Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

Hatzinger, Paul B.; Palmer, Patrick M.; Smith, Richard L.; Peñarrieta, Cecilia T; Yoshinari, Tadashi

doi:10.1016/s0167-7012(02)00132-x

Cited by 75 publications

(63 citation statements)

References 42 publications

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“…In vivo INT-reduction analysis-In this investigation, we followed the protocol described by Hatzinger et al (2003) with some modifications. Sample volume ranged from 20 to 280 mL, depending on the expected ETS activity level.…”

Section: Materials and Proceduresmentioning

confidence: 99%

In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities

Martínez‐García

Fernández

Aranguren-Gassis

et al. 2009

Limnology & Ocean Methods

108

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Oxygen consumption during in vitro dark bottle incubations is the most common method to estimate planktonic respiration. This method is time consuming and labor instensive and, consequently, the database of planktonic respiration rates is scarce. Electron Transport System (ETS) activity measurement has gained acceptance as a routine technique to estimate respiration due to its high sensitivity. However, the in vitro ETS assay commonly used yields potential rates. Hence, the empirically determined ratio between in situ respiration and potential ETS activity is not constant, varying over two orders of magnitude, highly limiting the routine application of this technique to estimate microbial respiration. We hypothesized that in vivo ETS activity should be a reliable estimator of in situ respiration in marine plankton communities. The aim of this study was to develop a methodology to estimate in vivo ETS activity rates by using tetrazolium salt 2‐para (iodo‐phenyl)‐3(nitrophenyl)‐5(phenyl)tetrazolium chloride (INT) as electron acceptor. We established a procedure to apply the in vivo INT reduction method to natural marine microplankton samples. Optimum incubation times were found to be lower than 2–6 h, even for oligotrophic waters. The method was tested in natural waters, covering a very wide range of respiration rates and trophic conditions. A significant linear relationship was found between in situ respiration and in vivo INT reduction. Moreover, the ratio between oxygen consumption and INT reduction was constant across contrasting environments, which reveals the INT reduction method as a simple, quick, sensitive, and robust technique suitable to substantially improve the microbial plankton respiration database.

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Section: Materials and Proceduresmentioning

confidence: 99%

In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities

Martínez‐García

Fernández

Aranguren-Gassis

et al. 2009

Limnology & Ocean Methods

108

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“…Due to this, together with their observation that CTC-staining kinetics greatly varied depending upon samples tested, these authors concluded that the CTC method is not valid to detect metabolically active bacteria. Hatzinger et al 17) reported that CTC was toxic to some groundwater isolates at concentrations normally used in viability assays. In this respect, we checked the effect of CTC on substrate-dependent oxygenuptake by P. pantotrophus.…”

Section: Discussionmentioning

confidence: 99%

“…Some investigators 17,36,43) reported that high concentrations of CTC had toxic effects on bacterial growth and metabolism. Therefore, we examined the effects of CTC on respiratory activity of activated sludge using an oxygen macroelectrode.…”

Section: Effects Of Ctc On Respiratory Activity and Growthmentioning

confidence: 99%

“…However, phosphate at a higher concentration than 10 mM was reported to have a concentrationdependent inhibitory effect on CTC reduction 39) . In addition, the toxic effect of high concentrations of CTC or its products on bacterial growth and metabolism was suggested 17,36,37,43) , although this issue should be more carefully addressed in relation to the relationship between CTC toxicity and CTC reduction efficiency. There are a number of reports on dehydrogenase-dependent histochemistry with exogenous electron transfer mediators such as phenazine methoxy sulfate (PMS) and 9-dimethylaminobenzo[a]-phenoxazin-7-ium chloride (Meldola's Blue) 4,5,25,42) .…”

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confidence: 99%

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An Improved Redox Dye-Staining Method Using 5-Cyano-2,3-Ditoryl Tetrazolium Chloride for Detection of Metabolically Active Bacteria in Activated Sludge

Yoshida

Hiraishi

2004

Microb. Environ.

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A fluorochrome-staining method using 5-cyano-2,3-ditoryl tetrazolium chloride (CTC) was improved to enhance the sensitivity of detection of metabolically active bacteria in activated sludge. The highest efficiency of CTC staining was obtained with 6 mM CTC in MOPS buffer at pH 6.5. CTC staining efficiency was also increased by adding a substrate mixture (0.05% peptone, 0.05% yeast extract and 1 mM glucose), Meldola's Blue as an electron transfer mediator and KCN. The bacterial counts detected by the CTC staining method thus improved accounted for 41-76% of the total counts in activated sludge with the average value being 54%. This value was approximately two-fold higher than those obtained by the traditional CTC staining assay which has been performed in phosphate buffer or phosphate-buffered saline without any addition of external substrate or electron transfer mediator. There was a high positive correlation between CTC-stained direct counts and the plate counts of aerobic chemoorganotrophic bacteria or viable cell counts measured using a LIVE/DEAD BacLight Bacterial Viability kit. The viable population in the activated sludges as measured by BacLight staining and plate counting accounted for 73±7.6% and 8.7±4.7% of the total population, respectively. These data suggested that most of the "viable but nonculturable" bacteria (i.e., BacLight-positive but unculturable population) potentially existing in the sludge system were so metabolically active as to be detectable by the improved CTC staining method.

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“…O sal de tetrazolium é reduzido por enzimas desidrogenases presentes no sistema transportador de elétrons de microrganismos aeróbicos, facultativos e anaeróbicos e, convertido em cristais azuis insolúveis (formazana) (Kansal et al, 1998;Hatzinger et al, 2003). Após o tempo de incubação foi aplicado 100 l tampão de lise bacteriana contendo 50% DMF (dimetilformamida), 20% SDS (dodecilsulfato de sódio) e água destilada estando a solução em pH 7,4, com posterior incubação a 37 ºC e 5% CO 2 durante a noite (Gomez-Flores et al, 1995;Kansal et al, 1998).…”

Section: Ensaio De Redução De Mttunclassified

Avaliação da virulência micobacteriana e modulação da resposta imune durante a infecção por isolados clínicos de Mycobacterium bovis e Mycobacterium tuberculosis.

Amaral¹

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Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

Cited by 75 publications

References 42 publications

In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities

In vivo electron transport system activity: a method to estimate respiration in natural marine microbial planktonic communities

An Improved Redox Dye-Staining Method Using 5-Cyano-2,3-Ditoryl Tetrazolium Chloride for Detection of Metabolically Active Bacteria in Activated Sludge

Avaliação da virulência micobacteriana e modulação da resposta imune durante a infecção por isolados clínicos de Mycobacterium bovis e Mycobacterium tuberculosis.

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