A rapid and simple sterility test method based on solid culture medium containing blood

Abstract

Current sterility test performed for most biological products takes 14 days. We evaluated solid medium, containing 5% blood for use in the membrane filtration (MF) and direct inoculation (DI) sterility test. Representative microorganisms prepared in a sample matrix at approximately 0.1, 1, 10 and 100 colony forming units were tested for growth by compendial MF sterility test using fluid thioglycolate medium and tryptic soy broth and also on the Schaedler blood agar (SBA). Sterility test performed on SBA was significantly more sensitive and faster in detecting various microorganisms than the compendial method, particularly for sample matrix containing 0.01% thimerosal (p < 0.05). SBA detected all microorganisms within 7 days. To implement solid medium in the DI sterility test, multiple BA plates were inoculated with the sample. All representative microorganisms were detected within 5 days. The sterility test using solid medium required 3 different incubation conditions, 30–35 °C aerobically and anaerobically to detect bacteria, and at 20–25 °C aerobically to detect mold and yeast. To eliminate aerobic incubation of solid medium at 20–25 °C, we evaluated representative species of mold and yeast for their growth at 30–35 °C and 20–25 °C in the sterility test performed on solid medium. Penicillium chrysogenum could not be detected at 30–35 °C consistently within 7 days. Sterility test performed on solid medium without any additional technology could be completed in 7 days, as compared to the 14 days required for the current compendial method.