Rapid detection of viable bacteria cells with a fluorescence-based method

Abstract:

[Background] Calcein UltraGreen? AM is a novel fluorescent dye used to label and monitor living cells. [Objective] A rapid detection method of viable bacteria was developed based on the characteristics of the Calcein UltraGreen? AM. This fluorogenic probe gives strong and stable fluorescence signal in living cells, its fluorescence intensity is proportional to the number of living cells. [Methods] The cells of bacteria, especially the viable cells, were stained with Calcein UltraGreen? AM, and then, its fluorescence intensity was measured. The standard curves of the fluorescence intensity value-viable bacteria number were established by simultaneously number counting of viable bacteria using the plate method. [Results] The optimum pH for staining viable cells of bacterial was 8.0. Under a specific staining temperature, the total number of viable bacteria can be readily detected within 20?30 min. The standard curves of the total number of both Gram-negative and Gram-positive bacteria, including Pseudomonas aeruginosa NY3, E. coli, Bacillus, Rhodococcus erythropolis FF, Staphylococcus aureus, and Bacillus subtilis, vs relative fluorescence intensity was established. A good linear relationship (R2>0.99) for the six bacteria were obtained when the OD600 of the bacterial suspensions were in the range of 0.01?0.30. [Conclusion] When the concentration of the sample suspension was ajusted to 105?109 CFU/mL, a rapid, convenient, accurate, reproducible and stable detection method was developed with good recovery and accuracy. This established method can be reaily used to measure the living bacteria in the fields of microbial experiments, solid bacteria fermentation, food hygiene and safety, and environmental mornitoring.