Quantification of Leuconostoc populations in mixed dairy starter cultures using fluorescence in situ hybridization

AIMS: Development of a rapid method to identify and quantify Leuconostoc populations in mesophilic starter cultures. METHODS AND RESULTS: 16S rRNA-targeted oligonucleotide probes were used in a whole cell in situ hybridization assay for the identification of the genus Leuconostoc and an undescribed Leuconostoc ribospecies. The probes were fluorescently labelled and used to quantify the Leuconostoc populations in five different mixed starter cultures. CONCLUSIONS: There was a good correlation between the results obtained using fluorescence in situ hybridization (FISH) with that of standard plate counting methods. SIGNIFICANCE AND IMPACT OF THE STUDY: To develop a FISH method capable of identifying and quantifying the Leuconostoc population in starter cultures within 1 day.     

Detection of ingested bacteria in benthic ciliates using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was applied to detect ingested natural bacteria within the food vacuoles of ciliates harvested from the natural sediment. In addition to this important qualitative aspect, FISH was also successfully used to measure the bacterivory of a culture of the ciliate Tetrahymena pyriformis on natural field sediment bacteria. In this feeding experiment, we compared the FISH technique with the only available alternative technique using fluorescently stained sediment (FS-sediment). The ingestion rate of unstained sediment bacteria determined by FISH was 4.6 bacteria per ciliate and hour. In contrast, Tetrahymena pyriformis cells that fed on bacteria from FS-sediment ingested 12.7 bacteria per ciliate and hour. Bacterial abundances in the sediment were equal in both sediment types (4 x 10(8) cells g sediment dry weight(-1)) when determined by DAPI counts. However, when analyzed using DTAF-counts, the number of bacteria in the FS-sediment increased to 9.7 x 10(8) cells g sediment dry weight(-1). From our findings we conclude that bacterivory by ciliates is overestimated when FS-sediment is used because DTAF stains bacteria as well as protein-containing detritus particles, which are also ingested by many ciliates. In contrast, FISH is a direct, a posteriori method that specifically stains phylogenetic lineages, e.g. eubacteria, after ingestion and thereby avoids a false determination of the number of ingested bacteria. Thus this method can also be used for the study of natural ciliate bacterivory in benthic systems.     

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.     

Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample

AIMS: A fluorescent in situ hybridization (FISH) technique using an Enterobacteriaceae-specific probe (probe D) to target 16S rRNA was improved in order to enumerate, within a single working day, Enterobacteriaceae present in food and environmental water samples. METHODS AND RESULTS: In order to minimize the time required for the FISH procedure, each step of FISH with probe D was re-evaluated using cultured Escherichia coli. Five minutes of ethanol treatment for cell fixation and hybridization were sufficient to visualize cultured E. coli, and FISH could be performed within 1 h. Because of the difficulties in detecting low levels of bacterial cells by FISH without cultivation, a FISH technique for detecting microcolonies on membrane filters was investigated to improve the bacterial detection limit. FISH with probe D following 6 h of cultivation to grow microcolonies on a 13 mm diameter membrane filter was performed, and whole Enterobacteriaceae microcolonies on the filter were then detected and enumerated by manual epifluorescence microscopic scanning at magnification of x100 in ca 5 min. The total time for FISH with probe D following cultivation (FISHFC) was reduced to within 7 h. FISHFC can be applied to enumerate cultivable Enterobacteriaceae in food (above 100 cells g-1) and environmental water samples (above 1 cell ml-1). CONCLUSIONS: Cultivable Enterobacteriaceae in food and water samples were enumerated accurately within 7 h using the FISHFC method. SIGNIFICANCE AND IMPACT OF THE STUDY: A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.     

Ontogeny of gonadotropin-releasing hormone (GnRH) neurons in hybrid striped bass Morone sp.: whole-mount in situ hybridization analysis

The ontogeny of gonadotropin-releasing hormone (GnRH) mRNA-producing neurons was studied in developing hybrid striped bass (white bass Morone chrysops female × striped bass Morone saxatilis male), 1–55 days post-fertilization (dpf), by whole-mount in situ hybridization. Neurons that produce salmon (s) GnRH were first detected at 32 h post-fertilization in the olfactory placodes. These neurons migrated posteriorly during development and reached their final position at the olfactory bulbs-telencephalon boundary, possibly the terminal nerve ganglion (TNg) by 11 dpf. First signal of chicken (c) GnRH-II neurons appeared in the midbrain 2 dpf and remained there throughout development. A signal of seabream (sb) GnRH mRNA was first detected at 21 dpf in the preoptic area (POA) and as a bilateral continuum along the ventral telencephalon at 32–55 dpf. The expression of all three forms of GnRH increased throughout development. These results suggest that cGnRH-II neurons originate in the mid-brain, and that sGnRH neurons originate in the olfactory placodes and migrate caudally to the TNg. Neurons that express sbGnRH seem to originate at the preoptic area and then to migrate anteriorly along the ventral telencephalon. An olfactory placodal origin of these neurons, however, cannot be ruled out.     

Ultrastructural reinvestigation of the trophosome in adults of Riftia pachyptila (Annelida, Siboglinidae)

Abstract. The trophosome of adults of Riftia pachyptila (Vestimentifera) was reinvestigated using 3-dimensional ultrastructural reconstruction and quantitative morphological analysis. The symbionts make up 24.1%, the symbiont-containing cells (bacteriocytes) are 70.5% of the trophosome's volume. The trophosome is composed of lobules that have a central axial blood vessel surrounded by a myoepithelium containing bacteriocytes, in turn surrounded by an apolar tissue of bacteriocytes. Part of the splanchnic peritoneum lining the trunk coelom encases the bacteriocytes and forms a ramifying network of peripheral blood vessels. Based on the morphology and ultrastructure of the adult, we hypothesize a mesodermal rather than endodermal origin of trophosome and its constitute bacteriocytes. Some of the central bacteriocytes are part of the myoepithelium surrounding the axial blood vessel and act as stem cells for a proliferating tissue produced in the center and ultimately degraded at the periphery of each lobule. Similarly, the rod-shaped symbionts in the center act as stem cells and exhibit a simple cell cycle. Differentiation into cocci takes place in the median and peripheral zone. Lysis of cocci occurs in the degenerative zone.     

荧光原位杂交技术及其在微生物生态学中的应用

综述了荧光原位杂交技术 (fluorescence in situ hybridization FISH)在微生物生态学领域的各种应用 ,同时就其发展过程、原理及种类做了介绍     

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.     

Identification of bacterial diversity in the oyster Crassostrea gigas by fluorescent in situ hybridization and polymerase chain reaction

AIMS: To carry out a rapid and reliable identification of bacterial diversity in the oyster Crassostrea gigas from Todos Santos Bay, México, in the current study we applied the molecular techniques of fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). In order to reach this goal, genus and group-specific oligonucleotides targeted to 16S rDNA/rRNA were used. METHODS AND RESULTS: Oysters were collected and different tissues were analysed by means of culture-independent methodologies. In the digestive glands and gonads gamma-Proteobacteria and Gram-positive bacteria with a low G+C content, were identified as metabolically active by FISH. In the oyster gills a higher active diversity was observed, including Gram-positive bacteria with a low and high G+C content, members of the Cytophaga/Flavobacterium cluster and gamma-Proteobacteria. Consistent with FISH analysis, the amplification of 16S rDNA genes fragments with genus and group-specific oligonucleotides confirmed the presence of the same groups, as well as members of the alpha- and beta-Proteobacterias, Pseudomonas spp. and Bacillus spp. CONCLUSIONS: The combination of accurate and very easy-to-apply molecular methods allowed us to carry out a rapid screening of high bacterial diversity in oysters. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is the first report about bacterial diversity in oyster tissues analysed by FISH and PCR, without using culture-dependent methods and allowed us to determine the phylogenetic diversity of the bacterial communities present in oyster cultures, including bacteria with and without metabolic activity, as well as uncultivable cells, which are generally underestimated by traditional identification.     

Fluorescence in situ hybridization for direct visualization of Gram-negative anaerobes in subgingival plaque samples

Abstract Fluorescent oligonucleotide probes complementary to variable regions of Porphyromonas gingivalis and Bacteroides forsythus 16S ribosomal RNA were used to identify these organisms in smears of formaldehyde-fixed subgingival plaque samples from patients suffering from periodontitis. Fluorescence in situ hybridization represents a useful method for assessing the microbial ecology of the periodontal flora.     

荧光原位杂交技术在医学微生物学中的应用

以16S rRNA 为靶序列的寡核苷酸探针荧光原位杂交技术已广泛应用于分析复杂环境中的微生物群落构成,包括监测和鉴定病原微生物以及未被培养微生物．通过对临床样品中微生物细胞的检测能提供微生物在人体中的种类、数量和空间分布等信息．其结果快速准确,较之传统的病原微生物诊断方法具有明显的优越性,在临床应用中有广泛的前景．     

Ultrastructural in situ hybridization: A review of technical aspects

Detection of nucleic acid sequence at the ultrastructural level has allowed us to better understand the expression of genes in some fields of application in cell biology. In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultrathin frozen section, or on ultrathin section of tissue embedded in hydrophilic resin such as Lowicryl. Before starting the detection of nucleic acid sequences at the electron microscope level, the experimenter has to choose various parameters: the type of tissue fixation, the probe and its label, and the in situ hybridization method, depending on the sensitivity, the resolution and the ultrastructural preservation required. This review of technical aspects, by describing the different methods of ultrastructural in situ hybridization, will help the experimenter to optimize each step of the hybridization procedure.     

Spatial distribution of bacterial phylotypes in the gut of the termite Reticulitermes speratus and the bacterial community colonizing the gut epithelium

The bacterial community colonizing the gut wall of the termite Reticulitermes speratus was characterized without cultivation. Analysis of 16S rRNA genes after fractionation of the gut revealed that the bacterial composition on the gut wall was diverse and significantly different from that able to move unconfined in the gut fluid or physically associated with the gut protists. Actinobacteria, Firmicutes and Bacteroidetes were dominant on the gut wall, but Spirochaetes and the Termite group 1 phylum, abundant in the gut lumen, were relatively rare. A sequence-specific probe enabled the in situ detection of a rod-shaped Actinobacteria member, abundantly colonizing the gut paunch epithelium.     

Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)

AIMS: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. METHODS AND RESULTS: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4',6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were < or =2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were < or =2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. CONCLUSIONS: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.     

Karyotype analysis of four Vicia species using in situ hybridization with repetitive sequences

Mitotic chromosomes of four Vicia species (V. sativa, V. grandiflora, V. pannonica and V. narbonensis) were subjected to in situ hybridization with probes derived from conserved plant repetitive DNA sequences (18S-25S and 5S rDNA, telomeres) and genus-specific satellite repeats (VicTR-A and VicTR-B). Numbers and positions of hybridization signals provided cytogenetic landmarks suitable for unambiguous identification of all chromosomes, and establishment of the karyotypes. The VicTR-A and -B sequences, in particular, produced highly informative banding patterns that alone were sufficient for discrimination of all chromosomes. However, these patterns were not conserved among species and thus could not be employed for identification of homologous chromosomes. This fact, together with observed variations in positions and numbers of rDNA loci, suggests considerable divergence between karyotypes of the species studied.     

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.     

斑马鱼整体原位杂交法研究MMP15a的发育相关表达的初步观察

克隆斑马鱼基质金属蛋白酶15a（MMP15a）基因,并研究其在斑马鱼胚胎早期发育中的时空表达状况。收集不同发育时期的斑马鱼胚胎,制备DIG标记的MMP15a RNA探针,采用全胚胎原位杂交方法研究MMP15a基因在胚胎斑马鱼的表达。结果MMP15a基因在胚胎受精后一个细胞时期就开始表达,从受精后24h起,在眼睛处表达明显,从受精后48h MMP15a在胸鳍和耳囊有特异性表达至到受精后96h。MMP15a在斑马鱼胚胎发育不同时期表达明显,且在胸鳍和耳囊处有持续表达。