结晶紫染色法检测活体细菌的实验研究

THE EXPERIMENTAL STUDY OF THE LIVING BACTERIA BY CRYSTAL VIOLET STAINING

目的探讨活体细菌着色检测的适宜条件。方法将金黄色葡萄球菌、黄色微球菌、大肠埃希菌和铜绿假单胞菌稀释浓度为5×107菌落形成单位/毫升(Colony forming unit,CFU/ml)。经不同浓度与作用时间的结晶紫(Crystal Violet,CV)着色细菌洗涤后,保存于甘油磷酸缓冲液。采用平皿倾注法测取细菌的CFU。结果与对照组相比,2%CV染色时,细菌CFU无显著性差异(P>0.05),染料大于此浓度染色时,CFU有差异性(P<0.05)。染色时间在3min内CFU无显著性差异(P>0.05)。30%甘油磷酸盐缓冲液保存着色活体细菌,在48h内活性无显著性差异(P>0.05),60h后有差异性(P<0.05)。结论实验方法适用于活体细菌显色法的形态学检测。

Objective To explore an appropriate condition for the examination of the living bacteria staining. Methods The culture of Staphylococcus aureus, Micrococcus flavus, Escherichia coli and Pseudomonas aeruginosa were collected and then centrifugally washed. The final concentration is 5 × 10^7 colony forming unit/milliliter （CFU/ml） diluted by normal saline（NS）. The four strains of bacteria were preserved in the glycerophosphate buffer after being dyed and laved by the different concentration and acting time of crystal violet. In order to know the effect of various factors such as staining solution concentration, acting time, and glycerin buffer fabrication upon the bacterial survival rate, and two methods were carried out. One was observed by the microscope, the other was put into the culture dish. Results Comparing with that of the control group, the bacterial CFU was had no statistical significance （ P 〉 0.05 ） dyed by 2% crystal violet. When the concentration of the dye - solution was higher than 2% , there showed the difference （P 〈 0.05）. In 3 rain dyeing time, CFU had no significant difference （P 〉 0.05）. The stained living bacteria preserved in the 30% glycerophosphate buffer within 48h have not shown significant difference （ P 〉 0. 05 ） , while after 60h, it showed significant difference （ P 〈 0.05 ）. Conclusion The method is quite suitable for the morphological detection of the living bacteria.