Broad diversity of viable bacteria in 'sterile' (0.2 microm) filtered water

Filtration of liquids through 0.2-microm filters is a common and often-used method for the removal of microorganisms from heat-sensitive solutions. Such 0.2 microm filtrations are frequently referred to as 'sterile filtration', reflecting the general belief that all living organisms are excluded from the filtrate. However, it has been demonstrated that a few bacterial species can pass through 0.2-microm filters. Despite these observations, it is generally assumed that most bacteria are unable to pass through these filters. In contrast to this assumption, a wide diversity of 19 bacterial taxa was isolated by the acclimatization method from 0.2 microm-filtrated freshwater samples. All isolates were able to grow on substrate-rich agar plates. The size of the isolated strains ranged from <1 to >10 microm in cell length. The obtained isolates belong to the Bacteroidetes, Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Spirochaetes. Previous studies which did not use the acclimatization method reported that only two out of the 19 taxa passed through 0.2- or 0.45-microm filters. The majority of the obtained isolates were more closely related to uncultured than to previously cultured bacteria. It is assumed that the obtained taxa represent only a small fraction of the bacteria able to pass through 0.2-microm filters, and it is concluded that microbiologists may have dramatically underestimated the diversity of 0.2 microm-filterable bacteria.