TTC IN PRESERVATIVE EFFICACY TESTING 173 The use of TTC in LB provided a more efficient and less costly means of detecting microbial growth than Alamar Blue fluorescence because microbial growth and con- comitant reduction in the redox potential of the culture media resulted in a change from colorless TTC (tetrazolium salt) to red/pink (reduced formazan product) that could be easily detected by visual examination of the microtiter plate wells following incubation. It is noteworthy that the miniaturized system with TTC in LB uses visual inspection of the 96-well plates for color change and does not require expensive instrumentation (spectrophotometers, fluorometers, etc.). The use of LB with TTC for preservative efficacy testing increased laboratory efficiency by decreasing the time required for preparation of media, plating on agar and counting colonies, and sterilizing materials prior to disposal. The system also used fewer materials than conventional plating methods consequently, it was environmentally friendly be- cause it generated less laboratory waste. This was noted in our earlier work using Alamar Blue in the miniaturized system (1). We have used TTC in the miniaturized assay system for over three years and have performed hundreds of preservative efficacy tests with it. We have found it to be reliable for detecting bacterial growth following enrichment. Finding virtually identical results with LB enrichment followed by addition of Alamar Blue and with LB containing TTC demonstrates that TTC did not interfere with the recovery of microorganisms used in these studies. Jin et al. (12) reported a dye reduction assay using tetrazolium salts for C. albicans. Although TTC could be used for preservative efficacy testing of yeasts in the miniaturized assay system, we elected to use conventional plating procedures for pre- servative efficacy testing with C. albicans and Aspergillus niger in our laboratory. There is increasing interest in the use of miniaturized, automated procedures in micro- biological testing (13), and it is believed that preservative efficacy testing using TTC in LB in 96-well microtiter plates would lend itself to automation. This miniaturized method is recommended for laboratories conducting large numbers of preservative ef- ficacy tests on aqueous cosmetic and drug products. REFERENCES (1) D. S. Orth and K. S. Delgadillo Eck, Preservative efficacy testing of aqueous cosmetics and drugs without counting colonies, Cosmet. Toiletr., 116(4), 41-50 (2001). (2) D.S. Orth and D. C. Steinberg, The safety factor in preservative efficacy testing, Cosmet. Toiletr., 118(4), 51-58 (2003). (3) D.S. Orth, K. S. Delgadillo, and C. Dumatol, Maximum allowable D-values for gram negative bacteria: Determining killing rates required in aqueous products, Cosmet. Toiletr., 113(9), 53-59 (1998). (4) United States Pharmacopoeia! Convention, "Microbiological Tests, Antimicrobial Preservatives- Effectiveness," in United States Pharmacopoeia XXV (United States Pharmacopoeia! Convention, Rock- ford, MD, 2002), pp. 1809-1811. (5) European Pharmacopoeia Commission, "Efficacy of Antimicrobial Preservation," in European Pharma- copoeia, 3rd ed. (Council of Europe, Strasbourg, 1996), pp. 286-287. (6) Preservation Subcommittee of the CTFA Microbiological Committee, A guideline for the determina- tion of adequacy of preservation of cosmetics and toiletry formulations, TGA Cosrnet. ]., 2, 20-23 (1970). (7) D.S. Orth, Linear regression method for rapid determination of cosmetic preservative efficacy,]. Soc. Cosrnet. Chern., 30, 321-332 (1979). (8) D.S. Orth, Standardizing preservative efficacy test data, Co57net. Toiletr., 106(3), 45-48, 51 (1991). (9) D.S. Orth, Putting the Phoenix phenomenon into perspective, Cosmet. Toiletr., 114(4), 61-66 (1999).

17 4 JOURNAL OF COSMETIC SCIENCE (10) Difeo Laboratories, Difeo Manual of Dehydrated Culture Media and Reagents for Microbiological and Clinical Laboratory Procedures, 9th ed. (Difeo Laboratories, Inc., Detroit, Michigan, 1953), pp. 192-199. (11) M. T. Ohara and T. Saito, Application of triphenyltetrazolium chloride in microbial limit test in pharmaceuticals and cosmetics: Multiple-tube method for yeasts detection, Boll. Chim. Farmaceutico, 136(1), 17-21 (1997). (12) Y. Jin, H.K. Yip, Y. H. Samaranayake, J. Y. Yau, and L. P. Samaranayake, Biofilm-forming ability of Candida albicans is unlikely to contribute to high levels of oral yeast carriage in cases of human immunodeficiency virus infection,]. Clin. Microbial., 41, 2961-2967 (2003). (13) K. C. Chapin and M. C. Musgnug, Validation of the automated reading and incubation system with Sensititre plates for antimicrobial susceptibility testing,]. Clin. Microbial., 41, 1951-1956 (2003).