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Abstract
The aim of this study was to establish a method for quantitative differential counting of live/dead anaerobic microorganisms. Artificial biofilms of Peptostreptococcus anaerobius 4330-1, one of the strictest anaerobes, were made on filters of 0.4 μm pore size. A commercial Live/Dead BacLight Bacterial Viability Kit using propidium iodide (PI) and SYTO9 fluorescent staining was found to be suitable for the facultatively anaerobic organism Propionibacterium acnes. However, P. anaerobius seemed to reduce SYTO9 and change fluorescent intensity, which made the kit unsuitable for this anaerobe. As an alternative method suitable for even P. anaerobius, a combination of phase-contrast microscopy and PI staining was adapted. Bacterial numbers could be counted in phase-contrast microscopic fields, and PI-positive organisms were counted as dead organisms in the same field. Only 3.8% of P. anaerobius cells in artificial biofilms were found to be PI-positive after exposure to air for 24 h, while 44% of planktonic P. anaerobius cells were PI-positive after exposure to air for 24 h. Similarly, 38% of P. anaerobius cells were PI-positive when organisms were collected onto filters to make bacterial masses and thereafter exposed to air for 24 h. These results indicate that P. anaerobius is highly aerotolerant in biofilms.