Specific detection and quantification of culturable and non-culturable mycobacteria in metalworking fluids by fluorescence-based methods.
Selvaraju-SB; Khan-IU; Yadav-JS
Lett Appl Microbiol 2008 Nov; 47(5):451-456
AIMS: To optimize and evaluate fluorescence microscopy assays for specific assessment of mycobacteria and co-contaminants, including culturable and non-culturable sub-populations, in metalworking fluids (MWF). Methods and RESULTS: Auramine-O-rhodamine (AR) staining and LIVE/DEAD BacLight Bacterial Viability staining (L/D staining) were adapted and evaluated for detection/quantification and differentiation (viable vs non-viable) of the MWF-associated mycobacteria and the background bacterial flora, respectively. The AR staining method was found to be specific to MWF mycobacteria with a minimum detection limit of 10 cells ml(-1) and was comparable to the QPCR in quantification efficiency in MWF matrix. The L/D staining-based microscopy allowed differential quantification of viable vs non-viable cells. In general, a 3-log difference was observed between the L/D microscopy count and culture count accounting for the presence of non-culturable fraction in the bacterial population in in-use MWF. The optimized AR staining- and the L/D staining-based microscopy methods have the potential for rapid, specific and differential assessment (viable vs non-viable) of MWF-associated mycobacteria and co-contaminants in field MWF. SIGNIFICANCE AND IMPACT OF THE STUDY: Early detection of MWF mycobacteria by rapid, low-cost, less-skill intensive and culture-independent fluorescence-based microscopy methods will facilitate timely intervention to protect the machine workers from occupational hazards.
Metalworking; Metalworking-fluids; Metalworking-industry; Occupational-exposure; Occupational-diseases; Occupational-health; Bacterial-cultures; Microbiology; Microscopic-analysis; Toxic-materials; Quantitative-analysis; Machine-operators; Worker-health; Workplace-monitoring;
Author Keywords: Epifluorescence microscopy; Live/dead baclight staining; Metalworking fluid; Mycobacterium immunogenum; Non-culturable; Real-time PCR; Viable but non-culturable
Jagjit S. Yadav, Environmental Genetics and Molecular Toxicology Division, Department of Environmental Health, College of Medicine, University of Cincinnati, Cincinnati, OH 45267-0056
Letters in Applied Microbiology
University of Cincinnati