Editor: Jeff Cole
Discrimination of live, anti-tuberculosis agent-injured, and dead Mycobacterium tuberculosis using flow cytometry
Article first published online: 24 MAR 2009
© 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiology Letters
Volume 294, Issue 1, pages 74–81, May 2009
How to Cite
Soejima, T., Iida, K.-i., Qin, T., Taniai, H. and Yoshida, S.-i. (2009), Discrimination of live, anti-tuberculosis agent-injured, and dead Mycobacterium tuberculosis using flow cytometry. FEMS Microbiology Letters, 294: 74–81. doi: 10.1111/j.1574-6968.2009.01549.x
- Issue published online: 2 APR 2009
- Article first published online: 24 MAR 2009
- Received 28 October 2008; accepted 10 February 2009.First published online 24 March 2009.
- flow cytometry;
- Mycobacterium tuberculosis;
Flow cytometry (FCM) using propidium iodide (PI)/bis-oxonol (BOX) staining can distinguish live, dead, and sublethally injured Escherichia coli by detecting intact vs. nonintact membranes (PI) and membrane potential (BOX). However, live bacteria, especially Mycobacterium tuberculosis, are not likely to be successfully discriminated from injured bacterium by FCM when utilizing the live/dead staining agents currently on the market. As injured cell membranes have integrity like that of live cells and are regarded as such by FCM, the distinction between live and injured cells has depended on the culture method, where injured bacteria cannot grow in general. We have previously shown that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro. In this study, we found that the chromosomal DNA of antibiotic-injured, but not live, M. tuberculosis could be cleaved within 2 h by EMA, and that the resultant decrease in the spaces of DNA base pairs could greatly inhibit the intercalation of SYTO9 in FCM. The percentage value of SYTO9+/PI− quadrant from antibiotic-injured M. tuberculosis after EMA treatment decreased by at least 80%, compared with that before EMA, but such a phenomenon did not take place in live cells. FCM (SYTO9/PI) following EMA treatment is a very rapid, simple, and effective method for discriminating live, antibiotic-injured, and dead M. tuberculosis without culture.