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Flow Cytometry at San Jose State University

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Introduction to Tetrahymena Nutrition, Fluorescence Microscopy, Flow Cytometry, and Experimental Microbiology.

 

Introduction to Tetrahymena

Tetrahymena are common holotrichic ciliates found in moist habitats. They are laboratory grown in pure liquid culture and obtain nutrients by absorption or by ingestion (phagocytosis) of smaller microbes. They have a specialized oral membrane and feeding orifice to take in a wide range of prey such as bacteria, fungi, or other protists. Tetrahymena is used as a model organism to study holozoic nutrition by feeding the Tetrahymena yeast or synthetic beads that have been “labeled” with a fluorescent dye. We will study the process of phagocytosis using fluorescence microscopy and flow cytometry. Both of these study techniques depend upon the interaction between high energy light and cells. 

 

Introduction to Optics

White light can be separated into distinct colors based on wavelength. The visible spectrum starts at short wavelength (high energy), violet light at about 400 nm and ends at long wavelength (low energy), red light at about 750 nm (Fig 1).

Fig. 1. Properties of light in the visible spectrum.

 

Special dyes called fluorochromes are used to stain cell components. Fluorescence occurs when a fluorochrome absorbs high energy (short wavelength) light, and emits energy (or fluoresces) lower energy (longer wavelength) light. Many different dyes can be used as fluorochromes. The widely used fluorochrome, fluorescein isothiocyanate (FITC), absorbs light at ~490nm and emits at ~555-570nm (Fig 2).

 

   

 

Fig. 2. Excitation and emission spectra of the FITC.

 

Fluorescence microscopy and flow cytometry utilyze special filters and dichroic mirrors to excite fluorochromes associated with a cell at the high energy, excitation (absorbtion) wavelength, and detect emission at lower energy, emission wavelengths. 

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