Thus, the CFU Nutlin-3a solubility dmso assay for mature hyphae is at best an under estimation of the total fungal biomass. Since our experiments were designed to compare untreated drug-free controls to drug-treated experimental groups, determination of the absolute fungal biomass was not essential for demonstrating

comparative effect of the drug treatment. Tetrazolium reduction assay In addition to CFU assay, we evaluated the effects of antimicrobial drugs on monomicrobial and polymicrobial biofilms of A. fumigatus and P. aeruginosa by the tetrazolium reduction assay [47, 48]. Briefly, monomicrobial and polymicrobial biofilms of A. fumigatus and P. aeruginosa were washed three times with sterile distilled water (1 ml each) and check details the excess water was removed by aspiration with a 1 ml micropipet. The washed adherent biofilm was overlaid with 1 ml fresh SD broth containing 100 mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide [MTT] and 0.2 mM menadione and incubated at 35°C for 3 h for the reduction of the tetrazolium compound. Under these conditions, the lightly yellowish MTT will be reduced to an insoluble blue tetrazolium salt accumulated within the mycelia.

At the end of the incubation period, the growth medium containing MTT was removed and the biofilm was washed three times (1 ml each) with sterile distilled water, and intracellular insoluble tetrazolium salt was dissolved in 1 ml 70% ethanol containing 0.1 N HCl for 30 min at 35°C. The amount of intracellular tetrazolium salts was quantified spectrophotometrically by measuring the absorbance of the solution at 570 nm. The accumulation of tetrazolium salt by the science reduction of MTT by cellular dehydrogenases is proportional

to the number of viable cells present in the biofilm. The effectiveness of the antimicrobial drug treatment was assessed on the basis of diminished tetrazolium reduction. Antimicrobial drugs Pharmaceutical grade cefepime (Sagent Pharmaceuticals, Schaumberg, IL, USA) and tobramycin pure powder were obtained from the Henry Ford Hospital Pharmacy and Sigma Chemical Company, St. Louis, USA, respectively. Stock solutions (1 mg/ml) of the antibiotics were prepared in sterile distilled water and stored as 0.25 ml aliquots at -20°C. selleck Voriconazole and posaconazole were obtained from Pfizer Pharmaceuticals (New York, NY, USA) and Schering-Plough Research Institute, Kenilsworth, NJ, USA (now part of Merck), respectively. The triazoles were dissolved in dimethylsulfoxide to obtain a stock solution of 10 mg/ml and stored as 0.25-ml aliquots at -20°C. The frozen stocks of the antimicrobial drugs were thawed at room temperature and used within 24 h. Where it is applicable, comparable concentrations of dimethylsulfoxide were used as control to examine its effect on the growth of the organism.