Chapter 2.2.2 : Assessment of Prokaryotic Biological Activity at the Single-Cell Level by Combining Microautoradiography and Fluorescence in situ Hybridization

Protocol overview. Major steps involved in labeling cells with FISH and MAR for interrogation about the capability of cells to be metabolic active when exposed to a generic (mixture of amino acids) or specific substrate in a natural mixed microbial community. doi:10.1128/9781555818821.ch2.2.2.f1

Imaging and quantification of group-specific microbial cells using STARFISH. Samples treated for STARFISH on a Teflon-coated cover glass can be analyzed by various fluorescence signals as well as transmitted light excitation. In this example, cells were tagged with three labels, (A) Cy3-tagged oligonucleotide probe targeting a group-specific prokaryote, (B) YO-PRO-1 DNA staining dye, and (C) 3H-substrate. Each label was detected and scored with a different light source. The Cy3-probe-labeled cells (Exmax 550 nm, Emmax 570 nm) provide records of the total number of cells belonging to the phylogenetic group the probe targets. YO-PRO-1 is a DNA-binding dye (Exmax 491 nm, Emmax 509 nm) that labels most viable cells, providing the total cell count (usually reported as cells/ml). Finally, the transmitted light shows the results from the radioactive nutrient incorporation into prokaryotic biomass, shown as the black markings on the photographic emulsion. By combining the three different microscopic field of views one can determine (i) (A + B) the percentage of a specific phylogenetic group in the community (counts in field A divided by counts in field B over the same area), in other words, the # Cy3-probe-labeled cells / # YO-PRO-1-labeled cells. In addition, (ii) (Panels B + C) the percentage of the total cells that take up the radioactive substrate can be quantified by the # 3H-positive cells / # YO-PRO-1-labeled cells. Finally, (iii) the distribution of uptake within each phylogenetic group ((# probe-labeled cells + # 3H-positive cells) / # YO-PRO-1-labeled cells). Notice that many of the radioactive marking (black dots) along the TM7 filaments are outside the cells themselves (Panels B + C). Such phenomenon is due to the radionuclide (beta participle from tritium) ability to travel a few microns away from the source. doi:10.1128/9781555818821.ch2.2.2.f2

Triple-labeled uncultured TM7 bacteria using STARFISH. The same field of view is shown in Fig. 2 and description of the three labels (a) Cy3-TM7905-probe targeting the 16S rRNA, (b) YO-PRO-1 DNA dye, and (b) trititated amino acids incorporated into bacterial biomass are described in detail in Fig. 2 legend. The black dots in Panel C indicate exposed silver grains in the photographic emulsion. Some silver grains do not seem to correlate to a bacterial cell labeled with either the YO-PRO-1 dye (b) or the Cy3-probe (a), possibly due to physical stress against the Kodak NTB2 liquid emulsion during the procedure as discussed in the text, which should be avoided. However, it is possible that the bacterial cells were small and not on the same focal plane as the silver grains since these clusters of cells form biofilms up to several microns in diameter. Images were captured at 1,000 × total magnification with a Zeiss Axioscope-A 1 epifluorescence microscope equipped with a Hamamatsu camera model ORCA R2 and AxioVision 4.7 image capturing software with frame averaging. Scale bar = 10 µm. doi:10.1128/9781555818821.ch2.2.2.f3