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.     

The use of acridine orange for testing blood platelet integrity

Two processes are involved in the accumulation of acridine orange in human blood platelets. One follows a diffusion like kinetics and is independent of the ATP level whereas the second one can be completely abolished by ATP depletion. The acridine orange incorporation rate seems to be a suitable parameter for testing platelet integrity. It reflects very sensitively the influence of the preparation method as well as of anticoagulating substances used on the stability of platelet suspensions. The rates of acridine orange incorporation and of aggregation were measured in platelet-rich plasma and in saline suspended platelets after gel filtration, respectively, over a period of 120 min storage. Both rates are influenced to a different degree by anticoagulating agents such as citrate, heparin and EDTA. When contact with anticoagulating agents during platelet preparation is avoided, platelets show a constant acridine orange incorporation and aggregation during storage and the smallest morphological alteration.     

Fluorescence decay and quantum yield characteristics of acridine orange and proflavine bound to DNA

Fluorescence properties (quantum yield, decay curve, lifetime and polarization) of acridine orange and proflavine bound to DNA were examined as a function of nucleotide to dye (P/D) ratio. First, mean fluoiescence lifetimes were determined by the phase-shift measurements. The lifetime and quantum yield of acridine orange increased in a parallel fashion with increasing P/D ratio. There was no parallel relation between the lifetime and quantum yield for proflavine; the lifetime showed a minimum around P/D = 10. Next, fluorescence decay curves were measured by the monophoton counting technique and analyzed with the aid of the method of moments and the Laplace transform method. The results showed that the fluorescence decay of bound acridine orange was exponential above P/D = 10. On the other hand, the decay of bound proflavine was exponential above P/D = 100, but markedly deviated from exponentiality with decreasing P/D ratio. The results of fluorescence polarization suggested that this phenomenon is the result of Förster energy transfer between proflavine molecules bound to the fluorescent site (AT pair) and bound to the quenching site (GC pair). Critical transfer distances were 26-4 and 37.0 Å, respectively, for bound proflavine and acridine orange.     

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

Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and muscopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracelluar dye contents as low as 5 X 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.     

Molecular Complexes of acridine orange and nucleosides

Summary The addition of various nucleosides to the aqueous AO solution brings about the red shift of the absorption band of AO monomer and the enhancement of the AO fluorescence emission. These phenomena are attributable to the formation of a kind of molecular complex between AO monomer and nucleoside.The absorption and fluorescence characteristics and their thermal behaviours enable us to determine the association constant and the binding energy. The binding energy of AO with purines is larger than that with pyrimidines, and the association constant between AO and the deoxyribonucleoside is larger than that between AO and the ribonucleoside. For the molecular complexes dealt with, the face-to-face arrangement of AO and nucleoside, linking of AO with sugar by hydrogen bridge, may be more preferential than the side-by-side arrangement with the direct linkage between AO and nucleic acid base by hydrogen bonding. The Van der Waals-London interactions may be one of the essential factors for the binding in these molecular complexes.The association constant and the binding energy for AO-DNA and -RNA systems were also determined. The magnitude of these quantities seems to reflect the difference in the structure of these nucleic acids. The rather open structure of RNA compared with DNA is in favour of affinity with AO and gives the larger association constant than that for AO-DNA. The binding energy is somewhat larger for AO-DNA complex than for AO-RNA complex, probably due to the structural difference of the base arrangement; the stacked base pairs for DNA and the stacked bases for RNA.This may explain the selective degradation [12] of guanine photo-sensitized by dyes either in the free state or when incorporated to DNA or RNA, as discussed elsewhere.     

Interaction between acridine orange and polyriboadenylic acid

The absorption spectra and circular dichroism of aqueous solutions of acridine orange mixed with polY(riboadenylic acid) [poly(rA)] have been measured for different mixing ratios at acid and neutral pH. The binding ratio of dye to poly(rA) has been determined by equilibrium dialysis. At acid pH where poly(rA) is in a double-stranded helix, monomeric dye molecules are intercalated between base pairs, first sparsely and then at neighbouring sites with mutual coupling, as the nucleotide-to-dye mixing ratio decreases. In the presence of excess dye, dimeric dye molecules of antiparallel type are bound to phosphate groups electrostatically and stack together to form linear sequences along a poly(rA) chain. At neutral pH where poly(rA) is single-stranded, isolated intercalation of monomeric dye molecules can occur in the helical parts. At intermediate mixing ratios, half-intercalated dimeric dye molecules are bound to adjacent sites and electronically coupled, inducing characteristic circular dichroism. In the presence of higher amounts of dye, external stacking of dimeric dye molecules of antiparallel type occurs along a poly(rA) chain. The binding of dye cations is suppressed to some degree at acid pH compared to that at neutral pH, owing to the repulsion exerted by protonated adenine bases.     

Validation of the mouse peripheral blood micronucleus assay using acridine orange supravital staining with urethane

The mouse peripheral blood micronucleus assay using acridine orange supravital staining was compared with the standard bone marrow assay using urethane (ethyl carbamate)-treated mice. Urethane was intraperitoneally injected to CD-1 and BDF₁ mice at doses ranging from 62 to 1000 and 62 to 250 mg/kg, respectively. Peripheral blood was collected from the tail 0, 24, 48, and 72 h and bone marrow cells were smeared at 24 and 42 h after the treatment. Although the response of micronucleus induction in peripheral reticulocytes was delayed by about 24 h compared to that in bone marrow polychromatic erythrocytes, the maximum frequencies of micronucleated young erythrocytes were comparable. Therefore, the peripheral blood micronucleus assay using the acridine orange supravital staining method may provide a good alternative to the conventional bone marrow assay.     

Enumeration of mycoplasmas after acridine orange staining

Samples of liquid mycoplasma cultures were mixed with equal part of a 0.01% solution of acridine orange and placed on agar plates. The number of fluorescing organisms per field was counted in an epifluorescence microscope at an X 1,000 magnification. When the number of fluorescing organisms per field was related to the number of colony-forming units per milliliter during the growth cycle, highly significant correlation was found in cultures with greater than or equal to 10(6) colony-forming units per ml during the exponential growth phase. The counts were weakly correlated during the stationary phase and not correlated during the death phase. This technique provides a mean to enumerate mycoplasmas in liquid cultures.