Rapid monitoring of bacteria in dialysis fluids by fluorescent vital staining and microcolony methods

Yamaguchi, Naohito; Baba, Takashi; Nakagawa, Sachiko; Saitō, Akira; Nasu, Masao

doi:10.1093/ndt/gfl675

Cited by 27 publications

(20 citation statements)

References 20 publications

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“…5,6) Thus, culture-independent methods should be used for microbiological monitoring in addition to culture-dependent methods. 7,8) Several molecular microbiological techniques have been developed to identify bacterial cells and analyze their diversity and community structure in natural environments without the need for isolation. In particular, denaturing gradient gel electrophoresis (DGGE) 9) has been used to determine the genetic diversity of natural microbial communities and to identify the phylogenetic affiliation of community members.…”

mentioning

confidence: 99%

16S Ribosomal RNA Gene-Based Phylogenetic Analysis of Abundant Bacteria in River, Canal and Potable Water in Bangkok, Thailand

Yamaguchi

Nishiguchi

Utrarachkij

et al. 2013

Biological & Pharmaceutical Bulletin

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In Southeast Asian countries, industrialization and urbanization is occurring rapidly, and water pollution in rivers and canals poses serious problems in some areas, especially in cities. Excess inflow of domestic, agricultural, and industrial wastewater to freshwater environments disturbs the aquatic microbial ecosystem, which can further pollute water by inhibiting biodegradation of pollutants. Therefore, monitoring of microbes in freshwater environment is important to identify changes in indigenous microbial populations and to estimate the influence of wastewater inflows on them. Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis is suitable for monitoring changes in microbial communities caused by human activities, but this method can be difficult in eutrophic freshwater samples that contain PCR inhibitors. In this study, we optimized DNA extraction procedures and PCR conditions for DGGE analysis of bacterial populations in freshwater samples (canal, river, and tap water) collected in Bangkok, Thailand. A simple freeze-thaw procedure was effective for extracting DNA from bacterial cells in the samples, and LA Taq with added bovine serum albumin provided the best PCR amplification. The PCR-DGGE approach revealed that the most common bacteria in freshwater samples belonged to Gammaproteobacteria, while a Gram-positive bacterium was present at Bangkok Noi Canal. Temporally and spatially continuous analyses of bacterial populations in Bangkok canals and rivers by PCR-DGGE approach should be useful to recognize disturbances of microbial ecosystems caused by excess inflows of wastewater.

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confidence: 99%

16S Ribosomal RNA Gene-Based Phylogenetic Analysis of Abundant Bacteria in River, Canal and Potable Water in Bangkok, Thailand

Yamaguchi

Nishiguchi

Utrarachkij

et al. 2013

Biological & Pharmaceutical Bulletin

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“…The present results indicate that on-chip T-RFLP analysis is an effective tool for "on-site" bacterial community profiling in freshwater environments, as well as freshwater used for medical 20,21) and industrial purposes. 1) Furthermore, people have activities in confined habitat such as Antarctic research bases and International Space Station.…”

Section: Bacterial Community Profiling In Hydroponics Solutionmentioning

confidence: 71%

Microchip-Based Terminal Restriction Fragment Length Polymorphism for On-Site Analysis of Bacterial Communities in Freshwater

Yamaguchi

Matsukawa

Shintome

et al. 2013

Biological & Pharmaceutical Bulletin

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Assessing microbiological quality assurance by monitoring bacteria in various sources of freshwater used for human consumption, recreation, and food preparation is important for a healthy life. Bacterial number and their community structure in freshwater should be determined as quickly as possible, and "realtime" and "on-site" microbiological methods are required. In this study, we examined the protocol for microchip-based terminal restriction fragment length polymorphism (T-RFLP) analysis, which uses microchip electrophoresis for rapid microbial community analysis. The availability of microchip-based T-RFLP was compared with conventional T-RFLP analysis, which uses a capillary electrophoresis system, with freshwater samples (spring water, river water, groundwater, and hydroponics solution). The detection limit of targeted bacteria by on-chip T-RFLP analysis was 1% (10 3 cells/mL). The fragment sizes determined by the two analysis methods were highly correlated (r 2 =0.98). On-chip T-RFLP analysis was completed within 15 min. T-RFLP profiles of nine hydroponics solution samples were analyzed by multidimensional scaling. Considerable changes and stability in bacterial community structure during hydroponic culture were detected by both analyses. These results show that on-chip T-RFLP analysis can monitor changes in bacterial community structure, as well as conventional T-RFLP analysis. The present results indicate that on-chip T-RFLP analysis is an effective tool for rapid and "on-site" bacterial community profiling in freshwater environments, as well as freshwater used for medical and industrial purposes.

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“…Although only 2 colonyforming units were visually confirmed Figure 2b, left , more microcolonies were detected by MFQ Figure 2b, right . These may also result in the detection of viable but non-culturable bacteria in the dialysis fluids Yamaguchi et al, 2007 . These microcolonies were capable of growing to a visually detectable size within a 14-day culture period, if the solution volume for the measurement could be reduced to avoid the inhibition by bacterial overload Osono and Hayama, 2010 .…”

Section: Acknowledgementmentioning

confidence: 99%

Rapid Detection of Microbes in the Dialysis Solution by the Microcolony Fluorescence Staining Method (Millflex Quantum)

Osono

Kobayashi

Inoue³

et al. 2014

Biocontrol Sci.

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A chemiluminescence system, Milliflex Quantum MFQ , to detect microcolonies, has been used in the pharmaceutical field. In this study, we investigated aquatic bacteria in hemodialysis solutions sampled from bioburden areas in 4 dialysis faculties. Using MFQ, microcolonies could be detected after a short incubation period. The colony count detected with MFQ after a 48-hour incubation was 92 39 , compared to that after the conventionally used 7-14-day incubation period in addition, the results also showed a linear correlation. Moreover, MFQ-based analysis allowed the visualization of damaged cells and of the high density due to the excessive amount of bacteria. These results suggested that MFQ had adequate sensitivity to detect microbacteria in dialysis solutions, and it was useful for validating the conditions of conventional culture methods.Key words Membrane filter method / Water bacteria / R2A / Hemodialysis / Validation.

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Rapid monitoring of bacteria in dialysis fluids by fluorescent vital staining and microcolony methods

Cited by 27 publications

References 20 publications

16S Ribosomal RNA Gene-Based Phylogenetic Analysis of Abundant Bacteria in River, Canal and Potable Water in Bangkok, Thailand

16S Ribosomal RNA Gene-Based Phylogenetic Analysis of Abundant Bacteria in River, Canal and Potable Water in Bangkok, Thailand

Microchip-Based Terminal Restriction Fragment Length Polymorphism for On-Site Analysis of Bacterial Communities in Freshwater

Rapid Detection of Microbes in the Dialysis Solution by the Microcolony Fluorescence Staining Method (Millflex Quantum)

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