Dynamics of biofilm formation in drinking water: phylogenetic affiliation and metabolic potential of single cells assessed by formazan reduction and in situ hybridization

The DNA of human chromosomes terminates in several kilobases of telomere repeats that are gradually lost with age and with replication in vitro. Defective telomere maintenance has been shown to be causally linked to cell cycle exit and apoptosis. In order to overcome the limitations imposed by Southern blotting, we have established a quantitative fluorescence in situ hybridization (Q-FISH) technique. This technique allows estimation of telomere length in specific chromosome arms from metaphase cell preparations. Furthermore, we have extended quantitative in situ hybridization to flow cytometry (flow FISH) in order to obtain information on the mean telomere repeat content in suspended cells. Telomere length in granulocytes, monocytes, CD8 and CD4 T lymphocytes and natural killer cells was found to differ slightly in the peripheral blood of adults. However, strikingly longer telomeres were observed in B lymphocytes (∼ 1.3 kb longer), suggesting a functional role for telomere maintenance in this cell subset. In summary, Q-FISH and flow FISH represent new methods for measuring telomere length in single cells and allow studies of telomere dynamics in haematopoietic subpopulations at various stages of normal and abnormal antigen responses.

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Simultaneous in situ detection of beta-interferon mRNA and DNA in the same cell.

Journal of Histochemistry & Cytochemistry ◽

10.1177/37.5.2703705 ◽

1989 ◽

Vol 37 (5) ◽

pp. 697-701 ◽

Cited By ~ 2

Author(s):

F J Tang ◽

P O Ts'o ◽

S A Lesko

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

In Situ Hybridization ◽

Single Cells ◽

Specific Gene ◽

Whole Cells ◽

Beta Interferon ◽

Ht1080 Cells ◽

In Situ Detection

We report a quantitative method that combines in situ mRNA hybridization with microfluorometric analysis of DNA content to detect gene expression in single cells of a heteroploid cell population. The model was a human fibrosarcoma HT1080 cell line which consisted of diploid and tetraploid cells that were induced with polyI:polyC for production of beta-interferon. The level of beta-interferon mRNA detected by in situ hybridization was found to be two to three times higher in tetraploid compared to diploid HT1080 cells, and correlated with beta-interferon activity in that a subclone of tetraploid HT1080 cells secreted two- to fivefold more beta-interferon than a subclone of diploid HT1080 cells. Interestingly, beta-interferon-related transcripts were detected during S-phase in uninduced tetraploid HT1080 cells. In addition, beta-interferon induced by polyI:polyC was expressed in all phases of the cell cycle as demonstrated with a human diploid fibroblast, HF926. The unique features offered by the combination of microfluorometry and in situ hybridization provide a valuable tool to investigate specific gene expression related to ploidy or cell-cycle stage in the same individual cell of an unsynchronized population. Since the method allows direct observation of morphology, one can be assured that all quantitative measurements were made on whole cells with intact nuclei.

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Detection of Streptococcus Suis by in Situ Hybridization, Indirect Immunofluorescence, and Peroxidase-Antiperoxidase Assays in Formalin-Fixed, Paraffin-Embedded Tissue Sections from Pigs

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063870001200305 ◽

2000 ◽

Vol 12 (3) ◽

pp. 224-232 ◽

Cited By ~ 16

Author(s):

Mette Boye ◽

Anne A. Feenstra ◽

Conny Tegtmeier ◽

Lars Ole Andresen ◽

Søren R. Rasmussen ◽

...

Keyword(s):

In Situ Hybridization ◽

Indirect Immunofluorescence ◽

Polyclonal Antibodies ◽

Single Cells ◽

Streptococcus Suis ◽

Tissue Sections ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1–31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.

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The natural history of encephalomyocarditis virus-induced myositis and myocarditis in mice. Viral persistence demonstrated by in situ hybridization.

Journal of Experimental Medicine ◽

10.1084/jem.168.5.1639 ◽

1988 ◽

Vol 168 (5) ◽

pp. 1639-1648 ◽

Cited By ~ 31

Author(s):

M E Cronin ◽

L A Love ◽

F W Miller ◽

P R McClintock ◽

P H Plotz

Keyword(s):

Skeletal Muscle ◽

In Situ Hybridization ◽

Nucleic Acid ◽

Single Cells ◽

Viral Persistence ◽

Encephalomyocarditis Virus ◽

High Yield ◽

Viral Nucleic Acid ◽

History Of

Picornaviruses can initiate chronic inflammation that persists after the virus can no longer be cultured from inflamed tissues. In an attempt to understand this transition we have sought evidence for viral persistence by methods that detect viral genome independent of whether or not whole competent virus is present. In mice infected with a myotropic variant of encephalomyocarditis virus, EMC-221A, virus can be cultured in high yield at 1 wk and in low yield at 2 wk from skeletal muscle, heart, and brain; a small number of plaque-forming units could be cultured from brain at 4 wk. By contrast, in situ hybridization detected viral nucleic acid at least a week or two thereafter, often in single cells. In the skeletal muscle, inflammation disappeared by 3 wk, but in heart it remained for the full 12 wk of observation. In the brain, microglial nodules, sometimes with associated viral nucleic acid, were present for a long period. Application of this technique allows a more accurate assessment of the role of viral persistence in the pathogenesis of virus-initiated but apparently autoimmune inflammation.

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