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Jump To: Page 1 Page 2 Page 3 Page 4 Page 5 Paint-A-Gate Tutorial
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.
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).
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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).
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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.