Factors affecting the selection and use of tetrazolium salts as cytochemical indicators of microbial viability and activity

Thom, Susan M.; Horobin, Richard W.; Seidler, E; Barer, Michael R.

doi:10.1111/j.1365-2672.1993.tb05151.x

Cited by 119 publications

(70 citation statements)

References 32 publications

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“…In a recent review, Ullrich et al (1996) describe a growing body of evidence which suggests that CTC is unsuitable for the enumeration of bacteria in environmental samples. The possible expla-nations for obtaining lower counts by CTC than by other direct methods of enumeration were (i) not all bacteria are capable of reducing tetrazolium salts (Thom et al 1993), (ii) the assay is not sensitive enough to detect bacteria with low rates of metabolic activity (Gasol et al 1995) and (iii) although CTC has been assumed to be non-toxic to bacteria (Rodriguez et al 1992), it has been reported that bacterial metabolic activity can be directly suppressed/inhibited by CTC, causing an under-estimation of the total number of bacteria within a sample (Ullrich et al 1996).…”

Section: Discussionmentioning

confidence: 99%

Application of laser scanning for the rapid and automated detection of bacteria in water samples

Reynolds

Fricker

1999

Journal of Applied Microbiology

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It is widely accepted that the heterotrophic plate count method may not support the growth of all viable bacteria which may be present within a water sample and that alternative procedures using ‘viability markers’ may yield additional information. In this study, ChemChrome B (CB), which is converted to a fluorescent product by esterase activity, was used to stain viable bacteria (captured by membrane filtration) from potable water samples. The labelled bacteria from each sample were subsequently enumerated using a novel laser scanning instrument (ChemScan). Analysis of 107 potable water samples using this procedure demonstrated the presence of a significantly greater number of bacteria than were detected by culture (z‐test, P < 0·05). The mean number of bacteria isolated by culture on R2A agar incubated at 22 °C for 7 d was only 25·2% of the total number of viable bacteria detected using the CB/ChemScan viability assay. Further analysis of 81 water samples using a 5‐cyano‐2,3,4‐tolyl‐tetrazolium chloride (CTC) viability assay also demonstrated the presence of many viable bacteria which were not capable of growth under the culture conditions employed in this study. However, the results indicate that ChemChrome B has the ability to stain a significantly greater number of heterotrophs than CTC (z‐test, P < 0·05). In contrast, six potable waters were identified in which the CTC viability assay resulted in counts greater than those obtained using CB. The ChemScan instrument was successfully used for rapid and accurate enumeration of labelled micro‐organisms, allowing information on the total viable microbial load of a water sample to be determined within 1 h. Furthermore, the ChemScan system has the potential for use in detecting specific organisms labelled with fluorescently‐labelled antibodies or nucleic acid probes.

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Section: Discussionmentioning

confidence: 99%

Application of laser scanning for the rapid and automated detection of bacteria in water samples

Reynolds

Fricker

1999

Journal of Applied Microbiology

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“…There is some evidence that CTC may be toxic to some bacteria (Kaprelyants and Kell, 1993a;Ullrich et al, 1996) at the concentrations used, and might not work in some circumstances (Thom et al, 1993), but has been satisfactorily used to stain active bacteria from cultures (Kaprelyants and Kell, 1993a;López-Amorós et al, 1995aMcFeters et al, 1995), freshwater plankton (del Giorgio et al, 1997b;Yamaguchi and Nasu, 1997) and marine plankton (López-Amorós et al, 1998;Sieracki et al, 1999). A double staining protocol with SYTO 13 and CTC has been devised but requires a flow cytometer with double laser capabilities (López-Amorós et al, 1998).…”

Section: Single-cell Activitymentioning

confidence: 99%

Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities

Gasol¹,

Giorgio²

2000

Sci. Mar.

790

717

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SUMMARY: Flow cytometry is rapidly becoming a routine methodology in aquatic microbial ecology. The combination of simple to use bench-top flow cytometers and highly fluorescent nucleic acid stains allows fast and easy determination of microbe abundance in the plankton of lakes and oceans. The different dyes and protocols used to stain and count planktonic bacteria as well as the equipment in use are reviewed, with special attention to some of the problems encountered in daily routine practice such as fixation, staining and absolute counting. One of the main advantages of flow cytometry over epifluorescence microscopy is the ability to obtain cell-specific measurements in large numbers of cells with limited effort. We discuss how this characteristic has been used for differentiating photosynthetic from non-photosynthetic prokaryotes, for measuring bacterial cell size and nucleic acid content, and for estimating the relative activity and physiological state of each cell. We also describe how some of the flow cytometrically obtained data can be used to characterize the role of microbes on carbon cycling in the aquatic environment and we prospect the likely avenues of progress in the study of planktonic prokaryotes through the use of flow cytometry.

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“…Thom et al 1993), whereas others have suggested temperature (Tabor & Neihof 1984, Robarts & Sephton 1988, Sommaruga & Conde 1997, lysis (Proctor & Fuhrman 1990), grazing (Gasol et al 1995) and resource availability (del Giorgio & Scarborough 1995, Choi et al 1999. In terms of resource availability, a wide variety of sources including phytoplankton excretion and lysis (Cole et al 1988, Maurin et al 1997) and grazing and release by zooplankton (e.g.…”

Section: Resale or Republication Not Permitted Without Written Consenmentioning

confidence: 99%

Variations in the number of active bacteria in the euphotic zone of a recently flooded reservoir

Jugnia¹,

Richardot²,

Debroas³

et al. 2000

Aquat. Microb. Ecol.

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Using the CTC (5-cyano-2, 3-ditolyl tetrazolium chloride) method, we estimated the active fraction of bacterioplankton in the euphotic zone of the recently flooded Sep Reservoir, France, with the aim of evaluating its importance and dynamics in relation to temperature and some components of dissolved organic matter (DOM) (i.e. dissolved combined amino acids: DCAA; dissolved free monosaccharides: DFCHO; dissolved combined monosaccharides: DCCHO), and its metabolic significance in conjunction with a bacterial growth indicator:14 C-glucose uptake. From our results, it appears that only a small fraction (0.04 to 3.23%) of total bacterial count in this reservoir was metabolically active. Recorded correlations and multivariate regression analysis suggest that temporal variations in the number of active bacteria are mainly (80%) governed by temperature, together with the high concentrations of those components of DOM, which were present in this ecosystem but which apparently were not always available for bacterial metabolism. We also found a positive relation between the number of active bacteria and the potential heterotrophic activity, which suggest that CTC-positive bacteria are responsible for the bulk of bacterial community metabolism in this recently formed reservoir.KEY WORDS: Active bacteria · CTC · Dissolved organic matter · Temperature · Bacterial activity · New reservoir Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 22: [251][252][253][254][255][256][257][258][259] 2000 fraction of bacterioplankton (e.g. Thom et al. 1993), whereas others have suggested temperature (Tabor & Neihof 1984, Robarts & Sephton 1988, Sommaruga & Conde 1997, lysis (Proctor & Fuhrman 1990), grazing (Gasol et al. 1995) and resource availability (del Giorgio & Scarborough 1995, Choi et al. 1999. In terms of resource availability, a wide variety of sources including phytoplankton excretion and lysis (Cole et al. 1988, Maurin et al. 1997) and grazing and release by zooplankton (e.g. Hygum et al. 1997) have been identified as producing dissolved organic matter (DOM) that fuels bacterioplankton metabolism. Inputs of allochthonous DOM and physical disaggregation of detrital particles (Hadas et al. 1990) are also likely to be significant energy sources for bacterial activities. However, nearly all these studies have been conducted in natural or stabilized artificial lake ecosystems. To our knowledge, no information is available on fluctuations in the number of active bacteria in recently flooded reservoirs that are expected to contain large quantities of terrestrial detritus and organic matter, which is known to efficiently regulate bacterial activity (Paterson et al. 1997).The aim of this study was to determine the importance of the active fraction of bacterioplankton in a recently flooded reservoir, the main variables controlling their abundance (especially temperature and DOM) and their metabolic significance in conjunction with a growth indicator:14 C-glucose uptake. The numbe...

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Factors affecting the selection and use of tetrazolium salts as cytochemical indicators of microbial viability and activity

Cited by 119 publications

References 32 publications

Application of laser scanning for the rapid and automated detection of bacteria in water samples

Application of laser scanning for the rapid and automated detection of bacteria in water samples

Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities

Variations in the number of active bacteria in the euphotic zone of a recently flooded reservoir

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