Fluorochrome and flow cytometry to monitor microorganisms in treated hospital wastewater

Abstract

Flow cytometry with a fluorescent technique (FCM/FL), epifluorescence microscopy with a fluorescent technique (EFM/FL), and a culture method were used and compared to study the microorganism population profiles in wastewater treatment. In the two non-culture methods (FCM/FL and EFM/FL), four fluorescent dyes [acridine orange (AO), 4′,6-diamino-2-phenylindole dihydrochloride (DAPI), propidium iodide (PI), and YOPRO-1] were used to determine the total concentration and viability of microorganisms in the wastewater samples. Results showed that the total cell concentrations (both the bacteria and fungi) determined by using the non-culture-based methods were 18 to 67 times higher than those by the culture method (p = 0.036): the total cell concentration ranged from 1.10 × 10⁷ to 2.44 × 10⁸ cells/mL determined by both FCM and EFM with AO-staining method, and from 1.02 × 10⁷ to 2.00 × 10⁸ cells/mL by EFM with DAPI-staining method, whereas the culturable concentration of bacteria and fungi ranged from 0 to 3.22 × 10⁶ CFU/mL and from 0 to 4.13 × 10⁵ CFU/mL, respectively. No difference in total concentrations between dyes (AO and DAPI) and methods (FCM and EFM) were observed. By using EFM method, the microorganism viability ranged from 0.24 to 0.86 with PI staining and from 0.09 to 0.74 with YOPRO-1 staining. In the FCM analysis, the microorganism viability ranged from 0.23 to 0.87 with PI staining and from 0.18 to 0.73 with YOPRO-1 staining. In addition, the cultivability of microorganism ranged from 0 to 0.105 by the culture method. The total concentrations and viabilities of microorganisms were highly underestimated by the culture method. Results also showed that the viabilities determined by using either EFM/FL or FCM/FL were significantly higher than the cultivabilities. In addition, significant difference in viability between PI and YOPRO-1 for both EFM and FCM analysis was observed. However, the difference in viability between EFM and FCM depended on dyes. In regard to the difference between bacteria and fungi, significant difference in total concentration, viability, and cultivability was observed. In conclusion, the EFM/FL and FCM/FL methods can effectively assess total concentration and viability of microorganisms in environmental samples.

Keywords:

• Fluorochrome
• flow cytometry (FCM)
• epifluorescence microscopy (EFM)
• viability
• cultivability.

Acknowledgments

This work was partially supported by grant NSC 93-2320-B-002-050- from the National Science Council, Republic of China. Pei-Shih Chen was supported by a graduate scholarship from the same grant during the part of this research effort.