Characterization of a p-nitrophenol-degrading mixed culture with an improved methodology of fluorescence in situ hybridization and confocal laser scanning microscopy

2011 ◽  
Vol 86 (11) ◽  
pp. 1405-1412 ◽  
Author(s):  
María Eugenia Suárez-Ojeda ◽  
Helena Montón ◽  
Mónica Roldán ◽  
Mariángel Martín-Hernández ◽  
Julio Pérez ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Mixed Culture ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser

Considerations in the use of fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy to characterize rumen methanogens and define their spatial distributions

2015 ◽  
Vol 61 (6) ◽  
pp. 417-428 ◽  
Author(s):  
Edith R. Valle ◽  
Gemma Henderson ◽  
Peter H. Janssen ◽  
Faith Cox ◽  
Trevor W. Alexander ◽  
...  
Keyword(s):  
Spatial Distribution ◽  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser

In this study, methanogen-specific coenzyme F420autofluorescence and confocal laser scanning microscopy were used to identify rumen methanogens and define their spatial distribution in free-living, biofilm-, and protozoa-associated microenvironments. Fluorescence in situ hybridization (FISH) with temperature-controlled hybridization was used in an attempt to describe methanogen diversity. A heat pretreatment (65 °C, 1 h) was found to be a noninvasive method to increase probe access to methanogen RNA targets. Despite efforts to optimize FISH, 16S rRNA methanogen-specific probes, including Arch915, bound to some cells that lacked F420, possibly identifying uncharacterized Methanomassiliicoccales or reflecting nonspecific binding to other members of the rumen bacterial community. A probe targeting RNA from the methanogenesis-specific methyl coenzyme M reductase (mcr) gene was shown to detect cultured Methanosarcina cells with signal intensities comparable to those of 16S rRNA probes. However, the probe failed to hybridize with the majority of F420-emitting rumen methanogens, possibly because of differences in cell wall permeability among methanogen species. Methanogens were shown to integrate into microbial biofilms and to exist as ecto- and endosymbionts with rumen protozoa. Characterizing rumen methanogens and defining their spatial distribution may provide insight into mitigation strategies for ruminal methanogenesis.

Monitoring the development of anaerobic biofilms using fluorescent in situ hybridization and confocal laser scanning microscopy

2000 ◽  
Vol 41 (12) ◽  
pp. 69-77 ◽  
Author(s):  
J. C. Araujo ◽  
G. Brucha ◽  
J. R. Campos ◽  
R. F. Vazoller
Keyword(s):  
In Situ Hybridization ◽  
Confocal Laser Scanning Microscopy ◽  
Fluorescent In Situ Hybridization ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Anaerobic Consortium

In this study we investigated the development of anaerobic biofilm using a laboratory reactor. We were especially interested in comparing the organization of anaerobic cells (particularly those that are very common in domestic sewage sludge) in a hydrophilic (glass) versus a hydrophobic (polypropylene) surface. Fluorescent in situ hybridization (FISH) with domain and group specific probes directed against 16S ribosomal RNA were used to quantify microbial composition in the biofilm. FISH and confocal laser scanning microscopy (CLSM) were used to elucidate spatial distribution of microbes in the biofilms. Two experiments were carried out, one with pure methanogenic organisms and the other with a microbial anaerobic consortium. The pure methanogen cultures, Methanobacterium formicicum (DSM 1535); Methanosaeta concilli (DSM 3671) and Methanosarcina barkeri (DSM 800) were used to seed the modified Robbins Device (MRD) to allow the development of biofilms on polypropylene and glass surfaces during the 9-days experiment. The results showed that all the three species were colonizing both surfaces after two and nine days of experimental period. In another experiment, with polypropylene coupons only, MRD was seeded with a microbial anaerobic consortium and biofilm formation was studied during 11 days. At the end of this period, the biofilms generated were of uneven thickness with areas of minimal or no surface coverage and areas where the biofilm attained a thickness of 7.0 to 9.0 μm as revealed by CLSM. The results showed that the modified Robbins Device together with the fluorescent in situ hybridization and confocal laser scanning microscopy are suitable tools to study anaerobic biofilm development in different kinds of support materials.

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