Fluorochrome and Fluorescent In Situ Hybridization to Monitor Bioaerosols in Swine Buildings

Abstract

Total microbial cell concentration, viability, and culturability of bioaerosols in swine buildings were monitored by using epifluorescence microscopy with fluorochrome (EFM/FL) with four fluorescent dyes (AO, DAPI, PI, and YOPRO-1) and by using fluorescent in situ hybridization (FISH) with five oligonucleotide probes (fl-Univ, fl-EUB, cy-EUK, fl-PSMg, and fl-NotEUB) probes. Results from these two non-culture-based methods were then compared with those using a commonly used culture method. The total microbial cell concentration measured using the non-culture-based methods was 10 to 200 times higher than that using the culture method; from 5.48 × 106 to 2.18 × 107 cells/m3 with AO staining and from 5.03 × 106 to 2.13 × 107 cells/m3 with DAPI staining, compared with the average concentration of 1.02 × 105 CFU/m3 for bacteria and 1.27 × 103 CFU/m3 for fungi by the culture method. The viability ranged from 0.27 to 0.76 by EFM/FL with PI staining, from 0.02 to 0.60 with YOPRO-1 staining, from 0.53 to 0.79 by FISH, and from 0.002 to 0.033 by the culture method. The viability by EFM/FL and FISH were much higher than the culturability. In summary, the total microbial cell concentration and viability were highly underestimated by the culture method. Based on the FISH results, eubacteria and eukaryotes were the dominant components of the bioaerosols. In conclusion, EFM/FL and FISH methods can effectively assess the total microbial cell concentration and viability of bioaerosols in environmental samples.