An improved method for collecting heterotrophic microorganisms living on pebbles in streams

In microbiological studies in streams, pebble samples have until now been taken out of the water following the conventional method. However, this allows the loss of microorganisms as a result of the removal of overlying water. In the present study, to minimize the loss of microorganisms, we have developed a new sampling method, called the submerged method, for collecting microorganisms living on pebbles in streams. The abundance of microorganisms on natural pebbles and artificial clay tiles, both of which had biofilms developing on their surfaces, was measured using both the conventional and submerged methods and the results from the two methods were compared. The cell densities of bacteria (0.10–14.00 × 10⁸ cells cm^–2), heterotrophic nanoflagellates (0.36–50.30 × 10⁴ cells cm^–2), and ciliates (0.071–88.27 × 10² cells cm^–2) measured by the submerged method tended to be higher than those obtained by the conventional method, although there were only a few cases in which a significant difference existed between microbial abundances determined by the two methods. Also supported by microscopic observation, these results suggest the presence of planktonic and/or weakly attached microorganisms on substrate materials in streams. Significant correlations between the concentration of chlorophyll a and the cell densities of heterotrophic microorganisms and significant correlations among heterotrophic microorganisms suggest the presence of active microbial food webs in streams.