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

Abstract

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.

Tackling the minority: sulfate-reducing bacteria in an archaea-dominated subsurface biofilm

“…Biofilms were supplemented with 100 ml spring water and 10 ml 50 mM CTC (5-cyano-2,3-ditoryl tetrazolium chloride; Stellmach, 1984;Stellmach and Severin, 1986;Yoshida and Hiraishi, 2004), which was prepared under anaerobic conditions (N 2 gas phase). After an anaerobic incubation of 2 h at 11 1C in a waterbath, the biofilms were removed and underwent fixation, FISH in suspension (Delta495a/b/c mix, RG, performed similar to Wallner et al, 1993) and subsequent DAPI (4 0 ,6-diamidino-2-phenylindole) staining.…”

“…The formation of CTC-formazan precipitates can be attributed to biological redox reactions, for example, respiratory electron transport, and thus provide evidence for the metabolic activity (Stellmach, 1984;Stellmach and Severin, 1986;Yoshida and Hiraishi, 2004).…”

Nitrate removal performance of Diaphorobacter nitroreducens using biodegradable plastics as the source of reducing power

“…The direct total cell count and total viable count in the reactors were measured by epifluorescence microscopy using a LIVE/DEAD® BacLight™ Bacterial Viability kit (Invitrogen Corporation, Carlsbad, USA) and an Olympus BX-50 phase-contrast/epifluorescence microsope equipped with an FD-120M CCD camera (Flovel Co., Tokyo, Japan) as described [21]. Cells attached on the surface of solid substrate were observed by epifluorescence microscopy with SYBR Green (Invitrogen) staining [21] and scanning electron microscopy (SEM) using a JEOL SEM system.…”

“…Cells attached on the surface of solid substrate were observed by epifluorescence microscopy with SYBR Green (Invitrogen) staining [21] and scanning electron microscopy (SEM) using a JEOL SEM system. Growth in liquid cultures was monitored by measuring OD 660 …”

A Great Leap forward in Microbial Ecology

“…It is now recognized that wastewater treatment processes harbor a vast variety of microorganisms, most of which are still yet-to-be cultured, and hence uncharacterized (3,39,168,173). However, a number of new exciting insights into microbial structure and function in wastewater treatment processes have been recently gained by applying culture-independent molecular approaches, which has significantly expanded our understanding of process design, operation and control.…”