The method of the invention is directed to the novel use of a diffusion chamber within which previously "uncultivatible" microorganisms can be isolated. Rather than attempting to replicate the natural environment of an unknown microorganism, the method of the invention provides for exposing dividing microorganisms to all the components of the original environment while simultaneously containing the resulting colonies so that they can be isolated. The method of the invention can take advantage of the recognition that the preponderance of difficult-to-grow microorganisms do not form colonies visible to the naked eye. Therefore, these organisms must be isolated under a compound microscope as "microcolonies." In addition, methods according to the invention permit the isolation of novel microorganisms capable of growing in artificial media only in co-culture in the presence of a companion microorganism.