Clostridium difficile continues to cause infections in healthcare and other settings. Its spores survive well indoors and require sporicidal chemicals for infection control. However, proper testing of disinfectants is impeded due to difficulties in obtaining viable spores of high enough quality and titers to meet current regulations for sporicidal claims. A new liquid medium (Clospore) has been developed, based on a systematic review of the compositions of 20 other available media. C. difficile spores grown in the new medium and treated with a mixture of lysozyme and trypsin yielded final suspensions with > 10(9) CFU/mL of viable spores, with a purity of > 91% as tested by spore-staining and phase-contrast microscopy. The spores showed a biological decay rate of about 0.1 log10/month when dried on metal disks and stored indoors (air temperature 23 +/- 2 degrees C; relative humidity 52.76 +/- 15.08%). Heating the purified spore suspensions to 70 degrees C for 10 min to inactivate any vegetative cells showed no spore activation or inactivation. The spores could be stored for at least 14 months either refrigerated (4 degrees C) or frozen (-20 or -80 degrees C) in 50% (v/v) ethanol with virtually no loss in viability. The resistance of the enzyme-treated spores to three levels of sodium hypochlorite (1000, 3000, and 5000 ppm), using a standardized quantitative carrier test, was almost identical to that of the spores concentrated by centrifugation alone. The described procedure has been successfully applied to four standard (ATCC) and six clinical strains of C. difficile.