European Society of Mycobacteriology - Instituto Nacional de Saúde ...

PP-106
RAPID DETECTION OF RESPIRATORY ACTIVE
MYCOBACTERIA BY AURAMINE O-CTC DOUBLE STAINING
Ichijo, Tomoaki; Izumi, Yoko; Yamaguchi, Nobuyasu; Nasu, Masao
Osaka University
Introduction
Clarifying the dynamics of nontuberculosis mycobacterial (NTM) and determining their hot spots in aquatic environments
are important to prevent their infection to human beings. For this purpose, methods for the detection of active
mycobacterial cells are required. Culture methods are widely used for detection of mycobacterial cells, but these methods
are rather difficult to detect mycobacteria quantitatively and rapidly. Mycobacterial cells are quantifiable under a
fluorescent microscope within several hours with acid-fast staining. In addition, several fluorochromes are available to
determine the bacterial activities. In this study, we attempted to detect mycobacteria with physiological activity rapidly
by combining Auramine O acid-fast staining with 5-cyano-2, 3-ditoryl tetrazolium (CTC) as an indicator of respiratory
activity (Auramine O-CTC).
Methods
Mycobacterium smegmatis was stained with CTC and fixed with formaldehyde. Fixed cells were stained with Auramine
O and observed under an epifluorescent microscope with blue excitation.
Results And Discussion
We firstly optimized the condition of the Auramine O-CTC double staining method to address the following problems;
(i) CTC formazan dissolved in the decolorization step and (ii) fluorescent intensity of Auramine O was decreased by
fluorescent resonant energy transfer (FRET). Specificity of this method was confirmed by staining non-targeted bacteria.
Results can be obtained within 90 min by the optimized procedure, while more than one week is required for the culture-dependent
approach. In conclusion, the Auramine O-CTC double staining method was useful for rapid detection of
respiratory active mycobacteria. This method may contribute to identifying dynamics of NTM in aquatic environments.
Acknowledgement
This work was supported by the JSPS Grant-in-Aid for Scientific Research (A)(20249007).
178 ESM 2009