分光光度法与活菌计数法测定副鸡禽杆菌菌液浓度的相关性研究

Study on the correlation between spectrophotometric method and live bacterial count method for determining the concentration of bacterium solution of Avibacterium paragallinarum

关键词:  副鸡禽杆菌  菌液浓度  分光光度法  活菌计数法

Key words:  Avibacterium paragallinarum  bacterial concentration  spectrophotometric method  viable count method

摘要:

为建立一种快速测定副鸡禽杆菌菌液浓度的方法，本研究选取副鸡禽杆菌血清A、B、C型代表性菌株（CVCC254株、CVCC257株和CVCC256株），培养制备菌液，分别在450nm、540nm、600nm、650nm波长测定其吸光度值（OD值），同时测定活菌计数值。回归分析两组数据，获取回归方程及标准曲线，分析回归方程的测定系数（R2）和标准曲线的点线离散度，确定最适测定波长。再选取不同培养时间点（10h、12h、14h、16h、18h、20h）的菌液，在最适波长下测定其OD值并代入回归方程获取活菌数，同时采用活菌计数法获取活菌计数值，将两组数据使用SPSS 17.0软件进行统计分析，进一步验证分光光度法与活菌计数法测定菌液浓度的相关性。结果显示，3株菌液在600nm波长测定的OD值与活菌计数值之间均呈最佳的线性关系，确定其菌液浓度最适测定波长均为600nm，其回归方程分别为y=14.302x－0.1441（R2=0.9962）、y=14.464x－0.2746（R2=0.995）和y=14.681x－0.01326（R2=0.9989），y为活菌计数值（×108 CFU/mL），x为OD600值。3株培养至衰退期前的菌液在600nm波长测定的OD值代入回归方程计算获得的活菌数值与其活菌计数值无显著差异（P＞0.05）。研究结果表明，衰退期前副鸡禽杆菌菌液的分光光度法与活菌计数法具有良好的相关性。因此，该分光光度法可用于快速测定衰退期前副鸡禽杆菌菌液浓度。

Abstract:

In order to establish a rapid method for determining the concentration of avibacterium paragallinarum in liquid form, representative strains of avibacterium paragallinarum serotypes A, B and C were selected in this study(strains CVCC254, CVCC257 and CVCC256)，The viable bacterial count value was determined at the same time. Regression analysis of the two sets of data, obtain the regression equation and the standard curve, analyse the coefficient of determination (R2) of the regression equation and the dispersion of the point line of the standard curve, and determine the optimal measurement wavelength.Then the bacterial solutions at different incubation time points (10h, 12h, 14h, 16h, 18h, 20h) were selected, and their OD values were measured at the optimum wavelength and substituted into the regression equation to obtain the number of viable bacteria，the viable bacteria counting method was also used to obtain the viable bacteria count values, and the two sets of data were statistically analysed using SPSS 17.0 software to further validate the correlation between the spectrophotometric method and the viable bacteria counting method to determine the concentration of bacterial fluids.The results showed that there was an optimal linear relationship between the OD values and the viable bacterial count values of the three strains of bacterial fluids measured at 600nm, and it was determined that the optimal wavelength for the determination of their bacterial concentration was 600nm for all of them.The regression equations were y=14.302x-0.1441 (R2=0.9962), y=14.464x-0.2746 (R2=0.995), and y=14.681x-0.01326 (R2=0.9989), respectively, with y as the viable bacteria count value (×108 CFU/mL) and x is the OD600 value.The values of viable bacteria obtained by substituting the OD values of the three strains cultured to the pre-decline stage measured at 600 nm into the regression equation were not significantly different from their viable count values (P > 0.05).The results of the study showed that the spectrophotometric method of pre-decline avibacterium paragallinarum bacterial fluids correlated well with the viable count method. Therefore, the spectrophotometric method can be used for the rapid determination of the concentration of avibacterium paragallinarum bacterial fluids before the decline period.