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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria.
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Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria.

机译：流式细胞仪和荧光原位杂交技术在空气传播细菌暴露评估中的应用。

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Current limitations in the methodology for enumeration and identification of airborne bacteria compromise the precision and accuracy of bioaerosol exposure assessment. In this study, flow cytometry and fluorescent in situ hybridization (FISH) were evaluated for the assessment of exposures to airborne bacteria. Laboratory-generated two-component bioaerosols in exposures chambers and complex native bioaerosols in swine barns were sampled with two types of liquid impingers (all-glass impinger-30 and May 3-stage impinger). Aliquots of collection media were processed and enumerated by a standard culture technique, microscopy, or flow cytometry after nucleic acid staining with 4',6-diamidino-2-phenylindole (DAPI) and identified taxonomically by FISH. DAPI-labeled impinger samples yielded comparable estimates of bioaerosol concentrations when enumerated by microscopy or flow cytometry. The standard culture method underestimated bioaerosol concentrations by 2 orders of magnitude when compared to microscopy or flow cytometry. In the FISH method, aliquots of collection media were incubated with a probe universally complementary to eubacteria, a probe specific for several Pseudomonas species, and a probe complementary to eubacteria for detection of nonspecific binding. With these probes, FISH allowed quantitative identification of Pseudomonas aeruginosa and Escherichia coli bioaerosols in the exposure chamber without measurable nonspecific binding. Impinger samples from the swine barn demonstrated the efficacy of the FISH method for the identification of eubacteria in a complex organic dust. This work demonstrates the potential of emerging molecular techniques to complement traditional methods of bioaerosol exposure assessment.
机译：空气中细菌计数和鉴定方法的当前限制损害了生物气溶胶暴露评估的准确性和准确性。在这项研究中，流式细胞仪和荧光原位杂交（FISH）被评估为评估空气传播细菌的暴露。用两种类型的液体撞击器（全玻璃撞击器30和5月3级撞击器）对实验室在暴露室产生的两组分生物气溶胶和猪舍中复杂的天然生物气溶胶进行采样。用4'，6-二dia基-2-苯基吲哚（DAPI）进行核酸染色后，通过标准培养技术，显微镜或流式细胞术对收集培养基的等分试样进行处理和计数，并通过FISH分类鉴定。当通过显微镜或流式细胞术计数时，DAPI标记的撞击器样品产生了可比的生物气溶胶浓度估算值。与显微镜或流式细胞术相比，标准培养方法低估了2个数量级的生物气溶胶浓度。在FISH方法中，将收集培养基的等分试样与普遍与真细菌互补的探针，对几种假单胞菌物种具有特异性的探针以及与真细菌互补的探针进行温育，以检测非特异性结合。使用这些探针，FISH可以在暴露室中定量鉴定铜绿假单胞菌和大肠杆菌生物气溶胶，而没有可测量的非特异性结合。来自猪舍的撞击器样品证明了FISH方法在复杂有机粉尘中鉴定真细菌的功效。这项工作证明了新兴的分子技术可以补充传统的生物气溶胶暴露评估方法。

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