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. © 2004, Japanese Society of Microbial Ecology & The Japanese Society of Soil Microbiology. All rights reserved.