Methods and sources for the enrichment and isolation of budding, nonprosthecate bacteria from freshwater.

Methods are described for the observation, enrichment and isolation (from various freshwater samples) of bacteria of the generaPlanctomyces andPirella. Because immature buds were easily dislodged by shearing forces, slide culture techniques and direct microscopy of the budding process are recommended. An "auxanographic" technique to detect possible stimulation by soluble substrates was based on the diffusion of these substrates from peripherally placed crystals into the agar of a slide culture. Nearly every water sample investigated contained representatives of the generaPlanctomyces, Pirella, andBlastobacter, as well as budding cocci. Six enrichment techniques were tried; some enrichment experiments lasted several months. Allowing samples without added substrate to stand for a long time or generally employing nutrient-poor media were most successful. The "petri dish method," taking advantage of attachment of many budding bacteria to glass surfaces, was especially useful for increasing the numbers ofPlanctomyces spp. Pure cultures obtained from freshwater samples were tentatively placed in the generaPlanctomyces, Pirella, andBlastobacter. One strain appeared to represent a new genus of gram-positive, budding, and nonprosthecate bacteria.