van der Waaij L A, Mesander G, Limburg P C, van der Waaij D
Department of Medical Microbiology, University of Groningen, The Netherlands.
Cytometry. 1994 Jul 1;16(3):270-9. doi: 10.1002/cyto.990160312.
We describe a flow cytometry method for analysis of noncultured anaerobic bacteria present in human fecal suspensions. Nonbacterial fecal compounds, bacterial fragments, and large aggregates could be discriminated from bacteria by staining with propidium iodide (PI) and setting a discriminator on PI fluorescence and by exclusion of events with large forward scatter. Since anaerobic bacteria, which account for over 99.9% of all fecal bacteria, die during sample preparation, a fixation step was not necessary. A second aim of this study was to investigate the technical possibility of measurement of in vivo IgA coating of fecal anaerobic bacteria as well as their bacterial size. Fecal samples of 22 healthy human volunteers were analyzed. The fluorescence distribution of IgA-coated bacteria labeled with fluorescein isothiocyanate (FITC)-anti-Hu-IgA had overlap with noncoated bacteria. However, with match region subtraction, detection of low levels of specific FITC fluorescence on IgA-coated bacteria was achieved. The median bacterial two-dimensional surface area was 1.0 microns2. To validate flow cytometry data, all samples were analyzed with an image analysis system as well. With this new method, a rapid evaluation of fecal flora with high sensitivity for specific FITC fluorescence is possible without culturing.
我们描述了一种用于分析人粪便悬液中未培养厌氧菌的流式细胞术方法。通过用碘化丙啶（PI）染色，并根据PI荧光设置鉴别器，以及排除具有大前向散射的事件，可以将非细菌性粪便化合物、细菌碎片和大聚集体与细菌区分开来。由于占所有粪便细菌99.9%以上的厌氧菌在样品制备过程中死亡，因此无需固定步骤。本研究的第二个目的是探讨测量粪便厌氧菌体内IgA包被情况及其细菌大小的技术可能性。对22名健康人类志愿者的粪便样本进行了分析。用异硫氰酸荧光素（FITC）-抗人IgA标记的IgA包被细菌的荧光分布与未包被细菌有重叠。然而，通过匹配区域减法，实现了对IgA包被细菌上低水平特异性FITC荧光的检测。细菌二维表面积的中位数为1.0平方微米。为了验证流式细胞术数据，所有样本也用图像分析系统进行了分析。使用这种新方法，可以在不进行培养的情况下，对粪便菌群进行快速评估，且对特异性FITC荧光具有高灵敏度。
