Application of the fluorescent-antibody technique to an ecological study of bacteria in soil

The fluorescent-antibody technique was used to identify cells and spores of Bacillus subtilis and cells of B. circulans from soil. From cells grown in three broth media of different nutrient status, i.e., a cold extracted soil medium (CSE), an unamended autoclaved soil extract (HSE), and nutrient broth (NB), antisera were produced with both quantitative and qualitative differences in antibody content. The specificities of antisera to two strains of each of the Bacillus species were determined. Antisera for B. subtilis O antigens were species-specific and showed no cross-reactions, whereas those for the B. circulans O antigens were strain-specific and in some cases showed cross-reactions with B. alvei. This cross-reaction was removed by absorption of the antiserum with B. alvei O antigen. Fluorescein isothiocyanate gamma-globulin conjugates prepared from these antisera showed the same specificity reactions. A method for staining bacteria on soil particles was developed, by use of small staining troughs. By mounting stained soil particles on slides and irradiating them with transmitted and incident ultraviolet blue light, bacteria on both mineral and organic particles, taken directly from soil, could be observed. Fluorescent antibodies against cells grown in CSE gave brighter fluorescence of stained bacteria on soil particles than did fluorescent antibodies against cells grown in either HSE or NB. Colonies of both Bacillus species were generally small and localized. Spore antisera, though not rigorously tested for specificity, were used to identify spores of B. subtilis on soil particles. The uses and implications of the technique in soil bacteriology are discussed.