A Combined Fluorochrome Method for Quantitation of Metabolically Active and Inactive Airborne Bacteria

Abstract

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 epi uorescent method to estimate the quantity and activity of airborne bacteria aerosolized into full-scale rooms. Midget impingers, lled with the uorescing 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 uorescein derivative and counted with an epi uorescent microscope. The choice of uorochrome stains allowed concurrent identi cation of active and inactive bacteria in the same microscopic eld. Airborne bacterial numbers determined by epi uorescence microscopy were compared to standard, nonselective colony counts cultured from midget impingers operated under identical conditions. Direct epi uorescent 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%.

Evaluation of a Methodology for Quantifying the Effect of Room Air Ultraviolet Germicidal Irradiation on Airborne Bacteria

“…These results are comparable to our preliminary results for UVGI effectiveness of 0¡ » 100%, for total UVGI, ventilation, and other removal rates of 2¡ » 6 h ¡ 1 . For a subset of our experiments, we tested a new method to directly measure the number of airborne bacteria without cultivation (Hernandez et al 1999). Using two different uorescing dyes to stain collected bacteria, this method counts all microbial cells that are present in the air and differentiates between those that are metabolically competent and those that are nonviable.…”

“…These experiments showed trends that deserve further exploration and identi ed aspects of the study design that could be improved. Revisions to our design include (1) using actual Mycobacterium species, (2) generating concentrations of bacterial aerosol that allow for epi uorescent microscopy techniques (Hernandez et al 1999), (3) sampling at a larger number of points with replicates, (4) installing ceiling-mounted lamps with improved louvers, (5) collecting more rened measurements of UVGI delivery to the test room (Rahn et al 1999), and (6) varying RH and UV irradiance levels. These changes have been incorporated in another series of tests that are currently underway.…”

“…For E. coli, higher concentrations were measured near the aerosol source (Figure 2). In the experiment with UVGI, this bacterium was not detected at any other sampling point even though total cell counts demonstrated that similar numbers of E. coli were present throughout the room (Hernandez et al 1999). Figure 3 compares the concentration measured near the aerosol source (near-eld) with the average concentration in the remainder of the room (far-eld).…”

Molecular Analysis of Bacterial and Circovirus Bioaerosols in Concentrated Animal Feeding Operations

“…Direct counts were calculated based on the average of all fields counted; in all cases, the coefficient of variation observed was less than 20% (Hernandez et al 1999). After testing for normality, a paired t-test, with a one-tailed distribution was applied at a 90% confidence level to test for significant differences between the direct counts recovered from each environment.…”

“…Aerosol samples were stained and enumerated using 4 6-diamidino-2-phenylindole (DAPI) (Sigma Chemicals, St. Louis, MO, USA) and previously described methods (Hernandez et al 1999). Samples were incubated with DAPI (20 μg/mL) for 5 min at 4…”