An evaluation of rhodamine-labeled lysozyme as a fluorescent stain for in situ soil bacteria

Rhodamine-labeled lysozyme, in conjunction with fluorescein isothiocyanate conjugated to gelatin as a counterstain, was evaluated as a fluorescent stain for resident soil microorganisms. Preparations were observed by light-diffraction microscopy, transmitted incandescent-light microscopy, and reflected ultraviolet-fluorescence microscopy using a modified light-diffraction microscope, and by conventional transmitted incandescent and ultraviolet-fluorescence microscopy. Laboratory-grown microbial cultures superimposed on the resident soil microflora, as well as the non-dormant resident soil microorganisms, usually fluoresced properly when the stain and counterstain were applied to soil, and the blocking test with non-conjugated lysozyme was effective. What were assumed to be semidormant cells fluoresced to a lesser degree, and the blocking test was only partially effective. In contrast, the dormant resident soil microorganisms usually did not become stained by the methods used. A possible explanation is that a peripheral structural component surrounding many of the resident dormant cells in soil may have rendered their cell-wall substrate inaccessible to the enzyme conjugate.