1887

Abstract

cannot be cultured, so ascertaining viability of the organism remains a major obstacle, impeding many avenues of investigation. This study tested a two-colour, Syto9 and propidium iodide, fluorescence assay, which scores for membrane damage in individual bacilli, to determine if a rapid direct-count viability-staining technique can be reliably applied to . A variety of experimental conditions were employed to validate this technique. This technique was also used to correlate the viability of with the course of athymic mouse foot pad infection to optimize the provision of viable as a research reagent. The data show that in untreated suspensions of there is a good correlation between the metabolic activity of leprosy bacilli and their membrane damage. Fixation of with ethanol, paraformaldehyde and gluteraldehyde completely suppressed their metabolic activity but showed little effect on their membrane integrity. The present study also showed that the metabolic activity of declines more than the extent of membrane damage at 37 °C within 72 h, but that they are not significantly affected at 33 °C. Irradiation at 10 Gy showed high numbers of dead bacilli by the staining method. The results show that the reliability of metabolic-activity data as well as viability-staining data is dependent on the method by which is killed. This staining method helped us predict reliably that the smaller -infected athymic mouse foot pad seen early in infection, between 4 and 5 months, yields markedly better quality leprosy bacilli than older, larger foot pad infections, as defined by their metabolic activity and membrane integrity.

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2005-03-01
2019-11-21
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