Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange

Summary Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and mucopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracellular dye contents as low as 5×10^–16 mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Fluorescence polarization of stretched polytene chromosomes stained with acridine orange

The molecules of the fluorescent dye acridine orange (AO) bind to DNA in such a way that the absorption and emission dipoles lie on a plane perpendicular to the DNA axis. For this reason, definite fluorescence polarization should correspond to each mode of spatial DNA packing. A chromosome, considered as an axially symmetrical ensemble of DNA, was characterized by two experimental parameters, P and P , i.e., by polarizations of fluorescence excited by light polarized parallel and perpendicular to the symmetry axis. In view of the sequential order in the packing levels of DNA fiber in a chromosome, it was suggested that, under mechanical stretching, the highest level is disrupted first, then the others, in the order of their sequence.Isolated chromosomes of Chironomus thummi were stained with AO and stretched with needles of a micromanipulator. From the changes of P and P measured during stretching it was concluded the polytene chromosome bands have three, at least, DNA packing levels, tentatively described as 100 å fiber, 250 å coil and chromomere.     

2-colored fluorescence of differentially condensed chromosomes stained with acridine orange]

Metaphase chromosomes of the Chinese hamster differentially-condensed under the influence of a) 5-bromdeoxyuridine, b) colcemide, and c) cold, were stained with acridine-orange (AO) in concentrations of 1.5 X 10(-7) to 3 X 10(-5) g/ml at pH 4.1 to 8.5. It was found that stretched chromosomal segments fluoresced in the orange/red part of the spectrum, whereas normally condensed ones--were green. The colour distribution along the chromosome depended mainly on the AO concentration and the exposure in the UV-light, and was independent of pH and molarity of the buffer. Apparently this phenomenon cannot be attributed to the uneven denaturation of the chromosomal DNA, but rather depends on structural and/or chemical differences between euchromatin and heterochromatin.     

The differential fluorescence of bacteria stained with acridine orange and the effects of heat.

Some factors affecting the fluorescence of bacteria stained with acridine orange and the direct epifluorescent filter technique (DEFT) were studied. When bacterial cells from a chemostat operated at dilution rates between 0.1 and 0.7/h were used the differential fluorescence observed in the DEFT related to cell 'activity' and the orange fluorescence, which was predominant at high growth rates, may be related to an increase in the RNA content of the cells. Heat affected the colour of cell fluorescence and this was dependent on the cell type and, in particular, age. Uptake of acridine orange into the cells was also found to be an important factor determining the colour of fluorescence. However, with heat-treated cells there was no correlation between the amount of uptake and colour of fluorescence. The relative amounts and degree of denaturation of the different types of nucleic acids remaining in the cells after heat treatment appeared primarily to determine the colour of fluorescence.     

The differential fluorescence of bacteria stained with acridine orange and the effects of heat

Some factors affecting the fluorescence of bacteria stained with acridine orange and the direct epifluorescent filter technique (DEFT) were studied. When bacterial cells from a chemostat operated at dilution rates between 0.1 and 0.7/h were used the differential fluorescence observed in the DEFT related to cell 'activity' and the orange fluorescence, which was predominant at high growth rates, may be related to an increase in the RNA content of the cells. Heat affected the colour of cell fluorescence and this was dependent on the cell type and, in particular, age. Uptake of acridine orange into the cells was also found to be an important factor determining the colour of fluorescence. However, with heat-treated cells there was no correlation between the amount of uptake and colour of fluorescence. The relative amounts and degree of denaturation of the different types of nucleic acids remaining in the cells after heat treatment appeared primarily to determine the colour of fluorescence.     

A laser-light pulse counting method for automatic and sensitive counting of bacteria stained with acridine orange

A laser-light pulse counting method was developed and investigated for its ability automatically to count acridine orange-stained bacteria and microcolonies of bacteria on the surface of polycarbonate membrane filters. The system consisted of an Argon laser which delivered a 5 μm diameter spot of coherent 488 nm wavelength light which was raster scanned across a 5×2 mm area of the membrane. Fluorescence pulses of orange light ( ca 650 nm) were detected via a dichroic mirror and barrier filter with a photomultiplier tube. For microcolony preparations a good relationship between the number of light pulses and cell density down to below 10²/ml was observed, but at lower cell densities counting was not reliable, probably because of fluorescent debris. The method was also able to count single cells, but background auto-fluorescence and photo-bleaching produced a high and variable background signal in some samples.