Assessing Microbe-Mineral Associations in Soil/Saprolite Using Confocal Laser-Scanning Microscopy and Image Analysis.

Microbe-mineral associations were evaluated with depth in soil/saprolite profiles from the Luquillo Experimental Forest of eastern Puerto Rico. Two cores of a ridgetop inceptisol were obtained by hand augering until point of refusal (4.7 m). Samples were aseptically removed for staining with BacLight and 3-D image capture by confocal laser scanning microscopy (CLSM). Images were processed with two different software programs. First, the COMSTAT package by A. Heydorn was used to calculate the area within each field showing above-threshold levels of green and red fluorescence, for live and damaged/dead cells respectively. Next, Image J from NIH was used to determine the area within each field occupied by mineral particles. We quantified microbe-mineral associations as the percent mineral area occupied by cells (%MAC) by dividing fluorescent-cell-area by mineral-surface-area. Live cell %MAC in soil (0-0.5 m), upper saprolite (0.5-2.6 m), and lower saprolite (2.6-4.6 m) were 13-17%, 1-4%, and <1%, respectively. In the deepest saprolite, immediately above weathering bedrock (4.56 m), the %MAC covered by live cells increased to 1%. Dead cell %MAC also decreased throughout the soil profile with 35.8 – 41.2%, 4.46 – 18.1%, and 0.74 – 2.9 %MAC in the soil, upper saprolite, and lower saprolite respectively. Dead %MAC increased to 6.0 % in saprolite above weathering bedrock. After visual scoring of minerals as biotite or quartz based on opacity/transparency, microbial cells were found to be consistently associated with biotite while the surfaces of quartz particles were virtually devoid of cells at each depth. Our procedure provides insights on patterns of microbe-minerals associations and has important implications regarding the use of microbial DNA rather than RNA for assessing community composition and function.