title: Specific detection and quantification of culturable and non-culturable mycobacteria in metalworking fluids by fluorescence-based methods.
abstract: OBJECTIVE
  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).
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Microbial Pathogenesis and Toxicology Laboratory, Department of Environmental Health, College of Medicine, University of Cincinnati, Cincinnati, OH 45267-0056, USA.
authors.forename: S B
authors.initials: SB
authors.lastname: Selvaraju
authors.forename: I U H
authors.initials: IU
authors.lastname: Khan
authors.forename: J S
authors.initials: JS
authors.lastname: Yadav
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D001576:Benzophenoneidum / Q000378:metabolism; D015169:Colony Count, Microbial / Q000379:methods; D007220:Industrial Waste; D050296:Microbial Viability; D008856:Microscopy, Fluorescence; D009161:Mycobacterium / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D014871:Water Microbiology
chemical_list: D007220:Industrial Waste; D001576:Benzophenoneidum