A fluorescence-based assay for measuring the viable cell concentration of mixed microbial communities in soil

Pascaud, Alice; Amellal, Samira; Soulas, Marie-Louise; Soulas, Guy

doi:10.1016/j.mimet.2008.09.016

Cited by 31 publications

(23 citation statements)

References 25 publications

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“…The inference is that LIVE/DEAD ® BacLight™ Bacterial Viability Kit may not be suitable for mixed populations of unknown species. This view is supported by a study which investigated the proportions of live and dead bacteria in soil samples which proved unsuccessful despite complex mathematical compensations and careful dilution procedures (Pascaud et al, 2009). …”

Section: Dye Based Methods For Viability and Vitalitymentioning

confidence: 99%

A review of practical tools for rapid monitoring of membrane bioreactors

2016

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The production of high quality effluent from membrane bioreactors (MBRs) arguably requires less supervision than conventional activated sludge (CAS) processes. Nevertheless, the use of membranes brings additional issues of activated sludge filterability, cake layer formation and membrane fouling. From a practical standpoint, process engineers and operators require simple tools which offer timely information about the biological health and filterability of the mixed liquor as well as risks of membrane fouling. To this end, a range of analytical tools and biological assays are critically reviewed from this perspective. This review recommends that Capillary Suction Time (CST) analysis along with Total Suspended and Volatile Solids (TSS/VSS) analysis is used daily. For broad characterisation, total carbon and nitrogen analysis offer significant advantages over the commonly used chemical and biological oxygen demand (COD/BOD) analyses. Of the technologies for determining the vitality of the microbial biomass the most robust and reproducible, are the second generation adenosine-5'-triphosphate (ATP) test kits. Extracellular polymer concentrations are best monitored by measurement of turbidity after centrifugation. Taken collectively these tools can be used routinely to ensure timely intervention and smoother operation of MBR systems.

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Section: Dye Based Methods For Viability and Vitalitymentioning

confidence: 99%

A review of practical tools for rapid monitoring of membrane bioreactors

2016

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“…Currently, there is a shift to applying fluorophore-based assays to environmental samples. The fluorescence light re-emitted from excited fluorophores may be measured using various forms of epifluorescence microscopy, flow cytometers, and microplate readers among others (Pascaud et al 2009;Cai et al 2014). Few studies have incorporated the use of microplate readers, although it has been proposed that rapid and accurate readings of fluorescence produced from fluorophores used in viability assays may be performed directly by using these readers (Pascaud et al 2009;Cai et al 2014).…”

Section: Introductionmentioning

confidence: 99%

“…The fluorescence light re-emitted from excited fluorophores may be measured using various forms of epifluorescence microscopy, flow cytometers, and microplate readers among others (Pascaud et al 2009;Cai et al 2014). Few studies have incorporated the use of microplate readers, although it has been proposed that rapid and accurate readings of fluorescence produced from fluorophores used in viability assays may be performed directly by using these readers (Pascaud et al 2009;Cai et al 2014). However, it has been reported that simply extrapolating previously established protocols for fluorophores in combination with microplate readers can cause poor results, and it is therefore necessary for samplespecific optimization (Pascaud et al 2009).…”

Section: Introductionmentioning

confidence: 99%

Assessment of microbial viability in municipal sludge following ultrasound and microwave pretreatments and resulting impacts on the efficiency of anaerobic sludge digestion

Cella

Akgül

Eskicioğlu

2015

Appl Microbiol Biotechnol

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A range of ultrasonication (US) and microwave irradiation (MW) sludge pretreatments were compared to determine the extent of cellular destruction in micro-organisms within secondary sludge and how this cellular destruction translated to anaerobic digestion (AD). Cellular lysis/inactivation was measured using two microbial viability assays, (1) Syto 16® Green and Sytox® Orange counter-assay to discern the integrity of cellular membranes and (2) a fluorescein diacetate assay to understand relative enzymatic activity. A range of MW intensities (2.17-6.48 kJ/g total solids or TS, coinciding temperatures of 60-160 °C) were selected for comparison via viability assays; a range of corresponding US intensities (2.37-27.71 kJ/g TS, coinciding sonication times of 10-60 min at different amplitudes) were also compared to this MW range. The MW pretreatment of thickened waste activated sludge (tWAS) caused fourfold to fivefold greater cell death than non-pretreated and US-pretreated tWAS. The greatest microbial destruction occurred at MW intensities greater than 2.62 kJ/g TS of sludge, after which increased energy input via MW did not appear to cause greater microbial death. In addition, the optimal MW pretreatment (80 °C, 2.62 kJ/g TS) and corresponding US pretreatment (10 min, 60 % amplitude, 2.37 kJ/g TS) were administered to the tWAS of a mixed sludge and fed to anaerobic digesters over sludge retention times (SRTs) of 20, 14, and 7 days to compare effects of feed pretreatment on AD efficiency. The digester utilizing MW-pretreated tWAS (80 °C, 2.62 kJ/g TS) had the greatest fecal coliform removal (73.4 and 69.8 % reduction, respectively), greatest solids removal (44.2 % TS reduction), and highest overall methane production (248.2 L CH4/kg volatile solids) at 14- and 7-day SRTs. However, despite the fourfold to fivefold increases in cell death upon pretreatment, improvements from the digester fed MW-pretreated sludge were marginal (i.e., increases in efficiency of less than 3-10 %) and likely due to a smaller proportion of cells (10-20 %) in the polymeric network and mixed sludge fed to digesters.

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“…It was concluded that the TBN values estimated by AO and BacLight staining are relatively accurate and interchangeable for quantitative interpretation and that IA provides better precision than does VC. As a prudent measure, it is suggested to avoid use of DAPI staining for comparative studies investigating accuracy of novel cell-counting methods.Bacterial abundance is an instrumental parameter in assessing the roles of bacteria in the environments (18,27,30,45). While a variety of techniques are available (1,30,53,60), staining bacterial cells with acridine orange (AO) (29) or 4Ј,6-diamidino-2-phenylindole (DAPI) (48) and counting them on black polycarbonate (PC) filters by epifluorescence microscopy have become the standard procedure for direct counting (9,18,30).…”

mentioning

confidence: 99%

“…Bacterial abundance is an instrumental parameter in assessing the roles of bacteria in the environments (18,27,30,45). While a variety of techniques are available (1,30,53,60), staining bacterial cells with acridine orange (AO) (29) or 4Ј,6-diamidino-2-phenylindole (DAPI) (48) and counting them on black polycarbonate (PC) filters by epifluorescence microscopy have become the standard procedure for direct counting (9,18,30).…”

mentioning

confidence: 99%

Agreement, Precision, and Accuracy of Epifluorescence Microscopy Methods for Enumeration of Total Bacterial Numbers

Seo

Ahn

2010

Appl Environ Microbiol

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To assess interchangeability of estimates of bacterial abundance by different epifluorescence microscopy methods, total bacterial numbers (TBNs) determined by most widely accepted protocols were statistically compared. Bacteria in a set of distinctive samples were stained with acridine orange (AO), 4-6-diamidino-2-phenylindole (DAPI), and BacLight and enumerated by visual counting (VC) and supervised image analysis (IA). Model II regression and Bland-Altman analysis proved general agreements between IA and VC methods, although IA counts tended to be lower than VC counts by 7% on a logarithmic scale. Distributions of cells and latex beads on polycarbonate filters were best fitted to negative binomial models rather than to Poisson or log-normal models. The fitted models revealed higher precisions of TBNs by the IA method than those by the VC method. In pairwise comparisons of the staining methods, TBNs by AO and BacLight staining showed good agreement with each other, but DAPI staining had tendencies of underestimation. Although precisions of the three staining methods were comparable to one another (intraclass correlation coefficients, 0.97 to 0.98), accuracy of the DAPI staining method was rebutted by disproportionateness of TBNs between pairs of samples that carried 2-fold different volumes of identical cell suspensions. It was concluded that the TBN values estimated by AO and BacLight staining are relatively accurate and interchangeable for quantitative interpretation and that IA provides better precision than does VC. As a prudent measure, it is suggested to avoid use of DAPI staining for comparative studies investigating accuracy of novel cell-counting methods.Bacterial abundance is an instrumental parameter in assessing the roles of bacteria in the environments (18,27,30,45). While a variety of techniques are available (1,30,53,60), staining bacterial cells with acridine orange (AO) (29) or 4Ј,6-diamidino-2-phenylindole (DAPI) (48) and counting them on black polycarbonate (PC) filters by epifluorescence microscopy have become the standard procedure for direct counting (9,18,30). The Live/Dead BacLight staining kit, which is widely accepted as a rapid measure of viability of individual cells, also provides a total count of bacteria (10). Currently, most studies reporting total bacterial numbers (TBNs) use one of the three staining methods described above. However, the basic question of which fluorochrome to use for a given samples still presents challenges, as comparative studies using two or more of these fluorochromes have often yielded conflicting results (10,17,20,34,37,40,49,52,54,57,58).A more perplexing question is whether TBN values based on different fluorochromes are interchangeable for a quantitative interpretation incorporating TBN data from different methods. A large-scale intersystem study, an analysis of long-term collection of longitudinal data, or a collaborative study by multiple laboratories often requires an amalgamated use of TBN values from different fluorochromes. Apart from the interc...

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A fluorescence-based assay for measuring the viable cell concentration of mixed microbial communities in soil

Cited by 31 publications

References 25 publications

A review of practical tools for rapid monitoring of membrane bioreactors

A review of practical tools for rapid monitoring of membrane bioreactors

Assessment of microbial viability in municipal sludge following ultrasound and microwave pretreatments and resulting impacts on the efficiency of anaerobic sludge digestion

Agreement, Precision, and Accuracy of Epifluorescence Microscopy Methods for Enumeration of Total Bacterial Numbers

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