Evaluation of the redox dye 5-cyano-2,3-tolyl-tetrazolium chloride for activity studies by simultaneous use of microautoradiography and fluorescence in situ hybridization

Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only 65% of the active Thiothrix cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells.     

Enumeration of Respiring Pseudomonas spp. in Milk within 6 Hours by Fluorescence In Situ Hybridization following Formazan Reduction

Respiring Pseudomonas spp. in milk were quantified within 6 h by fluorescence in situ hybridization (FISH) with vital staining. FISH with an oligonucleotide probe based on 16S rRNA sequences was used for the specific detection of Pseudomonas spp. at the single cell level. 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) was used to estimate bacterial respiratory activity. The numbers of respiring Pseudomonas cells as determined by FISH with CTC staining (CTC-FISH) were almost the same or higher than the numbers of CFU as determined by the conventional culture method.     

Novel Epibiotic Thiothrix Bacterium on a Marine Amphipod

Comparative analysis of the 16S rRNA gene and fluorescent in situ hybridization (FISH) was used to identify epibiotic filamentous bacteria living on the marine amphipod crustacean Urothoe poseidonis. The epibionts belong to the gamma proteobacteria and represent a novel marine phylotype within the genus Thiothrix. FISH and denaturing gradient gel electrophoresis revealed that the Thiothrix filaments are present on the majority of the amphipods examined.     

Enumeration of respiring Pseudomonas spp. in milk within 6 hours by fluorescence in situ hybridization following formazan reduction

Respiring Pseudomonas spp. in milk were quantified within 6 h by fluorescence in situ hybridization (FISH) with vital staining. FISH with an oligonucleotide probe based on 16S rRNA sequences was used for the specific detection of Pseudomonas spp. at the single cell level. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used to estimate bacterial respiratory activity. The numbers of respiring Pseudomonas cells as determined by FISH with CTC staining (CTC-FISH) were almost the same or higher than the numbers of CFU as determined by the conventional culture method.     

5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction in a mesophilic anaerobic digester: measuring redox behavior,differentiating abiotic reduction,and comparing FISH response as an activity indicator

The tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) has been widely applied to assess microbiological activity in environmental samples. CTC reduction has previously been quantified in a variety of anaerobic systems (i.e., fermentative, nitrate reducing, sulfate reducing) using direct microscopy, solvent extraction, and flow cytometry. In this work, extracellular CTC reduction was observed and distinguished from its intercellular counterparts by the amorphous character and near uniform fluorescence of the resulting formazan precipitates (CTF). Fluorescence yielded by non-cellular-associated formazan precipitates bleached much more rapidly than CTF formed within cells under identical UV exposure (<2 min). Dehydrogenase activity assays and fluorescent in situ hybridization (FISH) were simultaneously carried out in microcosms containing active anaerobic digester biomass, propylene glycol, and settled sewage centrate for direct comparison. In substrate limited microcosms, quantitative FISH measurements remained well above their detection limit indicating sustained intercellular ribosomal RNA concentrations over a 5-day period, while dehydrogenase assays (CTC) decreased to background levels within 14 h of substrate limitation. Results from this work suggest that CTC reduction in cell-free samples may impede accurate enzyme activity measurements, particularly when quantification involves solvent extraction, flow cytometry, or software-aided counting. In addition, activity assessment in anaerobic digesters using FISH and CTC reduction assays may be comparable until substrate becomes limited.     

Microbial Diversity within Early-Stage Cultured Panulirus ornatus Phyllosomas

A thorough understanding of the microorganisms and pathogens associated with the larval stage of the tropical ornate rock lobster, Panulirus ornatus, is required to overcome disease outbreaks that currently block aquaculture attempts. This study used microscopy in addition to culture and molecularly based microbiological techniques to characterize the bacterial community associated with cultured, developmental stage PI to PII P. ornatus phyllosomas. Scanning electron microscopy demonstrated colonization of phyllosomas by filamentous, rod-shaped, and coccus-shaped bacteria. A clone library constructed from dead phyllosomas sampled from the larval rearing tank on day 10 was dominated by Thiothrix-affiliated sequences (56% of clones). A comparable library from live phyllosomas also contained Thiothrix-affiliated sequences, though these only represented 19% of clones within the library. Fluorescent in situ hybridization (FISH) confirmed identification of the filamentous bacteria as Thiothrix sp., being present on dead phyllosomas. FISH also identified Leucothrix sp. and Vibrio sp., as well as a range of other rod- and coccus-shaped bacteria, colonizing both live and dead phyllosomas. The development of the microbial community associated with phyllosomas was monitored through a standard larval rearing run using denaturing gradient gel electrophoresis (DGGE). Vibrio sp.-affiliated bands dominated the profiles of live animals through the rearing period and dead phyllosomas sampled on selected days. The population of Vibrio sp. associated with phyllosomas was monitored with culture-based analysis on selective media and demonstrated to increase significantly on day 7, coinciding with the beginning of the larval molt. An isolated Vibrio harveyi strain demonstrated an identical 16S rRNA sequence with retrieved DGGE and clone library sequences. Colonization of phyllosomas with filamentous bacterial species potentially hinders the ability of the animals to molt and, combined with the added stress of the molt process, likely results in reduced immune function, allowing opportunistic pathogenic Vibrio sp. to cause larval mortalities.     

In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.     

Isotope Labeling and Microautoradiography of Active Heterotrophic Bacteria on the Basis of Assimilation of 14CO2

Most heterotrophic bacteria assimilate CO₂ in various carboxylation reactions during biosynthesis. In this study, assimilation of ¹⁴CO₂ by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient ¹⁴CO₂ during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of ¹⁴C-labeled organic substrates. Experiments with E. coli showed that ¹⁴CO₂ was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO₂-MAR, was evaluated by targeting metabolic active filamentous bacteria, including “Candidatus Microthrix parvicella” in activated sludge. “Ca. Microthrix parvicella” was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [¹⁴C]oleic acid. However, the new HetCO₂-MAR approach indicated that “Ca. Microthrix parvicella,” did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO₂⁻, whereas the addition of O₂ or NO₃⁻ initiated growth, as indicated by detectable ¹⁴CO₂ assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO₂-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO₂-MAR results were supported by stable isotope analysis of ¹³C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of ¹³CO₂. In conclusion, the novel HetCO₂-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.     

Phenotypic Properties and Microbial Diversity of Methanogenic Granules from a Full-Scale Upflow Anaerobic Sludge Bed Reactor Treating Brewery Wastewater

Methanogenic granules from an anaerobic bioreactor that treated wastewater of a beer brewery consisted of different morphological types of granules. In this study, the microbial compositions of the different granules were analyzed by molecular microbiological techniques: cloning, denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH), and scanning and transmission electron microscopy. We propose here that the different types of granules reflect the different stages in the life cycle of granules. Young granules were small, black, and compact and harbored active cells. Gray granules were the most abundant granules. These granules have a multilayer structure with channels and void areas. The core was composed of dead or starving cells with low activity. The brown granules, which were the largest granules, showed a loose and amorphous structure with big channels that resulted in fractured zones and corresponded to the older granules. Firmicutes (as determined by FISH) and Nitrospira and Deferribacteres (as determined by cloning and sequencing) were the predominant Bacteria. Remarkably, Firmicutes could not be detected in the brown granules. The methanogenic Archaea identified were Methanosaeta concilii (70 to 90% by FISH and cloning), Methanosarcina mazei, and Methanospirillum spp. The phenotypic appearance of the granules reflected the physiological condition of the granules. This may be valuable to easily select appropriate seed sludges to start up other reactors.     

Phylogenetic in situ/ex situ analysis of a sulfur mat microbial community from a thermal sulfide spring in the north Caucasus

A phylogenetic in situ/ex situ analysis of a sulfur mat formed by colorless filamentous sulfur bacteria in a thermal sulfide spring (northern spur of the main Caucasian ridge) was carried out. Nine phylotypes were revealed in the mat. Thiothrix sp. and Sphaerotilus sp. were the dominant phylotypes (66.3% and 26.3%, respectively). The 16S rRNA gene nucleotide sequence of Sphaerotilus sp. phylotype from the clone library was identical to the sequences of the seven Sphaerotilus strains isolated from the same source. A very high degree of similarity of Sphaerotilus strains revealed by ERIC-PCR fingerprints indicated little or no population diversity of this species in the mat. Thiothrix phylotype from the clone library and two Thiothrix strains isolated from the same mat sample differed in one to three nucleotides of 16S rRNA genes; this is an indication of this organism’s population variability in the mat. 16S rRNA genes of the strains and clones of Thiothrix sp. exhibited the highest similarity (ca. 99%) with Thiothrix unzii; the strains and clones of Sphaerotilus had 99% similarity with the type species Sphaerotilus natans (the only species of this genus) and therefore can be assigned to this species. The minor seven components belong to the phylotypes from the Proteobacteria (3%), as well as the Chlorobia, Cyanobacteria, Clostridia, and Bacteroidetes phylogenetic groups, each of them constituting not more than 1%. Intracellular accumulation of elemental sulfur by Sphaerotilus similar to other filamentous sulfur bacteria was demonstrated for the first time (both in the population of the sulfur spring and in cultures with sulfide). Although mass growth of Sphaerotilus and Thiothrix is typical of bacterial populations of anthropogenic ecosystems (the activated sludge of treatment facilities), stable communities of these bacteria have not been previously found in the sulfur mats or “threads” of natural sulfide springs.     

Bacterial Activity at −2 to −20°C in Arctic Wintertime Sea Ice

Arctic wintertime sea-ice cores, characterized by a temperature gradient of −2 to −20°C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4′,6′-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O₂-based respiration), the abundances of total, particle-associated (>3-μm), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (−2 to −20°C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and Archaea. Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (−20°C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to −20°C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.     

Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes

In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The mcrA gene and the apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (> 98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the mcrA or apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences.     

Changes in Activity and Community Structure of Methane-Oxidizing Bacteria over the Growth Period of Rice

The activity and community structure of methanotrophs in compartmented microcosms were investigated over the growth period of rice plants. In situ methane oxidation was important only during the vegetative growth phase of the plants and later became negligible. The in situ activity was not directly correlated with methanotrophic cell counts, which increased even after the decrease in in situ activity, possibly due to the presence of both vegetative cells and resting stages. By dividing the microcosms into two soil and two root compartments it was possible to locate methanotrophic growth and activity, which was greatest in the rhizoplane of the rice plants. Molecular analysis by denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH) with family-specific probes revealed the presence of both families of methanotrophs in soil and root compartments over the whole season. Changes in community structure were detected only for members of the Methylococcaceae and could be associated only with changes in the genus Methylobacter and not with changes in the dominance of different genera in the family Methylococcaceae. For the family Methylocystaceae stable communities in all compartments for the whole season were observed. FISH analysis revealed evidence of in situ dominance of the Methylocystaceae in all compartments. The numbers of Methylococcaceae cells were relatively high only in the rhizoplane, demonstrating the importance of rice roots for growth and maintenance of methanotrophic diversity in the soil.     

Identification of active denitrifiers in full‐scale nutrient removal wastewater treatment systems

Denitrification is essential to the removal of nitrogen from wastewater during treatment, yet an understanding of the diversity of the active denitrifying bacteria responsible in full‐scale wastewater treatment plants (WWTPs) is lacking. In this study, stable‐isotope probing (SIP) was applied in combination with microautoradiography (MAR)‐fluorescence in situ hybridization (FISH) to identify previously unrecognized active denitrifying phylotypes in a full‐scale WWTP with biological N and P removal. Acknowledging that different denitrifiers will have specific carbon source preferences, a fully ¹³C‐labelled complex substrate was used for SIP incubations, under nitrite‐reducing conditions, in order to maximize the capture of the potentially metabolically diverse denitrifiers likely present. Members of the Rhodoferax, Dechloromonas, Sulfuritalea, Haliangium and Thermomonas were represented in the 16S rRNA gene clone libraries from DNA enriched in ¹³C, with FISH probes optimized here for their in situ characterization. FISH and MAR confirmed that they were all active denitrifiers in the community. The combined approach of SIP and MAR‐FISH represents an excellent approach for identifying and characterizing an un‐described diversity of active denitrifiers in full‐scale systems.     

Molecular phylogeny and taxonomy of colorless, filamentous sulfur bacteria of the genus Thiothrix

Comprehensive investigation combining molecular genetic techniques and comparative studies of morphological and physiological properties made it possible to resolve the disputed issue of the taxonomic status of the groups ??T. nivea?? and ??Eikelboom type 021N?? of the genus Thiothrix. The phylogenetic trees constructed on the basis of 16S rRNA and gyrB gene sequences demonstrated that members of the genus Thiothrix formed a cluster within the order Thiotrichales. According to the ??ribosomal?? tree, the cluster of the genus Thiothrix was divided into two main groups, I and II, corresponding to the groups ??T. nivea?? and ??Eikelboom type 021N??. The levels of similarity between the 16S rRNA gene sequences of Thiothrix species reached 88.9?C100%. On the contrary, in the ??gyrase?? tree, these species were not divided into ??T. nivea?? and ??Eikelboom type 021N?? groups. The levels of similarity between the amino acid sequences of the gyrB gene fragments of Thiothrix species varied from 74.5 to 99.2%. Importantly, members of the groups ??T. nivea?? and ??Eikelboom type 021N?? formed very similar 16S rRNA secondary structures in the variable region V3, where a 30-nucleotide deletion characteristic of all Thiothrix species was detected. Phenotypic analysis of the studied bacteria revealed some morphological and physiological properties shared by the groups ??T. nivea?? and ??Eikelboom type 021N??. The data obtained indicate that members of the groups ??T. nivea?? and ??Eikelboom type 021N?? are phenotypically and genetically heterogeneous species within the single monophyletic genus Thiothrix..     

In situ substrate conversion and assimilation by nitrifying bacteria in a model biofilm

Local nitrification and carbon assimilation activities were studied in situ in a model biofilm to investigate carbon yields and contribution of distinct populations to these activities. Immobilized microcolonies (related to Nitrosomonas europaea/eutropha, Nitrosomonas oligotropha, Nitrospira sp., and to other Bacteria) were incubated with [14C]-bicarbonate under different experimental conditions. Nitrifying activity was measured concomitantly with microsensors (oxygen, ammonium, nitrite, nitrate). Biofilm thin sections were subjected to fluorescence in situ hybridization (FISH), microautoradiography (MAR), and local quantification of [14C]-bicarbonate uptake (beta microimaging). Nitrifying activity and tracer assimilation were restricted to a surface layer of different thickness in the various experiments (substrate or oxygen limitation). Excess oxygen uptake under all conditions revealed heterotrophic activity fuelled by decay or excretion products during active nitrification. Depth limits and intensity of tracer incorporation profiles were in agreement with ammonia-oxidation activity (measured with microsensors), and distribution of incorporated tracer (detected with MAR). Microautoradiography revealed a sharp individual response of distinct populations in terms of in-/activity depending on the (local) environmental conditions within the biofilm. Net in situ carbon yields on N, expressed as e- equivalent ratios, varied between 0.005 and 0.018, and, thus, were in the lower range of data reported for pure cultures of nitrifiers.     

Isotope labeling and microautoradiography of active heterotrophic bacteria on the basis of assimilation of 14CO(2)

Most heterotrophic bacteria assimilate CO(2) in various carboxylation reactions during biosynthesis. In this study, assimilation of (14)CO(2) by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient (14)CO(2) during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of (14)C-labeled organic substrates. Experiments with E. coli showed that (14)CO(2) was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO(2)-MAR, was evaluated by targeting metabolic active filamentous bacteria, including "Candidatus Microthrix parvicella" in activated sludge. "Ca. Microthrix parvicella" was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [(14)C]oleic acid. However, the new HetCO(2)-MAR approach indicated that "Ca. Microthrix parvicella," did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO(2)(-), whereas the addition of O(2) or NO(3)(-) initiated growth, as indicated by detectable (14)CO(2) assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO(2)-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO(2)-MAR results were supported by stable isotope analysis of (13)C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of (13)CO(2). In conclusion, the novel HetCO(2)-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.     

Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. β-, γ-, δ-, and -proteobacteria in sulfur-cycling clades accounted for ≥75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating δ-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous γ-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of -proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.     

Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.