A combined fluorochrome method for quantitation of metabolically active and inactive airborne bacteria

Abstract

To better understand the ecology of microorganisms in the environment and to quantify the concentrations of airborne microorganisms, methods are needed to count all microbial cells that are present and differentiate between those that are metabolically competent and those that are nonviable as they exist in situ. We developed and tested a direct epifluorescent method to estimate the quantity and activity of airborne bacteria aerosolized into full-scale rooms. Midget impingers, filled with the fluorescing redox dye 5-cyano-2, 3-ditolyl tetrazolium chloride (CTC), were used to capture and stain metabolically active bacteria from room air. Both active and inactive bacteria were counterstained with a fluorescein derivative and counted with an epifluorescent microscope. The choice of fluorochrome stains allowed concurrent identification of active and inactive bacteria in the same microscopic field. Airborne bacterial numbers determined by epifluorescence microscopy were compared to standard, nonselective colony counts cultured from midget impingers operated under identical conditions. Direct epifluorescent estimates of total airborne bacteria were higher than concurrent plate counts. However, estimates of active airborne bacteria were lower than concurrent plate counts. Variability experiments showed that the direct-count method was repeatable to within 35%.