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The total bacteria count (TBC) of a substance is a quantitative estimate of the number of microorganisms present in a sample, which is an important and straightforward indicator to evaluate the biological quality of the targeted habitat. Due to the rapid speed and automated sample handling, FCM has been widely used in the basic research of microbiology, routine monitoring of food safety, and quality control of drinking water. A good discrimination of the small sizes of bacterial cells from abiotic particles and background signal is the prerequisite for the accurate counting of bacterial cells using FCM.