Flow cytometry for the rapid detection of bacteria in cell culture production medium

The rapid and sensitive detection of microbial contaminants is critical for timely contamination investigations and monitoring of process control for in vitro protein production systems. A flow cytometric method was developed to monitor two types of cell culture media used in large scale protein production. The process used a DNA dye, thiazole orange, which binds to nucleic acids of viable and nonviable organisms. To ensure a representative sample was tested and to enhance the detection limit, a concentration step before staining and analysis included a double centrifugation. In addition, a washing step was included to eliminate background fluorescence caused by material in the media formulations. The staining and analysis of concentrated and washed samples takes approximately 0.5 h and provides objective results. The feasibility of the method was demonstrated by spiking sterile media with six bacterial species that represent the most commonly encountered bacterial contaminants. In addition to the bacterial spiking study, 164 lots of large scale production medium were tested by the flow cytometric method in parallel with conventional culture using Trypticase Soy Broth (TSB). In the bacterial spiking study, the concentration met...Continue Reading