CTC staining and counting of actively respiring bacteria in natural stone using confocal laser scanning microscopy.

A method was established for staining and counting of actively respiring bacteria in natural stone by using the tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride (CTC) in combination with confocal laser scanning microscopy (CLSM). Applying 5 mM CTC for 2 h to pure cultures of representative stone-inhabiting microorganisms showed that chemoorganotrophic bacteria and fungi-in contrast to lithoautotrophic nitrifying bacteria-were able to reduce CTC to CTF, the red fluorescing formazan crystals of CTC. Optimal staining conditions for microorganisms in stone material were found to be 15 mM CTC applied for 24 h. The cells could be visualized on transparent and nontransparent mineral materials by means of CLSM. A semi-automated method was used to count the cells within the pore system of the stone. The percentage of CTC-stained bacteria was dependent on temperature and humidity of the material. At 28 degrees C and high humidity (maximum water holding capacity) in the laboratory, about 58% of the total bacterial microflora was active. On natural stone exposed for 9 years at an urban exposure site in Germany, 52-56% of the bacterial microflora was active at the east, west, and north side of the specimen, while only 18% cells were active at the south side. This is consistent with microclimatic differences on the south side which was more exposed to sunshine thus causing UV and water stress as well as higher temperatures on a microscale level. In combination with CLSM, staining by CTC can be used as a fast method for monitoring the metabolic activity of chemoorganotrophic bacteria in monuments, buildings of historic interest or any art objects of natural stone. Due to the small size of samples required, the damage to these objects and buildings can be minimized.

Immunological detection of Nitrospira-like bacteria in various soils.

Chemolithotrophic nitrite oxidizers were enriched from five different soils including freshwater marsh, permafrost, garden, agricultural, and desert soils and monitored during the cultivation procedure. Immunoblot analysis was used to identify the nitrite oxidizing organisms with monoclonal antibodies, which recognize the key enzyme of nitrite oxidation in a genus-specific reaction [Bartosch et al. (1999) Appl Environ Microbiol 65:4126-4133]. The morphological characteristics of the enriched nitrite oxidizers were additionally studied using transmission electron microscopy (TEM) and fluorescence microscopy. By means of the antibodies and TEM analysis Nitrospira could be clearly identified in enrichment cultures derived from freshwater marsh and from permafrost soil. Nitrospira cells were enriched simultaneously with cells of the genus Nitrobacter when nitrite concentrations of 0.2 g of NaNO2 L(-1) were used. However, in enrichment cultures containing 2 g of NaNO2 L(-1) Nitrobacter was exclusively detected. During fluorescence microscopic observations of DAPI stained samples microcolonies were found in enrichment cultures from freshwater marsh, permafrost, garden, and agricultural soil. They had a similar morphology to Nitrospira-like microcolonies from activated sludge. In conclusion, Nitrospira seems to be not only a common aquatic but also a usual soil bacterium.

Identification of nitrite-oxidizing bacteria with monoclonal antibodies recognizing the nitrite oxidoreductase.

Immunoblot analyses performed with three monoclonal antibodies (MAbs) that recognized the nitrite oxidoreductase (NOR) of the genus Nitrobacter were used for taxonomic investigations of nitrite oxidizers. We found that these MAbs were able to detect the nitrite-oxidizing systems (NOS) of the genera Nitrospira, Nitrococcus, and Nitrospina. The MAb designated Hyb 153-2, which recognized the alpha subunit of the NOR (alpha-NOR), was specific for species belonging to the genus Nitrobacter. In contrast, Hyb 153-3, which recognized the beta-NOR, reacted with nitrite oxidizers of the four genera. Hyb 153-1, which also recognized the beta-NOR, bound to members of the genera Nitrobacter and Nitrococcus. The molecular masses of the beta-NOR of the genus Nitrobacter and the beta subunit of the NOS (beta-NOS) of the genus Nitrococcus were identical (65 kDa). In contrast, the molecular masses of the beta-NOS of the genera Nitrospina and Nitrospira were different (48 and 46 kDa). When the genus-specific reactions of the MAbs were correlated with 16S rRNA sequences, they reflected the phylogenetic relationships among the nitrite oxidizers. The specific reactions of the MAbs allowed us to classify novel isolates and nitrite oxidizers in enrichment cultures at the genus level. In ecological studies the immunoblot analyses demonstrated that Nitrobacter or Nitrospira cells could be enriched from activated sludge by using various substrate concentrations. Fluorescence in situ hybridization and electron microscopic analyses confirmed these results. Permeated cells of pure cultures of members of the four genera were suitable for immunofluorescence labeling; these cells exhibited fluorescence signals that were consistent with the location of the NOS.