Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate ofBacteroides spp. in drinking water

2005 ◽  
Vol 92 (3) ◽  
pp. 356-363 ◽  
Author(s):  
Olga Savichtcheva ◽  
Noriko Okayama ◽  
Tsukasa Ito ◽  
Satoshi Okabe
Keyword(s):  
Drinking Water ◽  
In Situ Hybridization ◽  
Survival Rate ◽  
Fluorescence In Situ Hybridization ◽  
Direct Fluorescence

scholarly journals Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

2002 ◽  
Vol 68 (8) ◽  
pp. 4081-4089 ◽  
Author(s):  
Sven Poppert ◽  
Andreas Essig ◽  
Reinhard Marre ◽  
Michael Wagner ◽  
Matthias Horn
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Step ◽  
Detection Methods ◽  
The Novel ◽  
Antibody Staining ◽  
Uncultured Bacteria ◽  
Direct Fluorescence ◽  
Probe Set

ABSTRACT 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.

Influence of phosphate and disinfection on the composition of biofilms produced from drinking water, as measured by fluorescence in situ hybridization

2003 ◽  
Vol 49 (12) ◽  
pp. 741-753 ◽  
Author(s):  
M Batté ◽  
L Mathieu ◽  
P Laurent ◽  
M Prévost
Keyword(s):  
Drinking Water ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Bacterial Populations ◽  
Health Related ◽  
Phosphate Treatment ◽  
The Stability ◽  
Eub338 Probe ◽  
Direct Counts

Biofilms were grown in annular reactors supplied with drinking water enriched with 235 µg C/L. Changes in the biofilms with ageing, disinfection, and phosphate treatment were monitored using fluorescence in situ hybridization. EUB338, BET42a, GAM42a, and ALF1b probes were used to target most bacteria and the alpha (α), beta (β), and gamma (γ) subclasses of Proteobacteria, respectively. The stability of biofilm composition was checked after the onset of colonization between T = 42 days and T = 113 days. From 56.0% to 75.9% of the cells detected through total direct counts with DAPI (4'-6-diamidino-2-phenylindole) were also detected with the EUB338 probe, which targets the 16S rRNA of most bacteria. Among these cells, 16.9%–24.7% were targeted with the BET42a probe, 1.8%–18.3% with the ALF1b probe, and <2.5% with the GAM42a probe. Phosphate treatment induced a significant enhancement to the proportion of γ-Proteobacteria (detected with the GAM42a probe), a group that contains many health-related bacteria. Disinfection with monochloramine for 1 month or chlorine for 3 days induced a reduction in the percentage of DAPI-stained cells that hybridized with the EUB338 probe (as expressed by percentages of EUB338 counts/DAPI) and with any of the ALF1b, BET42a, and GAM42a probes. The percentage of cells detected by any of the three probes (ALF1b+BET42a+GAM42a) tended to decrease, and reached in total less than 30% of the EUB338-hybridized cells. Disinfection with chlorine for 7 days induced a reverse shift; an increase in the percentage of EUB338 counts targeted by any of these three probes was noted, which reached up to 87%. However, it should be noted that the global bacterial densities (heterotrophic plate counts and total direct counts) tended to decrease over the duration of the experiment. Therefore, those bacteria that could be considered to resist 7 days of chlorination constituted a small part of the initial biofilm community, up to the point at which the other bacterial groups were destroyed by chlorination. The results suggest that there were variations in the kinetics of inactivation by disinfectant, depending on the bacterial populations involved.Key words: biofilm, phosphate, chlorine, monochloramine, FISH, drinking water.

Direct and sensitive fluorescence in situ hybridization of 45S rDNA on tomato chromosomes

Genome ◽  
1994 ◽  
Vol 37 (6) ◽  
pp. 1062-1065 ◽  
Author(s):  
Jie Xu ◽  
E. D. Earle
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Chromosome 2 ◽  
45S Rdna ◽  
Plant Chromosomes ◽  
Signal Sensitivity ◽  
Direct Fluorescence ◽  
Hybridization Protocol ◽  
Tomato Genotypes

We describe a direct and sensitive fluorescence in situ hybridization protocol for plant chromosomes. We labelled 45S rDNA with fluorescein-12-dUTP and hybridized to somatic chromosomes of four tomato genotypes. This technique does not require posthybridization immunocytochemical amplifications. The improved signal sensitivity with this technique allowed identification of new rDNA loci on three pairs of chromosomes, in addition to the previously known locus on chromosome 2. We discuss favorable features of direct fluorescence in situ hybridization for chromosomes fixed on a slide and chromosomes or cells in suspension.Key words: direct fluorescence in situ hybridization, 45S rDNA, tomato chromosomes.

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