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Quantitative Cell Culture Techniques
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Sometimes it is important to know exactly how many cells are present in a liquid culture or adherent monolayer. Sometimes only the number of cells relative to a control culture or an earlier time point is what is required. At other times, it is important to distinguish the fractions of live cells versus dead, live versus apoptotic, differentiated versus undifferentiated, or labeled versus unlabeled. In all of these cases, the cells must be counted somehow. There are three general approaches to doing this: directly counting cells by eye; using a measurable parameter (absorbance, impedance) as a surrogate for cell count; or placing the cells into a specialized flow system that permits computerized cell counting. All of these methods are very commonly used, and each has its advantages and drawbacks. In this chapter, we discuss some of the most well-established ways for quantifying populations of bacteria and mammalian cells and provide some of the protocols we have developed that are designed to help sidestep the pitfalls that can occur. At the end of this chapter, you should be comfortable obtaining a bacterial IC50 growth curve; performing an end-point mammalian toxicity assay; and setting up a simple fluorescence-activated cell sorting (FACS) experiment. We also briefly introduce some of the emerging techniques, such as real-time impedance measurements for mammalian cells, imaging cytometry, and microfluidic techniques.
The application of molecular tools to study the drinking water microbiome – Current understanding and future needs
Published in Critical Reviews in Environmental Science and Technology, 2019
An alternative and most direct way to quantify microbial density is cell counting. Cells in water samples can be directly counted using microscopy or stained with fluorescent dyes and counted under an epifluorescence microscope or flow cytometry (FCM) as total cell count. Fully automated online FCM can be useful in discerning temporal bacterial dynamics at high frequency (Besmer et al., 2016; Besmer & Hammes, 2016). The usage of FCM in DSs is still limited to systems without residual disinfectants. For drinking water containing residual disinfectants, pretreatment using membrane filtration to concentrate bacteria at an appropriate density is required due to low cell number and the interference of bacteria-like particles (Besmer et al., 2016; Lautenschlager et al., 2013; Van Nevel et al., 2017), which is time-consuming and subjective (Santic, Krstulovic, & Solic, 2007).