Detection of nitrifying bacteria in activated sludge by fluorescent in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for eubacteria (EUB338), ammonium-oxidizing bacteria (Nsm156) and nitrite-oxidizing bacteria (Nb1000) were used for the rapid detection of nitrifying bacteria in the activated sludge of a pilot nitrifying reactor by whole-cell, fluorescent in situ hybridization (FISH). Emission scanning and synchronous scanning fluorescence spectrometry were used to measure the hybridization. The binding of the probes at a temperature significantly lower than the melting temperature of the hybrids was conventionally considered as non-specific. Total binding of the probes at a temperature significantly higher than the melting temperature of the hybrids was conventionally considered as the sum of non-specific and specific binding (hybridization). Non-specific binding of the oligonucleotide probes with a biomass of activated sludge was 37% of the total binding of the EUB338 probe, 54% of the total binding of the Nsm156 probe, and 69% of the total binding of the Nb1000 probe. The ratio of the specific binding of the Nsm156 and Nb1000 probes was 2.3:1. The ratio of the numbers of ammonium-oxidizing bacteria to nitrite-oxidizing bacteria, determined by microbiological methods, was 2.4:1. Measuring fluorescent in situ hybridization by fluorescence spectrometry appears to be a practical tool for monitoring the microbial communities that contain nitrifying bacteria. However, a method that accounts for the non-specific binding of the probes more easily and reliably should be developed for practical application.     

Cellular localization of a pollen-specific mRNA by in situ hybridization and confocal laser scanning microscopy

Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen.     

Fluorescence in situ hybridization with specific oligonucleotide rRNA probes distinguishes the sibling species Stylonychia lemnae and Stylonychia mytilus (Ciliophora, Spirotrichea)

Based on morphological and morphogenetic characters alone, the sibling species Stylonychia lemnae and Stylonychia mytilus, members of the Stylonychia mytilus complex, can hardly be distinguished. However, biochemical investigations of the isoenzyme pattern of different enzymes showed a distinct differentiation between these two species. In the last few years, fluorescence in situ hybridization (FISH) techniques have become a suitable and reliable tool for identification and differentiation of closely related species of protozoa, such as ciliates. To distinguish the sibling species, a set of specific oligonucleotide probes were developed. In the present study, the SSU rDNA of 7 clones of Stylonychia lemnae and 13 clones of Stylonychia mytilus, isolated from different geographic regions, were sequenced. Comparing all SSU rDNA sequences of both species, only one single difference within the whole gene was detected. Based on this difference, a set of two oligonucleotide probes, targeting the SSU rRNA of each species (Stylonychia mytilus and Stylonychia lemnae) was designed. These probes were successfully tested by applying the FISH techniques on preserved cells of different clones of both species.     

Specific detection of Pseudomonas spp. in milk by fluorescence in situ hybridization using ribosomal RNA directed probes

AIMS: Pseudomonas spp. are considered the most important milk spoilage organisms. Here we describe development of a fluorescence in situ hybridization (FISH) probe specific for detection and enumeration of Pseudomonas spp. in milk. METHODS AND RESULTS: 16S rRNA sequences were analysed to develop specific oligonucleotide probe for the genus Pseudomonas. Twenty different Pseudomonas spp. and 23 bacterial species from genera other than Pseudomonas (as negative controls) were tested. All tested Pseudomonas spp. yielded a positive FISH reaction, whereas negative controls showed no FISH reaction except for Burkholderia cepacia that showed a relatively weak FISH reaction. The FISH assay specifically stains Pseudomonas in milk when the milk contains a mixture of other bacterial species. The FISH assay takes 2 h and compares favourably with current culturing methods, which take a minimum of 48 h. Specificity of the probe was validated using polymerase chain reaction to selectively amplifying the Pseudomonas rDNA gene and sequencing the gene products. CONCLUSIONS: The method presented in this study allows simultaneously detection, identification and enumeration of Pseudomonas spp. in milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and accurate enumeration of Pseudomonas facilitates the identification of specific contamination sources in dairy plants, the accurate validation of pasteurization treatments and the prediction of shelf life of processed milk.     

In situ identification and localization of bacteria associated with Gyrodinium instriatum (Gymnodiniales,Dinophyceae) by electron and confocal microscopy

The presence of intracellular bacteria in the dinoflagellate Gyrodinium instriatum Freudenthal & Lee has previously been described but the bacterial flora associated with this species has not been characterized. In this study, new results of transmission electron microscopy (TEM) and in situ hybridization using several bacterial group-specific oligonucleotide probes are presented. The long-term association of endocytoplasmic and endonuclear bacteria with G. instriatum has been confirmed. All endonuclear and most of the endocytoplasmic bacteria labelled were identified as belonging to the betaproteobacteria. Large clusters of Cytophaga-Flavobacterium-Bacteroides (CFB) were labelled and observed in the cytoplasm of the dinoflagellate cells, but were absent from the nucleus. Gammaproteobacteria were only observed outside the dinoflagellates. No alphaproteobacteria were detected either free-living or intracellular. Empirical observation of intracellular CFB reflected a degradation process of moribund dinoflagellate cells, whereas the systematic colonization of dinoflagellate nucleoplasm by betaproteobacteria suggested a true symbiotic relationship. Natural colonization may have occurred, perpetuated by vertical transmission of intracellular bacteria to the dinoflagellate daughter cells, via a pool of bacteria sequestered within the nucleus. Dividing bacteria were observed in the nucleus and equilibrium may be maintained by release of endonuclear bacteria to the cytoplasm through nuclear envelope constrictions.     

Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques

A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.     

Substrate uptake in extremely halophilic microbial communities revealed by microautoradiography and fluorescence in situ hybridization

The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR approach) was applied to brine samples of a solar saltern crystallizer pond from Mallorca (Spain) where the simultaneous occurrence of Salinibacter spp. and the conspicuous square Archaea had been detected. Radioactively labeled bicarbonate, acetate, glycerol, and an amino acid mixture were tested as substrates for the microbial populations inhabiting such brines. The results indicated that hitherto uncultured 'square Archaea' do actively incorporate amino acids and acetate. However, Salinibacter spp. only showed amino acid incorporation in pure culture, but no evidence of such activity in their natural environment could be demonstrated. No glycerol incorporation was observed for any component of the microbial community.Communicated by W.D. Grant     

A simple method for the production of bacterial controls for immunohistochemistry and fluorescent in situ hybridization

Immunohistochemical and fluorescent in situ hybridization (FISH) assays are useful diagnostic methods for the identification of bacteria on formalin fixed paraffin embedded histological sections. To validate an anti-bacterial antibody or an oligonucleotide probe and to ensure fidelity during subsequent analyses, suitable positive and negative controls are necessary. Suspensions of fixed bacteria are often used, but ideally, these controls should be fixed, embedded and processed in the same way of tissue samples under analysis. Herein, we describe a simple method for the production of bacterial histological control samples: the sandwich. The sandwich is composed of two external layers of equine lung parenchyma and a central layer of the target bacterium. We prepared sandwiches containing Escherichia coli, Campylobacter jejuni, and Arcanobacterium pyogenes and tested them with appropriate antibodies and Eub338 FISH probe. The sandwich is an effective and simple method to prepare bacterial histological controls fixed and processed in the same way as the diagnostic tissues.     

Localization of the T-DNA on marker chromosomes in transformed tobacco cells by in situ hybridization

Summary Chromosome and molecular analyses were conducted on tobacco cells which had been transformed by the T-DNA of the Ti-plasmid. These analyses showed that there were specific chromosome rearrangements in the transformed cells (marker chromosomes). There was a positive correlation between the number of marker chromosomes per cell and the oncogenic potential of the transformed cells. However, we show, using the Southern hybridization method, that the T_L fragment of T-DNA, but not the T_R, clearly hybridizes with nuclear DNA. In situ hybridization was used to locate the insertion site of T-DNA: the hybridization signal was found on a small metacentric chromosome. This chromosome may occur single or translocated onto other chromosomes, to make marker chromosomes. Thus, by locating the T-DNA, we have confirmed the correlation between the marker chromosomes and the oncogenic potential.     

Design of species-specific oligonucleotide probes for the detection of Bacteroides and Parabacteroides by fluorescence in situ hybridization and their application to the analysis of mouse caecal Bacteroides-Parabacteroides microbiota

Aims: To develop species‐specific monitoring techniques for rapid detection of Bacteroides and Parabacteroides inhabiting the mouse intestine by fluorescence in situ hybridization. Methods and Results: The specificity of oligonucleotide probes was evaluated by fluorescence whole‐cell hybridization. Oligonucleotide probes specific for each species hybridized only with the target bacteria. Using these probes, caecal Bacteroides–Parabacteroides microbiota of conventional mice and specific pathogen‐free (SPF) mice from three different breeders were analysed. It was shown that Bacteroides acidifaciens Group‐1, Group‐2 and Group‐3 were dominant in conventional mice and SPF mice from two out of three breeders. Bacteroides vulgatus and Parabacteroides distasonis were detected in one of these two SPF breeding colonies in addition to Bact. acidifaciens. SPF mice of the remaining breeder harboured characteristic Bacteroides–Parabacteroides microbiota, consisting of Bacteroides sp. ASF519 and Bacteroides caccae. Conclusions: Bacteroides acidifaciens is the dominant and most typical species in the mouse Bacteroides–Parabacteroides microbiota. The Group‐3 was identified as a novel group and revealed to occupy a major niche together with Bact. acidifaciens Group‐1 and Group‐2. Significance and Impact of the Study: The species‐specific probe set developed in this study was the efficient tool for rapid detection of target bacterial groups inhabiting the mouse intestine. The results of this study provide important new information on the mouse Bacteroides–Parabacteroides community.     

Development and field application of rRNA-targeted probes for the detection of Cochlodinium polykrikoides Margalef in Korean coastal waters using whole cell and sandwich hybridization formats

The dinoflagellate, Cochlodinium polykrikoides Margalef, has been responsible for mass mortalities of both wild and farmed fish along the Korean coast on virtually an annual basis since 1982. Economic impacts to the fishing and aquaculture industries are extensive, with a loss of USD $95 million reported in 1995 alone. The use of taxon-specific molecular probes for harmful algal species is recognized as a promising approach for the early detection of bloom formation and as part of an effective mitigation strategy. We have developed and successfully applied large subunit ribosomal RNA (LSU rRNA)-targeted probes in both whole cell and sandwich hybridization assay (SHA) formats for the species-specific detection of C. polykrikoides in Korean coastal waters. Sequences of the D1–D3 variable regions used to design probes were identical between five Korean and one Hong Kong C. polykrikoides isolates, while sequences for several N. American Cochlodinium isolates differed to varying degrees from the former. The automated SHA detected C. polykrikoides at levels as low as 1–3 cells/L in the field, demonstrating its suitability for detecting the target species at pre-bloom concentrations. This method should thus prove valuable to existing monitoring programs aimed at providing aquaculture interests with an early warning of frequently devastating bloom events.     

Ultrastructural localization of fibronectin mRNA in chick embryo by in situ hybridization using 35S or biotin labeled cDNA probes

We have studied the expression of the fibronectin gene in 7 day-old chick embryo (stage 32) by in situ hybridization at the light and electron microscope levels, using a 397 base-pairs chicken cDNA, labeled by radioisotope or biotin-11dUTP. Cryostat sections of whole chick embryos displayed a selective label on the upper layer of the dermis, fibrous sclera and mesenchymal cells but not on cartilagenous sclera cells. These results show that the expression of the fibronectin gene varies in relation to the morphogenetic events. Hybridization at the ultrastructural level on thin sections of sclera embedded in Lowicryl K4M showed a selective labelling on various cell compartments. Biotin-11dUTP and radiolabeled probes were compared. The labeling was found precisely on the membrane of the rough endoplasmic reticulum and on the nuclear envelope. A few silver grains were located on the nucleus and in the perinucleolar region. This study shows that the postembedding in situ hybridization is a powerful procedure to study the expression of the extracellular protein genes and gives further information on the localization of mRNA.     

Quantification of whole-cell in situ hybridization with oligonucleotide probes by flow cytometry of Escherichia coli cells

The use of fluorescence in situ hybridization (FISH) in conjunction with flow cytometry is a popular method of analysing environmental microbial populations. However, false-positive results can be produced if the specificity of oligonucleotide probe binding is not considered. An aim of this research was to evaluate the specificity of labelled oligonucleotide probe binding in FISH by flow cytometry. An excess of unlabelled probe was used to competitively inhibit the specific binding of labelled probe. Comparisons were made between the mean cell fluorescence and the number of fluorescently stained cells in a pure culture of Escherichia coli ATCC 53323. Specific binding of species-specific probes for the detection of E. coli was in the range 47–70% of total binding. A eukaryote probe and a nonsense probe, used as negative controls, had no specific binding with cells of E. coli. The significance of the results obtained is that the enumeration of specifically probe-bound microbial cells by FISH and flow cytometry must be made by an application of labelled and unlabelled probes to distinguish specifically stained cells. This is also a more practical method for the analysis of environmental samples compared to washing of excess non-specifically bound probe, due to the reduction of cell loss from the analysis.     

Cellular localization of heat shock gene expression in rabbit cerebellum by in situ hybridization with plastic-embedded tissue

The major cell types in rabbit cerebellum which engage in the expression of a heat shock gene (hsp 70) after hyperthermia were identified. This required in situ hybridization on thin sections derived from plastic-embedded tissue. All classes of cerebellar neurons which were examined (Purkinje, granule, and stellate cells) responded by induction of hsp 70 mRNA within 1 hr after hyperthermia. Prominent induction of hsp 70 mRNA was also observed in oligodendroglia in the deep white matter.     

Specific detection and quantitative enumeration of Listeria spp. using fluorescent in situ hybridization in combination with filter cultivation (FISHFC)

Aims:  To design a rapid specific method for enumeration of viable Listeria spp. using the fluorescence in situ hybridization with filter cultivation (FISHFC) method.
Methods and Results:  The probe, Lis-1400, was designed from the 23S rRNA region of the Listeria genome, and labelled with 5'-carboxy-tetramethyl-rhodamine- N - hydroxy-succinimide-ester. Fluorescence was observed for all Listeria species but not for any organisms from the other genera, suggesting Lis-1400 is highly specific for Listeria spp. For purposes of filter cultivation prior to hybridization, hydrophilic polypropylene membrane filters gave better contrast between fluorescing colonies and background fluorescence. This was because of a high S/N ratio (fluorescence intensity of each microcolony/fluorescence intensity of background noise) after FISH treatment. Results were achievable in 14 h using Lis-1400-aided FISHFC as compared with 4–7 days required for confirmation of Listeria spp. by conventional plate count methods. Moreover, viable Listeria counts in selected food samples showed no significant differences between Lis-1400-aided FISHFC and conventional methods.
Conclusions:  The Lis-1400-aided FISHFC method is more efficient than conventional methods for enumeration of viable Listeria spp. in food samples.
Significance and Impact of the Study:  For enumeration of Listeria spp., Lis-1400-aided FISHFC method is equally accurate yet faster than conventional plate count methods, and can be valuable in the control of listeriosis.     

Detection of chromosome variation in interphase by in situ hybridization with repetitive DNA probes: potential applications to cytogenetic analysis and mutagenicity testing

Individual chromosomes can be identified by means of in situ hybridization with DNA probes for chromosome-specific repetitive sequences. The efficiency and sensitivity of the method are strictly dependent on the characteristics of the probes and the experimental conditions. Using three probes with different copy numbers, we demonstrated that the target chromosomes can be visualized in interphase when the homologous sequences are repeated at least 50 times.Possible applications of interphase analysis to clinical cytogenetics and mutagenicity testing are discussed.