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Volume 13, No. 6, December 2013, Pages 1755-1767 PDF(1.49 MB)  
doi: 10.4209/aaqr.2012.10.0293   

Applicability of LIVE/DEAD BacLight Stain with Glutaraldehyde Fixation for the Measurement of Bacterial Cell Concentration and Viability in the Air

Kotaro Murata, Daizhou Zhang

Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto 862-8502, Japan

 

Abstract

 

Data on the concentration and viability of microorganisms in the atmosphere provide crucial information with regard to their dispersion, roles in environmental changes, and effects on ecosystems. In this study, we investigated the application of the LIVE/DEAD® BacLight™ Bacterial Viability Kit (BacLight stain) with regard to the enumeration of viable and non-viable bacterial cells in the air, with the 4',6-diamidino-2-phenylindole (DAPI) stain used as the control of total cell counts. Two cultured bacterial strains isolated from an air sample, Bacillus subtilis and Micrococcus sp., were used in laboratory experiments. The results of BacLight staining agreed well with those of DAPI staining in detecting total cell counts, and the detection efficiency of Bacillus subtilis was 76–112%. Bacterial viability (number ratio of viable cells to total cells) showed consistency among repeated experiments with the same sample replicates, indicating the high confidence of counting viable cells, as well as total cells with the stain. Fixation of samples with glutaraldehyde prevented fluorescence bleaching at the exposure to fluorescence and increased detection accuracy. Application of the BacLight stain with and without fixation to air samples that were collected with a bio-sampler at an urban site proved the effectiveness of this approach in determining the cell concentration and viability of airborne bacteria at a 1-hour time resolution. A combination of BacLight staining and glutaraldehyde fixation treatment, in parallel with experiments without this treatment in field measurements, is proposed to ensure the detection accuracy. The method described here is able to measure the concentration and viability of bacterial cells in the air, and their variation with weather conditions.

 

 

Keywords: Bacterial abundance; Viability; Epifluorescent microscopy; BacLight stain; Bio-sampler.

 

 

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