Evaluation of the Redox Dye 5-Cyano-2,3-Tolyl-Tetrazolium Chloride for Activity Studies by Simultaneous Use of Microautoradiography and Fluorescence In Situ Hybridization

Abstract

Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only 65% of the active Thiothrix cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells.

Cytometric evidence reconciling the toxicity and usefulness of CTC as a marker of bacterial activity

“…1) indicates that not all cells are killed instantly, but rather many continue to function hours after exposure to CTC. In a study combining microautoradiography, fluorescent in situ hybridization and CTC, Nielsen et al (2003) found that all cells labeled with CTC were also labeled as positive by the other 2 techniques, thus discrediting the idea that CTC toxicity would impair the ability of scoring these cells as positive.…”

“…We suspect that these procedures caused unbalanced bacterial growth and high respiration rates in small bacteria, resulting in a CTF granule that broke the cell. Large bacteria, such as the benthic bacteria that Epstein & Rossell (1995) used for their experiments, or most coastal and freshwater bacteria may well be large enough to accommodate a large CTF granule (or even >1 crystal cell -1 if they are sufficiently large; Nielsen et al 2003) without cell breakage. It is important to mention here that the relationship between 'missing cells' and red-only particles reported in Fig.…”

The use of fluorescent probes to assess viability of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis by flow cytometry

“…Various fluorescent probes have the ability to explore different properties of the cell, such as enzyme activity (Kaneshiro et al, 1993;Diaper & Edwards, 1994a, 1994bEndo et al, 1997;Bunthof et al, 1999;Bunthof et al, 2001;Ben-Amor et al, 2002;Luppens et al, 2003); cytoplasmic membrane permeability (Magariños et al, 1997;Porter et al, 1997;Williams et al, 1998;Gregori et al, 2001;Hewitt & Nebe-Von-Caron, 2001), membrane potential (Mason et al, 1995;Langsrud & Sundheim, 1996;Lopes-Amoros et al, 1997;Ludovico et al, 2001), respiratory activity (Kaprelyants & Kell, 1993a, 1993bGunasekera et al, 2003;Yamaguchi et al, 2003;Nielsen et al, 2003), relative DNA content (Sgorbati et al, 1996;Bernander et al, 1998) and pH gradient (Breeuwer et al, 1996;Chitarra et al, 2000). Several of these parameters can be used as indicators of viability.…”

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

“…The CTC reduction method has been used to detect viable or respiring cells of microorganisms in purified and drinking water 10,23,31,32) , groundwater 17) , aquatic ecosystems 9,14,26,28,[34][35][36][37][38] , sediment 16,28) , soil 7,33,45,46) and bioreactors 11) . This method also has been applied to wastewater and activated sludge systems 27,44) . In addition, it has been shown that the viability of anaerobic bacteria is detectable by CTC staining 6,15) .…”

“…Recently, the CTC staining method was also compared to microautoradiography and fluorescence in situ hybridization (FISH) to detect viable bacteria in activated sludge 27) . In most cases, the CTC staining method was shown to give unstable scores as being positive or to have low staining efficiency compared to other viability tests.…”