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.
Laboratory for Cytochemistry, Department of Anatomy, School of Medicine, Tokushima University, Kuramoto, Tokushima 770, Japan
Publication date: January 1, 1986
More about this publication?
The Society publishes the internationally recognized, peer reviewed journal, Applied Spectroscopy, which is available both in print and online. Subscriptions are included with membership or can be purchased by institutional or corporate organizations. Abstracts may be viewed free of charge. Previously published as Bulletin (Society for Applied Spectroscopy)