DIRECT FLOW-CYTOMETRY OF ANAEROBIC-BACTERIA IN HUMAN FECES

We describe a flow cytometry method for analysis of noncultured anaero bic bacteria present in human fecal suspensions. Nonbacterial fecal co mpounds, bacterial fragments, and large aggregates could be discrimina ted from bacteria by staining with propidium iodide (PI) and setting a discriminator on PI fluorescence and by exclusion of events with larg e forward scatter. Since anaerobic bacteria, which account for over 99 .9% of all fecal bacteria, die during sample preparation, a fixation s tep was not necessary. A second aim of this study was to investigate t he technical possibility of measurement of in vivo IgA coating of feca l anaerobic bacteria as well as their bacterial size. Fecal samples of 22 healthy human volunteers were analyzed. The fluorescence distribut ion of IgA-coated bacteria labeled with fluorescein isothiocyanate (FI TC)-anti-Hu-IgA had overlap with noncoated bacteria. However, with mat ch region subtraction, detection of low levels of specific FITC fluore scence on IgA-coated bacteria was achieved. The median bacterial two-d imensional surface area was 1.0 mu m(2). To validate flow cytometry da ta, all samples were analyzed with an image analysis system as well. W ith this new method, a rapid evaluation of fecal flora with high sensi tivity for specific FITC fluorescence is possible without culturing. ( C) 1994 Wiley-Liss, Inc.