Improved permeabilization protocols for fluorescence in situ hybridization (FISH) of mycolic-acid-containing bacteria found in foams

Carr, E.; Eales, Kathryn L.; Soddell, Jacques A.; Seviour, Robert J.

doi:10.1016/j.mimet.2004.10.023

Cited by 54 publications

(47 citation statements)

References 20 publications

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“…Several studies discussed below have shown that the Mycolata in foaming plants are often FISH negative against the EUBmix probes targeting all Bacteria. This suggests they might be moribund or dead, a view often supported by their response to stains like Live/Dead T and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) (Carr et al 2005;Kragelund et al 2007b). These stains measure physiologically different parameters associated with cell viability.…”

Section: Methods For Studying the Ecophysiology Of Activated Sludge Pmentioning

confidence: 99%

Ecophysiology of the Actinobacteria in activated sludge systems

Seviour

Kragelund

Kong

et al. 2008

Antonie van Leeuwenhoek

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This review considers what is known about the Actinobacteria in activated sludge systems, their abundance and their functional roles there. Participation in processes leading to the microbiological removal of phosphate and in the operational problems of bulking and foaming are discussed in terms of their ecophysiological traits. We consider critically whether elucidation of their nutritional requirements and other physiological properties allow us to understand better what might affect their survival capabilities in these highly competitive systems. Furthermore, how this information might allow us to improve how these processes work is discussed.

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Section: Methods For Studying the Ecophysiology Of Activated Sludge Pmentioning

confidence: 99%

Ecophysiology of the Actinobacteria in activated sludge systems

Seviour

Kragelund

Kong

et al. 2008

Antonie van Leeuwenhoek

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“…After FISH hybridization, cells were immersed in 10 ppm DAPI for 10 min before being rinsed in water and air dried. Viability staining with the Live/Dead system (Molecular Probes) followed the method of Carr et al (7). Staining for PHA was performed by the method of Ostle and Holt (41), with some modifications.…”

Section: Methodsmentioning

confidence: 99%

Ecology of the Microbial Community Removing Phosphate from Wastewater under Continuously Aerobic Conditions in a Sequencing Batch Reactor

Ahn

Schroeder

Beer

et al. 2007

Appl Environ Microbiol

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109

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All activated sludge systems for removing phosphate microbiologically are configured so the biomass is cycled continuously through alternating anaerobic and aerobic zones. This paper describes a novel aerobic process capable of decreasing the amount of phosphate from 10 to 12 mg P liter ؊1 to less than 0.1 mg P liter ؊1(when expressed as phosphorus) over an extended period from two wastewaters with low chemical oxygen demand. One wastewater was synthetic, and the other was a clarified effluent from a conventional activated sludge system. Unlike anaerobic/aerobic enhanced biological phosphate removal (EBPR) processes where the organic substrates and the phosphate are supplied simultaneously to the biomass under anaerobic conditions, in this aerobic process, the addition of acetate, which begins the feed stage, is temporally separated from the addition of phosphate, which begins the famine stage. Conditions for establishing this process in a sequencing batch reactor are detailed, together with a description of the changes in poly-␤-hydroxyalkanoate (PHA) and poly(P) levels in the biomass occurring under the feed and famine regimes, which closely resemble those reported in anaerobic/aerobic EBPR processes. Profiles obtained with denaturing gradient gel electrophoresis were very similar for communities fed both wastewaters, and once established, these communities remained stable over prolonged periods of time. 16S rRNA-based clone libraries generated from the two communities were also very similar. Fluorescence in situ hybridization (FISH)/microautoradiography and histochemical staining revealed that "Candidatus Accumulibacter phosphatis" bacteria were the dominant poly(P)-accumulating organisms (PAO) in both communities, with the phenotype expected for PAO. FISH also identified large numbers of betaproteobacterial Dechloromonas and alphaproteobacterial tetrad-forming organisms related to Defluviicoccus in both communities, but while these organisms assimilated acetate and contained intracellular PHA during the feed stages, they never accumulated poly(P) during the cycles, consistent with the phenotype of glycogen-accumulating organisms.High levels of phosphate in effluents from activated sludge systems not designed to remove it can lead to toxic cyanobacterial blooms in receiving bodies of water. Consequently, efforts have been directed towards removing phosphate during treatment by microbiological means with a process called enhanced biological phosphorus removal (EBPR), where phosphate is removed from the wasted biomass as intracellular poly(P) (5, 37, 45). Such treatment processes are based on the underlying principle that the biomass needs to be recycled repeatedly through alternating anaerobic and aerobic stages (37), a requirement regarded as crucial for successful EBPR operation. Only after repeated recycling are poly(P)-accumulating organisms (PAO) thought to have a selective advantage over other populations, eventually allowing them to become dominant (5,37,45). In the anaerobic (feed) stage, PAO are belie...

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“…The gene encoding the enzyme has been cloned (19). Treatment with lysozyme and/or other murein-degrading enzymes is known to be effective for FISH analysis of some gram-positive bacteria, since thick cell walls of some grampositive bacteria hinder oligonucleotide probes, which would otherwise permeate into cytoplasms (1,2,4). In this study, we attempted to apply pseudomurein endoisopeptidase to FISH analysis for clearly visualizing the methanogens in the family Methanobacteriaceae.…”

Section: Ing ␤-(133)-linked N-acetyl-d-glucosamine and ␤-(133)-linkedmentioning

confidence: 99%

Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae

Nakamura

Terada

Sekiguchi

et al. 2006

Appl Environ Microbiol

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In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H 2 -producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.

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Improved permeabilization protocols for fluorescence in situ hybridization (FISH) of mycolic-acid-containing bacteria found in foams

Cited by 54 publications

References 20 publications

Ecophysiology of the Actinobacteria in activated sludge systems

Ecophysiology of the Actinobacteria in activated sludge systems

Ecology of the Microbial Community Removing Phosphate from Wastewater under Continuously Aerobic Conditions in a Sequencing Batch Reactor

Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae

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