The fluorescent dyes TO-PRO-3 and TOTO-3 iodide allow detection of microbial ...
15.09.11
Fluorescence microscopy with nucleic acid.specific dyes, such as 4′,6-diamidino-2-phenylindole (DAPI) or acridine orange, is used widely to visualize microbial cells in environmental samples independently of their physiological state or phylogenetic placement ( 1 ). However, inefficient staining, nonspecific binding to sample components, and autofluorescence of mineral particles under UV excitation often interfere with efficient cell detection, in particular in soil and sediment samples ( 2 ). Novel high-affinity stains, such as SYBR Green, Sytox, or Syto dyes, promise better signal-to-background ratios due to their increased fluorescence when bound to DNA ( 3 - 5 ). Nevertheless, the general issue of nonspecific background Fluorescence remains problematic for many sample types. Excitation of nucleic acid–specific dyes with monochromatic light (e.g., in confocal laser scanning microscopy; CLSM) can reduce such background fluorescence, and CLSM is now often used for direct cell counts and fluorescence in situ hybridization (FISH) applications in environmental studies ( 2 , 6 ). However, many commonly used dyes require excitation in the UV range, for which confocal microscopes often are not properly equipped. In contrast, the monomeric TO-PRO-3 iodide (T P3; 642/661 nm excitation/emission peaks) and the dimeric TOTO-3 iodide (TT3; 6 42/660 nm) can be excited with a standard CLSM He-Ne 633 nm laser. While not assessed in the present study, they can also be used with light micro-scopes that are equipped for detection of red fluorescence (630–700 nm). These cyanine dyes display a high affinity for double-stranded nucleic acids in fi xed cells, fluorescing strongly as DNA-dye complexes but only weakly prior to DNA intercalation. TP3 is widely applied to stain DNA in eukaryotic cells (e.g., for flow cytometry, DNA quantification, or as a FISH counterstain) ( 7 - 9 ). Even though reports on using TP3 to stain prokaryotic cells are limited to flow cytometry of pure cultures ( 10 , 11 ), the weak fluorescence of unbound and nonspecifically bound dye molecules and the lower levels of autofluo-rescence with high wavelength excitation make TP3 and TT3 promising alterna-tives for the visualization of microbial cells in natural ecosystems. Here we present our results on the use of these dyes as both general nucleic acid stains for detecting microorganisms in mineral-rich soils and as FISH counterstains.
Source: BioTechniques.com