分光光度法快速测定副鸡禽杆菌菌液浓度的应用

Spectrophotometry for rapid determination of the concentration of Avibacterium paragallinarum

摘要:

【背景】感染副鸡禽杆菌(Avibacterium paragallinarum)会引起一种鸡的急性上呼吸道疾病——传染性鼻炎。一般情况下该病潜伏期短、发病急且传染性强，易给养殖场造成巨大的经济损失。【目的】比较分光光度法(spectrophotometry,SP)与平板菌落计数法(plate colony count,PCC)测定副鸡禽杆菌菌液浓度的准确性，探究SP替代PCC的可行性。【方法】无菌生理盐水悬浮副鸡禽杆菌菌液沉淀物，用SP测定不同稀释度菌悬液吸光度值(OD值)，用PCC测定活菌数。以不同波长(450、540、600和650 nm)测定的OD值为自变量，活菌数为因变量，获得标准曲线和线性方程，比较分析确定最适测定波长。设置胰蛋白胨大豆肉汤培养基(tryptone soy broth,TSB)组和无菌生理盐水(sterile physiological saline solution,SPSS)组，分别以TSB和SPSS为悬浮液和稀释液，用SP和PCC法分别测定不同稀释度的副鸡禽杆菌菌悬液，通过标准曲线和线性方程比较分析两种悬液对OD值与菌液浓度相关性的影响。取不同培养阶段(10、12、14、16和18 h)菌液，以测定OD值直接输入线性方程获得菌液浓度，同时进行平板菌落计数，采用SPSS 17.0软件统计分析以验证SP法适用性。【结果】在波长600 nm下测定的OD值与PCC结果线性关系最佳(R²>0.995)。在该波长下，TSB组与SPSS组测定的OD值与PCC结果无显著差异(R²_差=0.000 5)，且TSB组略优于SPSS组(R²_TSB=0.998 9>R²_SPSS=0.998 4)。培养10、12、14 h时SP与PCC法测定结果差异不显著(P>0.05)，而培养16 h和18 h时差异显著(P<0.05)。【结论】在对数生长期及稳定期，采用分光光度法测定OD₆₀₀值可快速确定副鸡禽杆菌菌液浓度。

Abstract:

[Background] The infection with Avibacterium paragallinarum (Apg) results in an acute upper respiratory disease known as infectious coryza in chickens. In general, the disease exhibits a short incubation period, an acute onset, and a high degree of infectivity, thereby causing substantial economic losses of breeding farms. [Objective] To assess the feasibility of spectrophotometry (SP) as a potential substitution of plate colony count (PCC) in determining the concentration of Apg. [Methods] The Apg precipitate was suspended in tryptone soy broth (TSB) or sterile physiological saline solution (SPSS), as two sets of diluents respectively. The absorbance (OD) values of the bacterial suspensions at different dilutions were determined by SP and the viable cell count was determined by PCC. With OD values measured at different wavelengths (450, 540, 600 and 650 nm) as the independent variables and the viable cell counts as the dependent variables, the standard curves and linear equations were established. Set up a tryptone soy broth (TSB) group and a sterile physiological saline solution (SPSS) group, using TSB and SPSS as suspensions and dilutions. Use SP and PCC methods to measure the suspension of different dilutions of Streptococcus parahaemolyticus, and compare and analyze the effect of the two suspensions on the correlation between OD value and bacterial concentration through standard curves and linear equations. The OD values of the bacterial suspension samples collected at different culture stages (10, 12, 14, 16 and 18 h) were measured and substituted into the linear equation to obtain the concentration of the bacterial suspension. PCC was carried out at the same time. SPSS 17.0 was used for statistical analysis to verify the applicability of SP. [Results] The OD value measured at 600 nm (OD₆₀₀) showed the best linear relationship with the PCC results (R²>0.995). At this wavelength, the OD values measured in the TSB and SPSS groups were not different from the PCC results (R²_difference=0.000 5), and the TSB group was superior to the SPSS group (R²_TSB=0.998 9> R²_SPSS=0.998 4). The results of SP and PCC showed no differences at the incubation time points of 10, 12, and 14 h (P>0.05) but had differences at the time points of 16 and 18 h (P<0.05). [Conclusion] During the logarithmic and stationary phases, SP could rapidly measure OD₆₀₀ to determine the concentration of Apg.