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PubMed1976

Aggregation of acridine orange: crystal structure of acridine orange tetrachlorozincate 2C17H19N3-2HCl-ZnCl2-CH3COOH.

The crystal structure of the biological stain, "acridine orange," has been determined. This compound, when crystallized from ethanol, is shown to be a zinc chloride double salt of acridine orange, containing, in addition, acetic acid of crystallization. These additional components are residuals from the method of preparation of acridine orange. This complex, 2 acridine orange-2HCl-ZnCl2-CH3COOH, (2C17H19N3-2HCl-ZnCl2-CH3COOH) crystallizes in the monoclinic space group P21, a = 9.965 (2), b = 21.507 (6), c = 9.645 (2) A, beta = 113.98 degrees (2), V = 1888.7 (8) A3, FW = 800.0, Z = 2, DX = 1.41 g-cm-3, Dobs = 1.43 (9) g-cm-3. Three-dimensional diffraction data were collected with CuKalpha radiation, and the structure refined to R = 0.065 for 1885 observed reflections. In the crystal structure hydrogen bonds are formed, via the protonated nitrogen atom of the central rings of two acridine orange cations, to two chloride ions in a ZnCl42- tetrahedral grouping. These two acridine orange molecules are stacked in parallel planes, approximately 3.4 A apart, with the long axes of the ring systems inclined at 26.5 to each other. Thus an apparent dimerization of the acridine, orange is facilitated by the anions present, resulting in the complex studied. The two -N(CH3)2 groups of each acridine orange molecule are not protonated in this crystalline form. The mode of molecular packing found here may be relevant to models for the external stacking of acridine orange around a DNA molecule. The importance of removing any zinc salt from acridine orange preparations prior to aggregation studies is stressed.

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PubMedDate not supplied

Screening for Trichomonas vaginalis infection by use of acridine orange fluorescent microscopy.

The acridine orange test for detection of Trichomonas vaginalis in smears has been adapted for delayed examination of specimens. Mailed-in slides stained by acridine orange were compared with on-site wet mounts; the acridine orange test detected 96% of all positives, whereas only 76% were detected by wet mounts. In a similar comparison with Papanicolaou smears, the acridine orange test detected 89% as compared with 67% detected by Papanicolaou smears.

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PubMed1978

Acridine orange.

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PubMed1978

The effects of acridine orange on deoxyribonucleic acid in Escherichia coli.

1. Acridine Orange inhibits growth of Escherichia coli K12 when incubated at pH 7.9, but not at pH 7.4.2. At a non-permissive temperature for DNA polymerase I, Acridine Orange inhibits growth of a temperature-sensitive strain and also increases the rate of elimination of the F'-Lac plasmid. 3. DNA isolated from cells treated with Acridine Orange under conditions that inhibit growth contains material of low molecular weight, which is absent from DNA isolated from cells treated under conditions in which growth is not impaired. 4. Cells incubated with Acridine Orange at both pH 7.4 and 7.9 suffer degradation of DNA, as shown by loss of labelled DNA from the acid-insoluble fraction, which is not observed with untreated cells at either pH. 5. The results suggest that elimination of the F'-Lac plasmid by Acridine Orange requires inactivation of repair processes.

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PubMed1979

Atomic resolution analysis of a 2:1 complex of CpG and acridine orange.

Cytidylyl-3', 5'-guanosine and acridine orange crystallize in a highly-ordered triclinic lattice which diffracts X-rays to 0.85 angstrom resolution. The crystal structure has been solved and refined to a residual factor of 9.5%. The two dinucleoside phosphate molecules form an antiparallel double helix with the acridine orange intercalated between them. The two base pairs of the double helical fragment have a twist angle of 10 degrees and it is found to have a C3' endo-(3', 5')-C2' endo mixed sugar puckering along the nucleotide backbone as has been observed for other simple intercalator complexes. Twenty-five water molecules have been located in the lattice together with a sodium ion. The intercalator double helical fragments form sheets which are held together by van der Waals interactions in one direction and hydrogen bonding interactions in the other. The crystal lattice contains aqueous channels in which sixteen water molecules are hydrogen bonded to the nucleotide, none to the intercalator, five water molecules are coordinated about the sodium ion and four water molecules bind solely to other water molecules. The bases in the base pairs have a dihedral angle of 7 to 8 degrees between them.

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PubMedDate not supplied

A new post-staining fixation technique for Acridine Orange.

A technique for Acridine Orange staining of cells for automated flow analysis involving post-staining fixation with Millonig's glutaraldehyde buffer is presented. Preliminary data indicate that with this protocol there is decreased background fluorescence and increased signal to noise ration in flow. In addition, stained cells may be stored for several months without alteration in cellular morphology or fluorescence characteristics. Finally, nuclear and cytoplasmic fluorescence do not appear to vary significantly with respect to cell concentration or distribution on a slide or filter when cells are stained according to the glutaraldehyde protocol.

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PubMed1979

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.

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PubMed1979

Distribution patterns of DNA template activity in the embryonic tooth organ: an acridine orange ultracytochemical study.

The ultracytochemical acridine orange (AO) method has been employed to demonstrate DNA template activity within embryonic incisor tooth organs excised from New Zealand White rabbits during the 25th day of gestation. Survey ultrastructural examination revealed characteristic distribution patterns of AO positive cell populations in the inner and outer enamel epithelium and the adjacent ectomesenchyme of the cervical loop region and in populations of fibroblast-like cells connecting as a band the outer AO positive cell groups. With increasing differentiation of ameloblasts and odontoblasts the number of AO positive cells as well as the number of AO chromatin interaction products per single cell nucleus decreased. Treatment with RNase prior to incubation with AO did not result in a noticeable loss of AO chromatin interaction products suggesting that RNA is negligible as a target for AO. The present results and comparison with other biological system indicated that interaction of AO with DNA sites coding for RNA appears to be restricted to specific templates in particular physiological conditions, e.g., stimulation of cells prior to differentiation by extracellular factors.

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PubMed1979

Acridine orange staining of the mammalian fibroblast cell coat.

Acridine orange selectively binds to glycos- and galactosaminoglycan (GAG) compounds in the presence of Na+ in low concentrations. We have worked out a cytochemical method, which is suitable for the specific demonstration of surface GAG components. The method consists of a glutaraldehyde prefixation, an acridine orange block-staining for 48 h and an OsO4 postfixation for some hours. The specificity of the staining was verified with the help of fluorimetric--in vitro--measurements and enzymatic digestions, respectively.

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PubMed1977

Differential staining of bacteria in clinical specimens using acridine orange buffered at low pH.

Optimal conditions for acridine orange staining of air dried and methanol fixed bacteria on glass slides were studied. The pH of the staining buffer did not influence the fluorescence of an S. aureus and an E. coli strain at dye concentrations of 25-50 mg per litre. 81 bacterial strains representing 15 different species were stained with acridine orange under standard conditions, all strains showing orange fluorescence. The pH of the buffer influenced markedly the staining patterns of human cells and tissue materials, as represented by smears of peripheral blood, buccal scrapings, urethral secretions and tracheal exudates. The fluorescence obtained ranged from low intensity green at low pH values to bright orange at neutral and alkaline pH. This variability indicated a possibility of designing conditions for a differential staining method for the detection of bacteria in clinical specimens. The differential staining effect with a low pH in the buffer was confirmed on smears of buccal scrapings, cerebrospinal fluid samples and urethral secretions, showing orange fluorescence of the bacteria present and green-to-yellow fluorescence of background material, cells and tissue debris.

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PubMedDate not supplied

A poststaining fixation technique for acridine orange: quantitative aspects.

A poststaining fixation technique was developed for acridine orange staining of cytologic specimens. A quantitative evaluation was performed to assess the suitability of this technique for routine use in analytic cytology and its potential for future use in automated screening equipment. Three major advantages of the poststaining fixation technique over conventional acridine orange staining methods were documented: (1) stained cells may be stored for several months without alteration of cellular morphology or fluorescence characteristics; (2) the protocol minimizes diffusion of stain out of cells; and (3) poststaining fixation reduces dependence of nuclear or total cell fluorescence on cell distribution or concentration on a slide or in flow.

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PubMed1978

A comparison of acridine orange and Feulgen cytochemistry of human tumor cell nuclei.

Specimens of cells derived from tumors of the human female genital tract plus normal cells as standards have been divided into aliquots and stained according to acridine orange or pararosanilin:Feulgen procedures. Acridine orange-stained cells were slit-scanned for 535 nm nuclear fluorescence; Feulgen-stained cells were comb-scanned for 580 nm nuclear absorbance. For each specimen examined, the tumor cell:normal cell ratio of mean nuclear fluorescence following acridine orange staining was greater than the tumor cell:normal cell ratio of mean nuclear absorbance following Feulgen staining. The tumor cell:normal cell ratio of mean nuclear fluorescence ranged from 2.3 for a nonkeratinizing squamous cell carcinoma to 3.9 for a keratinizing squamous cell carcinoma. The tumor cell:normal cell ratio of mean nuclear absorbance ranged from 1.4 for a mixed mesodermal sarcoma to 2.3 for a small cell squamous cell carcinoma. These results indicate that the elevated nuclear fluorescence intensity from acridine orange-stained tumor cells cannot be explained solely on the basis of elevated Feulgen:DNA content. An alternative hypothesis, consistent with these results, is that DNA is the principal binding substrate for intranuclear acridine orange and that the DNA of certain tumor cells is more accessible to acridine orange than is the DNA of normal cells.

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PubMed1976

Inheritance of acridine orange R variants in human acrocentric chromosomes.

Sequential Q and acridine orange R banding were performed on 50 normal individuals to determine those individuals with the maximum number of variants by acridine orange reverse banding. In three individuals from different families, one of the two homologs of each of pairs of acrocentric chromosomes was classified as variant. The variant present in two individuals enabled unambiguous determination of the paternal or maternal origin of all 10 acrocentric chromosomes. In the third family, the variants in the third generation were sufficient to determine the origin of homologs in 5 of 10 acrocentric chromosomes. Overall, the parental origin of 36 of 50 acrocentric chromosomes (72%) could be determined. In no case was the pattern of variants found in offsprings and parents incompatible with Mendelian inheritance.

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PubMed1979

Mass action and acridine orange staining: static and flow cytofluorometry.

We present results involving an approach to acridine orange staining of intact cells based on basic physicochemical considerations. We show by static microfluorometry of several in vitro and in vivo cell lines that the important parameters for such staining are the molar ratio (Formula: see text), and molar concentration of acridine orange. Differential nuclear DNA and cytoplasmic RNA staining are totally controlled by these two parameters. We show this by a physicochemical model of cell-dye interaction. Finally, we use the method to study the growth parameters of complex in vivo cell populations by automated multiparameter flow microfluorometry. We have explored also, both by static and flow systems, the effect on AO-cell staining of various cell pretreatments such as Triton X-100 and chelating agents.

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PubMed1979

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.

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PubMed1975

Variation in human acrocentric chromosomes with acridine orange reverse banding.

Twenty-five normal subjects were studied by acridine orange reverse (RFA) banding in order to obtain a preliminary estimate of the type and frequency of variations in color and length. Color variations were classified into 1 of 6 colors and size variations into 1 of 5 levels. The same cells were also studied by Q banding. Acridine orange reverse banding was found to be more useful than Q banding for characterizing variations in chromosomes 14, 15, 21 and 22. In addition, it was found that there was no consistent relationship between pale or bright Q banding and the various colors observed with RFA banding. For the optimal characterization of a chromosomal variation, multiple banding technics, including RFA banding, are necessary.

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PubMed1976

[Lethal and mutagenic photodynamic effect of acridine orange on bacteriophage cd].

Both acridine-sensitized inactivation and mutagenesis in phage sd have been studied and compared with the effect of other mutagens. Inactivation curve was not stictly exponential, with a small shoulder at short light doses and deviation of survival lower than 10(-5). The lethal effect was not reactivated by multiplicity reactivation. Photodynamic damage in phage sd was accompanied by the increase of the rise (but not of the latent) period in the one-step curve of phage multiplication and increase of the burst size. Experiments were carried out at dye concentration of 1-10(-5) M or lower; a strong dark effect of the dye being observed under 2-fold increase of the dye concentration. Plaque-type mutants were formed up to the maximum approximately 1% at the survival approximately 2--10(-5); in comparison with other mutagens photo-sensitized mutagenesis in phage sd was lower. The significant increase in the number of plaque mutants was observed after the illumination of phage fraction surviving the pre-treatment with higher acridine orange concentration (greater than or equal to 2-10(-5)M).

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PubMed1978

Analysis of glycosaminoglycans in urine by using acridine orange fluorescence.

The fluorescence technique described here utilizes the electrostatic interaction between the polyanionic sites of glycosaminoglycans and the cationic dye Acridine Orange to analyse urinary glycosaminoglycans from patients suffering from mucopolysaccharidoses. The basis of the titration is the decrease in the fluorescence of free Acridine Orange that occurs when it is bound to polyanions. The effect of the presence of possible interfering materials such as salt, proteins and trace materials in urine was evaluated. This fluorescence technique is technically simple.

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