Development of a fluorescence in situ hybridization method for cheese using a 16S rRNA probe

A 16S rRNA fluorescence in situ hybridization (FISH) method for cheese was developed to allow detection in situ of microorganisms within the dairy matrix. An embedding procedure using a plastic resin was applied to Stilton cheese, providing intact embedded cheese sections withstanding the hybridization reaction. The use of a fluorescein-labelled 16S rRNA Domain Bacteria probe allowed observation of large colonies of microbial cells homogeneously distributed in the cheese matrix. FISH experiments performed on cheese suspensions provided images of the different microbial morphotypes occurring. The technique has great potential to study the spatial distribution of microbial populations in situ in foods, especially where the matrix is too fragile to allow manipulation of cryosections.     

Development of a PNA probe for fluorescence in situ hybridization detection of Prorocentrum donghaiense

Prorocentrum donghaiense is a common but dominant harmful algal bloom (HAB) species, which is widely distributed along the China Sea coast. Development of methods for rapid and precise identification and quantification is prerequisite for early-stage warning and monitoring of blooms due to P. donghaiense. In this study, sequences representing the partial large subunit rDNA (D1-D2), small subunit rDNA and internal transcribed spacer region (ITS-1, 5.8S rDNA and ITS-2) of P. donghaiense were firstly obtained, and then seven candidate DNA probes were designed for performing fluorescence in situ hybridization (FISH) tests on P. donghaiense. Based on the fluorescent intensity of P. donghaiense cells labeled by the DNA probes, the probe DP0443A displayed the best hybridization performance. Therefore, a PNA probe (PP0443A) analogous to DP0443A was used in the further study. The cells labeled with the PNA probe displayed more intensive green fluorescence than that labeled with its DNA analog. The PNA probe was used to hybridize with thirteen microalgae belonging to five families, i.e., Dinophyceae, Prymnesiophyceae, Raphidophyceae, Chlorophyceae and Bacillariophyceae, and showed no visible cross-reaction. Finally, FISH with the probes PP0443A and DP0443A and light microscopy (LM) analysis aiming at enumerating P. donghaiense cells were performed on the field samples. Statistical comparisons of the cell densities (cells/L) of P. donghaiense in the natural samples determined by FISH and LM were performed using one-way ANOVA and Duncan's multiple comparisons of the means. The P. donghaiense cell densities determined by LM and the PNA probe are remarkably higher than (p<0.05) that determined by the DNA probe, while no significant difference is observed between LM and the PNA probe. All results suggest that the PNA probe is more sensitive that its DNA analog, and therefore is promising for the monitoring of harmful algal blooms of P. donghaiense in the future.     

Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe

AIMS: Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum. METHODS AND RESULTS: FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h. CONCLUSIONS: Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.     

Seasonal dynamics of particle-associated and free-living marine Proteobacteria in a salt marsh tidal creek as determined using fluorescence in situ hybridization

The seasonal distributions of salt marsh free-living and particle-associated bacteria belonging to three subdivisions of the Proteobacteria were determined by fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). More than 66% (median = 78%) of total bacterial cells that were stainable with the fluorescent DNA stain Yo-Pro-1 were also detected using the bacterial probe EUB338. The alpha-Proteobacteria, especially those from the marine Rhodobacter group, were abundant on suspended particles and as free-living cells all year round. The marine Rhodobacter group constituted more than 25% of the particle-associated bacteria and more than 18% of the free-living bacteria. Probes specific for three subgroups within the marine Rhodobacter group detected more than 49% of the total marine Rhodobacter group cells. These subgroups displayed different seasonal dynamics. The marine Rhodobacter group is clearly a widespread, diverse and important bacterial lineage in bacterioplankton and particle-associated assemblages in south-eastern United States salt marshes at all times of the year.     

Improved 16S rRNA-targeted probe set for analysis of sulfate-reducing bacteria by fluorescence in situ hybridization

An updated dataset of in silico specificities for 54 previously published 16S rRNA-targeted oligonucleotides was assembled to provide guidance for reliable fluorescence in situ hybridization (FISH) analysis of sulfate-reducing bacteria. Additionally, six new FISH probes were developed for major deltaproteobacterial taxa, including a probe trio targeting most Deltaproteobacteria and Gemmatimonadetes.     

Quantification of methanogens by fluorescence in situ hybridization with oligonucleotide probe

To monitor anaerobic environmental engineering system, new method of quantification for methanogens was tested. It is based on the measurement of specific binding (hybridization) of 16S rRNA-targeted oligonucleotide probe Arc915, performed by fluorescence in situ hybridization (FISH) and quantified by fluorescence spectrometry. Average specific binding of Arc915 probe was 13.4±0.5 amol/cell of autofluorescent methanogens. It was 14.3, 13.3, and 12.9 amol/cell at the log phase, at stationary phase and at the period of cell lysis of batch culture, respectively. Specific binding of Arc915 probe per 1 ml of microbial sludge suspension from anaerobic digester linearly correlated with concentration of autofluorescent cells of methanogens. Coefficient of correlation was 0.95. Specific binding of oligonucleotide probe Arc915 can be used for the comparative estimation of methanogens during anaerobic digestion of organic waste. Specific binding of Arc915 probe was linear function of anaerobic sludge concentration when it was between 1.4 and 14.0 mg/ml. Accuracy of the measurements in this region was from 5 to 12%.     

A new multicolor fluorescence in situ hybridization probe set directed against human heterochromatin: HCM-FISH

A new multicolor fluorescence in situ hybridization (mFISH) probe set is presented, and its possible applications are highlighted in 25 clinical cases. The so-called heterochromatin-M-FISH (HCM-FISH) probe set enables a one-step characterization of the large heterochromatic regions within the human genome. HCM-FISH closes a gap in the now available mFISH probe sets, as those do not normally cover the acrocentric short arms; the large pericentric regions of chromosomes 1, 9, and 16; as well as the band Yq12. Still, these regions can be involved in different kinds of chromosomal rearrangements such as translocations, insertions, inversions, amplifications, and marker chromosome formations. Here, examples are given for all these kinds of chromosomal aberrations, detected as constitutional rearrangements in clinical cases. Application perspectives of the probe set in tumors as well as in evolutionary cytogenetic studies are given.     

Characterization of Robertsonian translocations by using fluorescence in situ hybridization.

Fluorescence in situ hybridization with five biotin-labeled probes (three alphoid probes, a probe specific for beta-satellite sequences in all acrocentric chromosomes, and an rDNA probe) was used to characterize 30 different Robertsonian translocations, including three t(13;13); one t(15;15), four t(21;21), three t(13;14), two t(13;15), two (13;21), two t(13;22), one t(14;15), eight t(14;21), two t(14;22), and two t(21;22). Of 8 de novo homologous translocations, only one t(13;13) chromosome was interpreted as dicentric, while 19 of 22 nonhomologous Robertsonian translocations were dicentric. The three monocentric nonhomologous translocations included both of the t(13;21) and one t(21;22). Two of 26 translocations studied using the beta-satellite probe showed a positive signal, while rDNA was undetectable in 10 cases studied. These results indicate that most homologous Robertsonian translocations appear monocentric, while the bulk of nonhomologous translocations show two alphoid signals. A majority of the breakpoints localized using this analysis seem to be distal to the centromere and just proximal to the beta-satellite and nuclear-organizing regions.     

Development of thermodynamic models for simulating probe dissociation profiles in fluorescence in situ hybridization

Stringency in ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH) is typically adjusted with formamide, and the optimum formamide concentration at which the probe can hybridize with the target rRNA, but not with rRNAs with mismatches, is to be found experimentally. This is a difficult task when target or closest non-target organisms are not available in pure culture, or when there are numerous non-targets of concern. The objective of this work was to formulate mechanistic models capable of simulating the effect of formamide on probe dissociation. Using a previously described equilibrium model of FISH [Yilmaz and Noguera (2004) Applied and Environmental Microbiology 70(12):7126-7139] as the basis, the effect of formamide on free energy changes of probe-target duplex formation (DeltaG(1)(0)) and folding of target region (DeltaG(3)(0)) was simulated to be linear, and models differing in the definitions of the slopes of these relationships (m(1) and m(3)) were calibrated using experimental dissociation profiles for 27 probes targeting the 16S rRNA of Escherichia coli (E. coli). A good level of predictive power was obtained when m(1) was linearly related to probe length and when m(3) was made proportional to DeltaG(3)(0). The effect of single mismatches on probe dissociation with formamide was also studied, although at a preliminary level. The expected changes in DeltaG(1)(0) with the introduction of mismatches were not sufficient to capture the overall trends of mismatched dissociation profiles. In conclusion, this study offers the first theoretical method to calculate dissociation profiles for perfectly matched probes, and suggests a direction to systematically evaluate the effect of formamide on mismatched probes.     

A fluorescence in situ hybridization technique for retrospective cytogenetic analysis

Fluorescence in situ hybridization (FISH) is being used increasingly in clinical practice; however, current FISH techniques require fresh material, and there is considerable variation in hybridization efficiency between laboratories. We have modified a FISH technique described by Pinkel et al. (1986) that works not only on freshly G-banded material but also on cytogenetic preparations ranging in age from 2 wk to 12 yr. We have tested this technique on several centromeric alphoid satellite probes (D1Z5, D7Z1, D17Z1, DXZ1, and DYZ3) and one noncentromeric minisatellite probe (D1Z2). Our average hybridization efficiency on freshly banded preparations for these probes is consistently greater than 90%. The combination of higher efficiency and the ability to perform hybridization on previously G-banded material makes this a valuable technique for retrospective analyses.     

Diagnosis of bovine freemartinism by fluorescence in situ hybridization on interphase nuclei using a bovine Y chromosome-specific DNA probe

A heifer co-twin to a bull, in most cases, is a sterile freemartin which needs to be identified and culled from replacement stock. Various methods are available for the diagnosis of freemartinism, but none is ideal in terms of speed, sensitivity, or specificity. The present study was thus conducted to develop and validate a satisfactory fluorescence in situ hybridization procedure on interphase nuclei (I-FISH) for identifying the bovine XX/XY-karyotypic chimerism, the hallmark of freemartinism. A 190-bp DNA FISH probe containing the bovine male-specific BC1.2 DNA sequence was synthesized and labeled with digoxigenin by PCR. The FISH was performed on metaphase spreads and interphase nuclei of blood lymphocytes. Upon FISH, the probe expectedly bound to the nucleus of the male cell or to a region of the p12 locus of the Y chromosome. Twenty-four young heterosexual twins (Holstein-Friesian and Korean Cattle breeds; 10 pairs and 4 singletons) were analyzed in the present study; all but three exhibited the XX/XY-karyotypic chimerism to varying extents in both I-FISH and karyotyping. One heifer was identified to have 100% XX cells by both analyses, whereas two bulls were judged as 100% XY- and XX/XY-chimeric karyotypes by karyotyping and I-FISH, respectively. Nevertheless, the ratios of the XY to XX cells in these animals were very similar between the two analyses. In conclusion, the present I-FISH was a rapid and reliable procedure that can be used for early-life diagnosis of bovine freemartinism.     

Telomere analysis by fluorescence in situ hybridization and flow cytometry.

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.     

Digoxigenin as a probe label for in situ hybridization on skeletal tissues.

Summary Non-specific staining was encountered using digoxigenin-labelled cDNA probes forin situ hybridization on sections of skeletal tissues. This staining was most pronounced in cartilaginous matrices. Experimental procedures indicate that the background staining is caused by antibody-binding to hydrophobic sites in the tissues revealed by proteolytic permeabilization. A protocol for minimizing this background is described.     

Chromatid segregation analysis in native human lymphocyte anaphases using sequential fluorescence in situ hybridization

A sequential multiprobe fluorescence in situ hybridization technique was developed to study the 13, 18, 21, X and Y chromatid segregation in human lymphocytes anaphases cultures without antimitotic treatment. This method was used to know if exist any different chromosomes segregation in lymphocytes from Down syndrome patients and compared it with controls. The results show that the prevalent sequence of centromere separation was X, 13, 21, Y and 18 in Down syndrome patients and Y, 13, X, 21 and 18 in controls. Chromatid segregation in early anaphase was asynchronic for all chromosome pairs studied. Late anaphase showed a frequency of non-disjunction of 4.5% in the controls, affecting only chromosomes 18 and Y; in the Down syndrome patients, the frequency was higher (20.3%) and affected all chromosomes studied. This technique could be applicated to know the incidence of non disjunction in couples with repetitive abortions or in cases with different aneuploidies in the offspring. This revised version was published online in September 2006 with corrections to the Cover Date.     

Localization of mRNA for pea legumin:In situ hybridization using a biotinylated cDNA probe

Summary In situ hybridization has been used to locate mRNA, for the storage protein legumin, in cotyledon storage parenchyma tissue of developing pea (Pisum sativum L.) seeds. The mRNA was hybridized with a biotinylated probe of cDNA in pBR 322 and subsequently located by avidin conjugates. Avidin-rhodamine was used for fluorescence microscopy localization at a tissue/cellular level and avidin-peroxidase (with DAB) and avidin-ferritin compared for localization at an ultrastructural level. Specific fluorescence associated with avidin-rhodamine was distributed unevenly throughout the cytosol but the cell walls, starch grains, vacuoles and protein deposits were unstained. The sizes and distribution of the regions of higher labeling within the cytosol suggest an association with elements of the endomembrane system. Following DAB reaction of the specifically localized avidin-peroxidase most, although not all, stain product was associated with the endoplasmic reticulum. The ER-associated reaction product was also accumulated within the ER lumen.Avidin-ferritin was also localized both in the cytosol and in association with the endoplasmic reticulum, although was less readily visualized in cells with a conventional ultrastructural appearance.Localization of avidin-ferritin was more readily visualized in cells which had undergone some limited structural damage during specimen preparation. In such cases ferritin was also shown to be specifically associated with the transition vesicles and trans-face peripheral vesicles of some dictyosomes.     

Automated image analysis for quantitative fluorescence in situ hybridization with environmental samples

When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental samples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample dispersion were optimized to reduce the interference of cell aggregates. An automated image analysis program that detects cells from 4',6'-diamidino-2-phenylindole (DAPI) micrographs and extracts the maximum and mean fluorescence intensities for each cell from corresponding FISH images was developed with the software Visilog. Intensity thresholds were not consistent even for duplicate analyses, so alternative ways of classifying signals were investigated. In the resulting method, the intensity data were divided into clusters using fuzzy c-means clustering, and the resulting clusters were classified as target (positive) or nontarget (negative). A manual quality control confirmed this classification. With this method, 50.4, 72.1, and 64.9% of the cells in two swine manure samples and one soil sample, respectively, were positive as determined with a 16S rRNA-targeted bacterial probe (S-D-Bact-0338-a-A-18). Manual counting resulted in corresponding values of 52.3, 70.6, and 61.5%, respectively. In two swine manure samples and one soil sample 21.6, 12.3, and 2.5% of the cells were positive with an archaeal probe (S-D-Arch-0915-a-A-20), respectively. Manual counting resulted in corresponding values of 22.4, 14.0, and 2.9%, respectively. This automated method should facilitate quantitative analysis of FISH images for a variety of complex environmental samples.     

The chromosome-based challenge assay using fluorescence in situ hybridization: a possible test for increased cancer susceptibility.

The challenge assay is a cytogenetic approach to measure the repair competence of cells. For in vitro studies, human lymphocytes are exposed to different substances and are irradiated simultaneously. To investigate subjects exposed occupationally or environmentally, untreated blood samples are directly irradiated without any further treatment. Certain substances like heavy metals reveal carcinogenic potential without well defined mechanism of action. While they are not mutagenic they may have an effect on DNA repair capacity. The challenge assay was successfully applied in vitro experiments with cadmium to detect an interaction of this heavy metal with the repair of X-ray-induced chromosome breaks. CdCl(2) alone had no effect on the formation of chromosome aberrations (CA), not even in the cytotoxic concentration (50 microM). However, cadmium showed an effect on the number of chromosomal rearrangements (CR) after X-ray challenge. For 0.5 microM CdCl(2), CA frequencies were significantly elevated compared to the rates for X-rays alone. For the two higher concentrations the rates showed a slight additional increase. Hence, the challenge assay appears suitable to test for chromosomal sensitivity induced by toxicants. Subsequently, a study of styrene exposed workers was initiated to address the question whether styrene exposure has an influence on the DNA repair. In addition, we investigated whether a polymorphism of genes coding for phase II detoxifying enzymes glutathione-S-transferases GSTM1 and GSTT1 had an influence on chromosomal sensitivity. First and preliminary data are presented. While there is a correlation of the rate of CR with cumulative lifetime exposure of styrene, the most recent styrene exposure had no effect. 'At risk' genotypes with higher incidence of CA could not be identified at this stage of the ongoing study. Conclusion: the challenge assay is able to detect enhanced susceptibility for CR caused by genetic predisposition for DNA repair deficiency. Our data indicate that environmental or occupational exposure to certain substances can interfere with DNA repair processes. As the process of induction of CR is associated with carcinogenesis, the challenge assay may provide a valuable biomarker for cancer epidemiology studies.     

Physical mapping of barley genes using an ultrasensitive fluorescence in situ hybridization technique.

The primary objective of this study was to elucidate gene organization and to integrate the genetic linkage map for barley (Hordeum vulgare L.) with a physical map using ultrasensitive fluorescence in situ hybridization (FISH) techniques for detecting signals from restriction fragment length polymorphism (RFLP) clones. In the process, a single landmark plasmid, p18S5Shor, was constructed that identified and oriented all seven of the chromosome pairs. Plasmid p18S5Shor was used in all hybridizations. Fourteen cDNA probes selected from the linkage map for barley H. vulgare 'Steptoe' x H. vulgare 'Morex' (Kleinhofs et al. 1993) were mapped using an indirect tyramide signal amplification technique and assigned to a physical location on one or more chromosomes. The haploid barley genome is large and a complete physical map of the genome is not yet available; however, it was possible to integrate the linkage map and the physical locations of these cDNAs. An estimate of the ratio of base pairs to centimorgans was an average of 1.5 Mb/cM in the distal portions of the chromosome arms and 89 Mb/cM near the centromere. Furthermore, while it appears that the current linkage maps are well covered with markers along the length of each arm, the physical map showed that there are large areas of the genome that have yet to be mapped.