alexa Flow Cytometric Measurement in Water and Environmental Engineering | OMICS International
ISSN: 2155-6199
Journal of Bioremediation & Biodegradation

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Flow Cytometric Measurement in Water and Environmental Engineering

Tiina Leiviskä*
Chemical Process Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, Finland
Corresponding Author : Tiina Leiviskä
Chemical Process Engineering Laboratory
Department of Process and Environmental Engineering
University of Oulu, Finland
E-mail: [email protected]
Received: June 20, 2012; Accepted: June 22, 2012; Published: June 24, 2012
Citation: Leiviskä T (2012) Flow Cytometric Measurement in Water and Environmental Engineering. J Bioremed Biodeg 3:e115. doi:10.4172/2155-6199.1000e115
Copyright: © 2012 Leiviskä T. This is an open-a ccess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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The flow cytometric (FCM) technique has been in use for a long time in biomedical research for counting and identifying cells. During the last few decades, various other applications have also been presented in research papers, such as measuring total bacterial cell concentrations in environmental samples (natural waters, ballast water) as well as in drinking water, milk and wine. In wastewater treatment, the viability and activity of activated sludge has been evaluated by FCM. In paper mills, FCM has been used to measure bacterial cells and pitch.
With the use of FCM, cells and particles can be calculated and differentiated as they flow in a fluid stream through a beam of laser light. The magnitude of forward-scattered light is proportional to the size of the particles, whereas the side-scattered light is caused by granularity and structural complexity inside the cell [1]. With selective fluorescent chemicals, particles of interest can be stained and calculated by detecting the fluorescent light at the proper wavelength.
Viable, dead and damaged cells can be distinguished by simultaneous DNA staining with SYBR Green I and Propidium Iodide (PI). SYBR Green I stains all cells producing green fluorescence, whereas PI is capable of staining only dead or damaged cells (penetrates only inactive cells membranes) producing red fluorescence. As the dead and damaged cells contain both SYBR Green I and PI, an energy transfer phenomenon occurs in which the fluorescent emission spectrum of SYBR Green I is absorbed by PI and is no longer visible [2]. In the damaged cells, the phenomenon is not complete and therefore both green and red fluorescence is emitted. From the cytogram presenting red versus green fluorescence, all three cases can be distinguished. Generally, total cell concentrations measured by FCM have been higher in comparison to heterotrophic plate counts [3,4]. The probable reason for this is the existence of cells that are not cultivable but are detected by FCM.
Colloidal wood extractives can be stained with Nile Red, which is an uncharged hydrophobic molecule. The fluorescence of Nile Red is influenced by the polarity of its environment. It fluoresces strongly in organic solvents or attached to hydrophobic compounds but is nonfluorescent in a water environment. When the amount of samples is large, FCM is a powerful technique for screening samples for more detailed and expensive analysis of extractives (e.g. extraction with methyl tert-butyl ether and GC-MS analysis).
Method development and optimization of a new sample type and new staining procedure is sometimes laborious, due to multiple variables such as sample pretreatment, selection of staining chemical, staining time and conditions, and the operation parameters in FCM measurement. Sample pretreatment usually include at least filtration with a large pore size because the sample must not include large particles, which could clog the analyzer. A systematic approach, however, repays the effort. Thereafter, the result is at hand in a few minutes depending on the sample treatment procedure.
We are currently using FCM for oily wastewaters to measure the amount of hydrophobic particles with Nile Red staining. Besides oil, these wastewaters also contain surfactants and metals. FCM provides more information about the selectivity of studied chemical treatment methods towards oil particles instead of using only e.g. chemical oxygen demand analysis. Additionally, FCM could be suitable for bioremediation studies on oil-contaminated sites and waters, for example in measuring the viability of microorganisms after being exposed to different contaminants. In fact, the FCM technique could be utilized a lot more in water and environmental engineering.

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