Vaahtovuo Jussi, Korkeamäki Mika, Munukka Eveliina, Viljanen Matti K, Toivanen Paavo
Turku University, Department of Medical Microbiology, Kiinamyllynkatu 13, 20520 Turku, Finland.
J Microbiol Methods. 2005 Dec;63(3):276-86. doi: 10.1016/j.mimet.2005.03.017. Epub 2005 Jun 1.
Hybridization of bacteria with fluorescent probes targeting 16S rRNA and inspection of hybridized bacteria with fluorescence microscopy (microscopy-FISH, i.e. fluorescence in situ hybridization) have constituted an accessible method for the analysis of mixed bacterial samples such as feces. However, microscopy-FISH is a slow method and prone to errors. Flow cytometry (FCM) enables analysis of bacteria more rapidly, accurately and reliably than microscopy. In this study, a FCM method for the analysis of 16S rRNA-hybridized and DNA-stained fecal bacteria was developed. The results of FCM-FISH were comparable to those of microscopy-FISH, and the coefficients of variation of the FCM analyses were extraordinarily low. In previous FCM-FISH studies, the Eub 338 probe, which is supposed to hybridize all bacteria, has been used to detect all bacteria present in the sample. We found that Eub 338 did not bind to all bacteria, which could be detected by DNA-staining; while SYTOX Orange DNA-stain detected all bacterial species tested and produced high fluorescence intensities enabling clear separation of bacteria from non-bacterial material. Thus, DNA-staining is a method of choice for the detection of all bacteria in FCM-FISH. We conclude that FCM of 16S rRNA-hybridized and DNA-stained bacteria is a rapid and reliable method for the analysis of mixed bacterial samples including feces.
用靶向16S rRNA的荧光探针使细菌杂交，并通过荧光显微镜检查杂交细菌（显微镜荧光原位杂交，即FISH），已成为一种可用于分析粪便等混合细菌样本的方法。然而，显微镜FISH是一种缓慢的方法，且容易出错。流式细胞术（FCM）比显微镜能更快速、准确和可靠地分析细菌。在本研究中，开发了一种用于分析16S rRNA杂交和DNA染色的粪便细菌的FCM方法。FCM-FISH的结果与显微镜FISH的结果相当，且FCM分析的变异系数极低。在以往的FCM-FISH研究中，假定能与所有细菌杂交的通用探针Eub 338已被用于检测样本中存在的所有细菌。我们发现Eub 338并非能与所有可通过DNA染色检测到的细菌结合；而SYTOX Orange DNA染色能检测所有测试的细菌种类，并产生高荧光强度，从而能够清晰地将细菌与非细菌物质区分开。因此，DNA染色是FCM-FISH中检测所有细菌的首选方法。我们得出结论，对16S rRNA杂交和DNA染色的细菌进行FCM分析是一种快速且可靠的方法，可用于分析包括粪便在内的混合细菌样本。
