scholarly journals Fluorescence In Situ Hybridization-Flow Cytometry-Cell Sorting-Based Method for Separation and Enrichment of Type I and Type II Methanotroph Populations

2006 ◽  
Vol 72 (6) ◽  
pp. 4293-4301 ◽  
Author(s):  
Marina G. Kalyuzhnaya ◽  
Rebecca Zabinsky ◽  
Sarah Bowerman ◽  
David R. Baker ◽  
Mary E. Lidstrom ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Cell Sorting ◽  
Type I ◽  
Type Ii ◽  
Specific Probe ◽  
Artificial Mixtures

ABSTRACT A fluorescence in situ hybridization-flow cytometry (FISH/FC)-based method was optimized using artificial mixtures of pure cultures of methanotrophic bacteria. Traditional oligonucleotide probes targeting 16S rRNAs of type I (MG84/705 probe) and type II (MA450 probe) methanotrophs were labeled with fluorescein or Alexa fluor and used for FISH, followed by fluorescence-activated FC analysis and cell sorting (FACS). The method resulted in efficient separation of target cells (type I or type II methanotrophs) from the artificial mixtures. The method was then applied for detection and enrichment of type I and type II methanotroph populations from a natural sample, Lake Washington sediment. Cells were extracted from the sediment, fixed, and subjected to FISH/FC/FACS. The resulting subpopulations were analyzed by reverse transcriptase PCR surveys of 16S rRNA, pmoA (encoding a subunit of particulate methane monooxygenase), and fae (encoding formaldehyde-activating enzyme) genes. The functional gene analysis indicated specific separation of the type I and type II methanotroph populations. 16S rRNA gene analysis revealed that type I methanotrophs comprised 59% of the subpopulation separated using the type I-specific probe and that type II methanotrophs comprised 47.5% of the subpopulation separated using the type II-specific probe. Our data indicate that the FISH/FC/FACS protocol described can provide significant enrichment of microbial populations of interest from complex natural communities and that these can be used for genetic tests. We further tested the possibility of direct whole-genome amplification (WGA) from limited numbers of sorted cells, using artificial mixtures of microbes whose genome sequences are known. We demonstrated that efficient WGA can be achieved using 104 or more cells separated by 16S rRNA-specific FISH/FC/FACS, while fewer cells resulted in less specific WGA.

scholarly journals Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris

2001 ◽  
Vol 67 (10) ◽  
pp. 4850-4857 ◽  
Author(s):  
Svetlana N. Dedysh ◽  
Manigee Derakshani ◽  
Werner Liesack
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Fluorescence Signal ◽  
Type I ◽  
Oligonucleotide Probes ◽  
Type Ii ◽  
Cell Counts ◽  
Wet Weight

ABSTRACT Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or theMethylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in aSphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 106 cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 104 type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 106 type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus andMethylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion ofMethylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (±0.2) × 106 cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population ofMethylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.

In situ hybridization reveals differential spatial distribution of mRNAs for type I and type II collagen in the chick limb bud

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 111-118 ◽  
Author(s):  
C.J. Devlin ◽  
P.M. Brickell ◽  
E.R. Taylor ◽  
A. Hornbruch ◽  
R.K. Craig ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Limb Development ◽  
Type I Collagen ◽  
Type Ii Collagen ◽  
Limb Bud ◽  
Type I ◽  
Type Ii ◽  
Mrna Accumulation ◽  
Rna Probes

During limb development, type I collagen disappears from the region where cartilage develops and synthesis of type II collagen, which is characteristic of cartilage, begins. In situ hybridization using antisense RNA probes was used to investigate the spatial localization of type I and type II collagen mRNAs. The distribution of the mRNA for type II collagen corresponded well with the pattern of type II collagen synthesis, suggesting control at the level of transcription and mRNA accumulation. In contrast, the pattern of mRNA for type I collagen remained more or less uniform and did not correspond with the synthesis of the protein, suggesting control primarily at the level of translation or of RNA processing.

scholarly journals Telomere analysis by fluorescence in situ hybridization and flow cytometry

1998 ◽  
Vol 26 (16) ◽  
pp. 3651-3656 ◽  
Author(s):  
M. Hultdin ◽  
E. Gronlund ◽  
K.- F. Norrback ◽  
E. Eriksson-Lindstrom ◽  
G. Roos ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization
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