6 48 h Mascara 6 48 h *Maximum enrichment time required to detect the presence of all 5 microorganisms in the respective products.

4 September 0 Results A total of 55 different product samples were inoculated and tested using the protocol described. Of these, 87% were positive for microbial contamination within 4 hours, 9% were positive within 0 hours, and 0% were positive within 48 hours (Table ). Inocula ranged from to 78 cfu. All inoculated samples were X the baseline post-enrichment. Area definitions were predefined in the BD FACSMicroCount software to distinguish between mold, bacteria, and yeast contamination events. All inoculated samples were positive for microbial contamination in their predefined area (Figure ). Pos Cont EC-4 hr 4/6/008 :48:06 AM 00 Pos Cont PSA-4 hr 4/6/008 :5:4 AM 00 Pos Cont SA-4 hr 4/6/008 :44:54 AM 00 Pos Cont CA-4 hr 4/6/008 :54:9 AM 00 Pos Cont AN-4 hr 4/6/008 :00: AM Cnts: Total: 5070 : 6900 /.9% : / 5.9% : 688 /.0% Cnts: Total: 84 : 4509 / 8.0% : 0099 / 5.% : 699 / 0.7% Cnts: Total: 9476 : 4970 /.7% : 870 / 79.6% : 9 / 0.9% Cnts: Total: 86 : /.0% : 08 / 7.7% : 8 / 8.% Cnts: Total: 68 : 565 / 90.0% : 4 / 5.4% : / 0.5% E. coli P. aeruginosa S. aureus C. albicans A. brasiliensis Area Neds NCI-4 hr 4/6/008 :0:8 AM 00 0 Area Area 0 00 Cnts: Total: 7 : / 5.9% : 8 / 47.% : 0 / 0.0% Broth Control Figure. Examples of BD FACSMicroCount intensity plots for positive control samples. Area (red) encompasses mold populations, Area (blue) encompasses bacterial populations, and Area (green) encompasses yeast populations.

5 September 0 Page 5 Discussion All 55 product samples inoculated for this study were positive for microbial contamination in 4 to 48 hours. The BD FACSMicroCount software contains three analysis boxes that allow the user to differentiate between mold, bacteria, and yeast contamination in a single sample. One analysis box (Area ) encompasses mold populations, one analysis box (Area ) encompasses bacterial populations, and one analysis box (Area ) encompasses yeast populations. The ability of this procedure to yield results in <48 hours is due in part to the ability of the BD FACSMicroCount system to detect mold filaments, which increases the sensitivity of the method. Conclusion The BD FACSMicroCount system and BD FACSMicroCount media kit product screening procedure provide a rapid and sensitive method for detecting the presence or absence of low levels of microorganisms in various emulsions, excipients, personal care, OTC, household, beverage, and cosmetic products.

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