粪便沙门菌、志贺菌和副溶血弧菌分离培养方法研究

Application study on isolation and culture methods of Salmonella,Shigella,and Vibrio parahaemolyticus in feces

目的通过优化粪便的分离培养方法,提高沙门菌、志贺菌和副溶血弧菌的检出率。方法分别对沙门菌、志贺菌和副溶血弧菌在不同放置温度(包括4℃和25℃、常温)、不同放置时间(0 h、2 h、3 h和6 h)及不同肉汤增菌时间(0 h、8 h、12 h和24 h)等实验条件下进行培养,记录检出率。结果将沙门菌、志贺菌、副溶血弧菌三种病原菌在4℃和25℃放置2 h,其检出率(95%和90%、60%和50%、85%和95%)比较差异均无统计学意义(P>0.05);将三种病原菌分别于常温放置0 h、2 h、3 h和6 h,沙门菌和副溶血弧菌的检出率无明显变化,志贺菌的检出率分别为75%、55%、20%、5%,呈下降趋势;三种病原菌增菌前(0 h)检出率均为0,增菌8 h、12 h和24 h,沙门菌的检出率分别为90%、90%和95%,志贺菌的检出率分别为50%、20%和5%,副溶血弧菌的检出率均为95%,增菌后检出率均有升高;使用沙门志贺肉汤,沙门菌的适宜增菌时间为8~24 h,志贺菌为8 h,使用碱性蛋白胨水,副溶血弧菌的适宜增菌时间为8~24 h;志贺菌在三组实验中的检出率均较低。结论粪便经增菌肉汤增菌至适宜时间接种选择性显色平板可显著提高沙门菌、志贺菌和副溶血弧菌的检出率;4℃或25℃放置,6 h内直接接种选择性平板,适用于沙门菌和副溶血弧菌菌量大于10^(6) cfu/mL的粪便样本;培养志贺菌不宜单独使用麦康凯琼脂平板。

Objective To improve the separation rate of Salmonella,Shigella,and Vibrio parahaemolyticus by optimizing the separation and culture method of feces.Methods The isolation rates of Salmonella,Shigella,and Vibrio parahaemolyticus were cultured at different storage temperatures(including 4℃and 25℃,normal temperature),different storage time(0 h,2 h,3 h,and 6 h),and different broth enrichment time(0 h,8 h,12 h and 24 h)were cul-tured and the detection rate was recorded.Results There was no significant difference in the separation rates of the three pathogens(Salmonella,Shigella),and Vibrio parahaemolyticus when they were placed at 4℃and 25℃for 2 h re-spectively:95%vs 90%,60%vs 50%,85%vs 95%,P>0.05.The isolation rates of Salmonella and Vibrio parahaemolyticus showed no significant change when the three pathogens were placed at room temperature for 0 h,2 h,3 h and 6 h,respec-tively,while the isolation rates of Shigella showed a decreasing trend(75%,55%,20%,5%).The detection rate of three pathogens before enrichment(0 h)was 0;after enrichment for 8 h,12 h and 24 h,the detection rates of Salmonella were 90%,90%,and 95%,respectively;the detection rates of Shigella were 50%,20%,and 5%,respectively;the detection rate of Vibrio parahaemolyticus was all 95%;the detection rate increased after enrichment.The optimum enrichment time was 8~24 h for Salmonella and 8 h for Shigella when using Samana Shigella broth.The optimum enrichment time of Vibrio parahaemolyticus in alkaline peptone water was 8~24 h.The isolation rates of Shigella in the three groups were all low.Conclusion The separation rates of Salmonella,Shigella,and Vibrio parahaemolyticus could be significantly increased by inoculation of selective chromogenic medium at appropriate time after inoculation with broth.Placed at 4℃or 25℃and directly inoculated with selective medium within 6 h are suitable for stool samples with salmonella and Vibrio para-haemolyticus bacteria content greater than 10^(6) cfu/mL.Macconkey medium is not suitable for Shigella culture alone.