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Polarization Fluorometry to Determine Cell Density with Fluorochrome for DNA

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Abstract:

There is a need for a rapid and sensitive technique to estimate a small number of cells. The total number of both living and dead cells suspended in a solution can be measured directly with the use of the Coulter counter or a flow-cytometer, but both of these measurements are inaccurate for small bacteria, such as an Escherichia coli cell (0.6 μm diameter × 2 μm length). The bacterial density is determined by the measurement of the turbidity of a cell suspension with a nepherometer or, more conveniently, with a spectrophotometer. However, foreign particles (e.g., dust, protein, or lipid) often increase turbidity and result in an error in the assay. Fluorometric assay for DNA content provides a sensitive estimation of the cell density free from turbidity changes. For example, Hill and Whatley used an antibiotic drug (mithramycin) as a labeling fluorochrome to measure cell DNA density with a lower limit of 0.5 μg. Recently, more sensitive quantification of DNA has been achieved with a bibenzimidazole dye, Hoechst 33258 (H33258). This dye reacts with the adenine-thymine-rich region of DNA, resulting in a high quantum efficiency for the determination. Downs and Wilfinger applied H33258 to quantify DNA measurement, and they achieved detection limits as low as 4 ng for extracted DNA from pituitary cells.

Keywords: Background correction; Determination of cell number; Fluorescence for DNA; Hoechst 33258 fluorochrome; Polarization fluorometry

Document Type: Short Communication

DOI: http://dx.doi.org/10.1366/0003702864815619

Affiliations: Laboratory for Cytochemistry, Department of Anatomy, School of Medicine, Tokushima University, Kuramoto, Tokushima 770, Japan

Publication date: January 1, 1986

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