结核分枝杆菌菌悬液光密度值与菌落计数的关联性

Correlation between optical density and colony forming units of Mycobacterium tuberculosis suspension

摘要:

目的建立一种基于光密度值计算结核分枝杆菌菌落数的可靠方法。方法利用低频超声和玻璃珠研磨两种方法制备H37Ra菌悬液，菌悬液2倍梯度稀释后，分别测定各个稀释度菌悬液在600 nm处的光密度值（OD600值），并分析OD600值与稀释倍数曲线，确定最佳的菌悬液制备方法，OD600线性范围，以及OD600值与CFU关联曲线。结果OD600值为0.1～0.6，线性范围内OD600值与稀释倍数线性回归分析结果显示，玻璃珠研磨法和低频超声法相关系数（R2）分别为0.98、1.00，均呈良好的相关性，且低频超声法比玻璃珠研磨法的相关性好，其菌液分散更均匀。OD600值与CFU值线性回归分析结果显示，玻璃珠研磨法和低频超声法回归方程分别是：CFU=2.35×107×OD600+4.42×105、CFU=3.26×107×OD600+6.89×105。结论低频超声法是一种较好的结核分枝杆菌菌悬液制备方法，结合OD600值测定，可成为一种可靠、快速的结核分枝杆菌定量方法。

Abstract:

ObjectiveTo establish a reliable approach for quantification of colony forming unit(CFU) of Mycobacterium tuberculosis(M.tb) by measuring optical density(OD).MethodsM.tb suspension H37Ra was prepared using lowpower ultrasonic or glass bead beating methods, and was twofold serially diluted, OD at 600nm (OD600) of each dilution ratio was measured respectively, OD600 and dilution curve were analyzed to determine the optimum approach for preparing bacterial suspension，linear range of OD600, as well as linear relationship between OD600 and CFU.ResultsOD600 was 0.1-0.6, linear regression analysis of OD600 and dilution ratio within linear range revealed that correlation coefficient (R2) of glass bead beating and lowpower ultrasonic methods were 0.98 and 1.00 respectively，both presented a good correlation, lowpower ultrasonic method was better than glass bead beating method, bacterial suspension dispersed more evenly. Linear regression analysis results of OD600 and CFU values showed that the regression equation of glass bead beating method and lowpower ultrasonic method were CFU=2.35×107×OD600+4.42×105 and CFU=3.26×107×OD600+6.89×105 respectively.ConclusionLowpower ultrasonic method is a good method for preparation of M.tb suspension，combined the measurement of OD600 value， it can be a reliable and rapid method for quantitative analysis of M.tb.