FISH-FCM方法检测酵母-细菌二元体系中微生物数量

Simultaneous enumeration of yeasts and bacteria in binary samples by FISH-FCM

以废水生物处理系统和生物发酵系统出现的酵母-细菌二元体系作为研究对象，探讨了二元体系生物样品的最佳超声分散条件以及同时检测酵母和细菌数量时荧光原位杂交（FISH）-流式细胞术（FCM）技术的测量精度。染色-FCM检测表明：同时适用于混合酵母样品（酵母假菌丝）和混合细菌样品（活性污泥絮体）的最佳超声分散条件为100W、60～90s，但过强超声条件（120s）对酵母Candida tropicalis纯培养物（或细菌Escherichia coli纯培养物）的超声粉碎效果完全不同于混合酵母样品（或混合细菌样品）。在此基础上，以C．tropicalis和E．coli分别作为酵母和细菌的模式微生物，采用双探针杂交的FISH—FCM技术检测了背景微生物（C．tropicalis或Ecoll）浓度为10^7个／ml时二元体系中目标微生物（10^2～10^7个／ml）的数量。流式细胞仪能够明显区分噪音和二元体系中的酵母和细菌，因而一次进样时能够同时分析两种微生物数量，并且目标微生物浓度可以精确到10^4个／ml，此时微生物含量仅为0．1％，其中细菌浓度甚至可以精确到10^3个／ml（相应含量仅为0．01％）。然而，当二元体系中目标微生物浓度过低时（〈10^4个酵母／ml或〈10^4个细菌／ml），背景微生物的存在会严重影响FISH—FCM技术的测量准确性。

In this study, the enumeration of yeast and bacterial cells was investigated using fluorescent in situ hybridization （FISH） in combination with flow cytometry method （FCM） after disruption of pseudomycelia or flocs by sonication. Firstly, mixed yeast samples with developed pseudomycelia and mixed bacteria samples, i.e. activated sludge flocs, were sonicated and investigated using the FCM following the staining with fluorescent dyes. The optimal sonication condition for both biological samples was 60-90 s at 100W. Excessive sonication treatment, i.e. for 120 s, had different effects on pure cultures of bacteria （or yeasts ） and mixed samples of bacteria （or yeasts ）. Subsequently, the simultaneous enumeration of yeasts and bacteria in binary samples was investigated by FISH-FCM using two probes （PF2 ,EUB338）. The binary samples were prepared using Candida tropicalis and Escherichia coli as model yeast and bacteria microorganisms, respectively, and contained 10^7 cells/ml of background microorganisms and varied concentrations of the target microorganisms ranging from 10^2- 10^7 cells/ml. Flow cytometry could clearly distinguish yeasts, bacteria and background microorganisms, and one injection provided enumeration results for both target microorganisms simultaneously. FISH-FCM effectively measured concentrations of the target microorganisms as low as 10^4 cells/ml for yeasts and 10^3 cells/ml for bacteria, corresponding to 1% and 0.1%, respectively. However, at concentrations 〈 10^4 cells/ml for yeasts and 〈 10^3 cells/ml for bacteria, the target microorganisms could not be accurately enumerated due to the presence of background microorganisms in the binary sample.