荧光法快速检测活菌数的方法研究及应用

【背景】Calcein UltraGreen~(TM)AM是一种新型荧光染料,用于标记和监测活细胞。【目的】基于该荧光染料的荧光特性及其在活细胞内的稳定特性,建立一种荧光定量快速检测活细菌总数的方法,并在实际样品中应用校正。【方法】通过应用荧光染料对细菌进行染色,再进行荧光强度检测,同时以平板计数法作平行对照,建立荧光强度值-活菌数标准曲线。【结果】确定了染色细菌的最佳pH值为8.0。该检测方法仅需固定染色温度,染色时间在20-30min范围即可快速检测。建立了革兰氏阴性菌铜绿假单胞菌NY3、大肠杆菌和革兰氏阳性菌芽孢杆菌、红平红球菌FF、金黄色葡萄球菌和枯草芽孢杆菌的细菌总数与相对荧光强度值标准曲线。当菌悬液OD600值在0.01-0.30范围内时,上述6种细菌与荧光信号强度呈良好的线性关系(R20.99)。【结论】当样品菌悬液浓度范围控制在105-109CFU/mL时,建立的荧光检测方法快速便捷,精密度、重复性、稳定性、回收率和准确度均较好,可应用于微生物实验、固体菌剂发酵、食品卫生与安全、环境检测等领域的活细菌总数现场快速检测。

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

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.