Mechanism of differential staining of BUdR-substituted Vicia faba chromosomes.

Chromosomes of the broad bean Vicia faba were isolated and air-dried on slides after incorporation of BUdR into DNA (BUdR substitution) for two rounds of replication. Then the preparations were embedded in a buffer solution containing trypsin as well as fluorescence dye (acridine orange or Hoechst 33258). We observed chromosomes with a fluorescence microscope at various times after embedding. After about 15 min one sister chromatid of some of the metaphase chromosomes showed enhanced darkening and disintegration within 1–4 min (melting effect) during observation. We suppose that fragmentation of BUdR-substituted DNA by the acridine orange-visible light system in acridine orange staining and by irradiation with wavelengths around the transition from UV to visible light in Hoechst 33258 staining is responsible for this phenomenon. The disintegration of one sister chromatid in BUdR-substituted chromosomes can also be produced by UV irradiation during trypsin treatment when fluorescence dyes are not present.     

A novel technique for viable cell determinations

We have developed a simple method to determine cell viability using two fluorescent dyes, Hoechst 33258 and acridine orange. When these dyes are used in combination, dead cells fluoresce brilliant blue and live cells fluoresce green. This method works over a range of dye concentrations (Hoechst 33258, 0.25-2 micrograms/ml; acridine orange, 1-5.0 micrograms/ml) and the fluorescence spectra of the two dyes are such that only one set of filters is required to visualize the effects of both dyes simultaneously. It is insensitive to a wide range of exogenous serum concentrations and is read with greater uniformity by different observers.     

A Novel Technique for Viable Cell Determinations

We have developed a simple method to determine cell viability using two fluorescent dyes, Hoechst 33258 and acridine orange. When these dyes are used in combination, dead cells fluoresce brilliant blue and live cells fluoresce green. This method works over a range of dye concentrations (Hoechst 33258, 0.25-2 μg/ml; acridine orange, 1-5.0 μg/ml) and the fluorescence spectra of the two dyes are such that only one set of filters is required to visualize the effects of both dyes simultaneously. It is insensitive to a wide range of exogenous serum concentrations and is read with greater uniformity by different observers.     

Effect of different fluorescent dyes on thermal stability of DNA and cell viability of the hyperthermophilic archaeon Aeropyrum pernix

The interactions of DNA-binding dyes (Hoechst 33258, DAPI, acridine orange) and DiBAC₄(3) with hyperthermophilic archaeon Aeropyrum pernix cells were investigated by the combination of calorimetric, spectroscopic and microscopic techniques. All of the dyes, studied here, affect the thermal stability of DNA in vivo and in vitro. Hoechst 33258 is highly DNA-specific probe, which does not affect the thermal transitions of other cellular components as can be detected by differential scanning calorimetry (DSC). Due to this unique property, it can be used as a potential DNA marker for in vivo DSC studies. The localization of the dyes in the cells and viability assay was revealed by fluorescence microscopy. Hoechst 33258, DAPI and acridine orange did not distinguish between viable and non-viable cells of Aeropyrum pernix. Only with the commercially available Live/Dead BacLight^TM kit we were able to discriminate viable and non-viable Aeropyrum pernix cells.     

A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidium-bromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.     

A comparison of fluorochromes for direct viable counts by image analysis

Total direct and direct viable counts of fresh and injured cultures of Escherichia coli were determined by image analysis in preparations stained with acridine orange, ethidium bromide and 4',6-diamidino-2-phenyl indole (DAPI). Cells stained with DAPI were not detected by image analysis. Fresh cultures stained with acridine orange or ethidium bromide gave comparable counts. Injured E. coli stained with ethidium bromide gave higher counts that with acridine orange. Injured cultures stained with acridine orange contain high proportions of green cells which are less easily detected than red cells in image analysis. In certain cases it may be better to use ethidium bromide, which stains all cells red, for direct viable counts by image analysis.     

Early and late replicative chromosomal banding patterns of Gallus domesticus.

Early and late replicating chromosomal banding patterns of Gallus domesticus were investigated by cell synchronization and incorporation of 5'-bromodeoxyuridine during early and late DNA synthesis. The early replicating chromosomal banding patterns observed, as revealed by either acridine orange or Hoechst 33258/propidium iodide staining, were similar to the structural G-banding patterns obtained by trypsin digestion and Giemsa staining. Late replicating chromosomal banding showed extensive reverse band complementarity to the G-banding pattern. Cell synchronization increased the number of prometaphase and metaphase plates available for analysis. G-banding obtained by Hoechst 33258/propidium iodide staining was investigated due to the fact that it is compatible with chromosomal in situ hybridization procedures that use nonisotopically-labeled DNA probes. Standard replicative G-banded and R-banded idiograms, as obtained after cell synchronization, are proposed.     

Fluorometric Determination of DNA in Aquatic Microorganisms by Use of Hoechst 33258

A method for the determination of microbial DNA in aquatic environments by the use of Hoechst 33258 has been developed. With unsophisticated instrumentation and simple extraction procedures, it is possible to detect from 0.05 to 10 μg of DNA in bacterial cultures or natural water samples. The method is specific for DNA; DNase I treatment of extracts of natural microbial populations removed 95 to 100% of the observed fluorescence. DNA content ranged from 165 ng ml⁻¹ for relatively eutrophic Potomac River water to 27 ng ml⁻¹ for coastal Atlantic Ocean water and was correlated to an acridine orange direct count (r = 0.90).     

Linear dichroism and polarized fluorescence of dye-complexed DNA fibers

Summary A simple method to obtain well orientated DNA fibers for studying the ordered binding of dyes and fluorochromes by linear dichroism and polarized fluorescence is described. The metachromatic dye toluidine blue and the intercalating fluorochromes ethidium bromide and acridine orange showed a perpendicular alignement to DNA; the minor groove binding fluorochromes 33258 Hoechst and DAPI appeared parallel. Thus, DNA fibers represent a suitable cytochemical test substrate for studying the orientation of bound dyes by polarization methods.     

A simple method for the fluorescence analysis of nucleic acid-dye complexes in cytological preparations

This communication describes a simple method for recording fluorescence emission spectra of cytological preparations using a conventional fluorescence spectrophotometer. The emission characteristics of "in situ" complexes between some basic fluorochromes (DAPI, 33258 Hoechst, acridine orange, pyronin Y, and ethidium bromide) and nucleic acid containing structures from smears of chicken blood and Ehrlich tumor cells (chromatin, basophilic cytoplasm) are briefly described.     

Study on the interaction of aromatic dyes with nucleic acids by means of UV, CD and NMR spectroscopies.

The interaction of several aromatic cationic dyes such as, ethidium bromide (EB), methylene blue (MB), acridine orange (AO), and Hoechst 33258 with calf-thymus DNA and poly(A)-poly(U) duplex was investigated. The different induced extrinsic Cotton effects (greater than 300 nm) were observed for DNA- and RNA-dye complexes. The binding properties of these complexes were examined by UV, CD, and NMR spectroscopies.     

Four fluorochromes for the demonstration and microfluorometric estimation of RNA

Microfluorometric estimates of total RNA were made in selected test material stained with berberine sulfate, acridine orange, and Hoechst 33258. These measurements were compared with those obtained with propidium iodide, which is known to interact only with double-stranded nucleic acids. It was observed that all of the fluorochromes, including propidium iodide, yielded very similar patterns of fluorescence in the various types of material tested. In isolated thymocyte and hepatocyte nuclei stained with either propidium iodide or Hoechst 33258 at pH 2, it was evident that RNA could be estimated only indirectly by measuring the amount of fluorescence before and after extraction with RNase. It was apparent that the total fluorescence of small thymocyte nuclei was affected much less by RNase extraction than that of 2c hepatocyte nuclei. Attempts to obtain direct estimates of RNA by exposing the preparations to DNase were not successful: the fluorescence of thymocyte nuclei dropped almost to zero, and hepatocyte nuclei could no longer be assigned to distinct ploidy classes. In addition, since the highly condensed chromatin of thymocyte nuclei was stained much more prominently than the looser chromatin of hepatocyte nuclei with Hoechst 33258, it was apparent that this fluorochrome - when used at pH 2 - has potential usefulness as a "probe" of organizational differences in chromatin.     

A new technique for retarding fading of fluorescence: DPX-BME

The antioxidant beta-mercaptoethanol (BME) used in conjunction with the permanent mountant DPX (DPX-BME) retarded fluorescent fading of mithramycin, acridine orange and Hoechst 33258 stained chicken erythrocytes, each to a varying degree. The initial fluorescence of all dyes examined was more intense with DPX-BME than with DPX alone. Specimens mounted in DPX-BME showed strong fluorescence and excellent morphology; if kept in the dark, they could be stored indefinitely without deterioration. Retarding fading of fluorescence with DPX-BME faciliated quantitation of DNA using fluorescence cytophotometry.     

A New Technique for Retarding Fading of Fluorescence: Dpx-Bme

The antioxidant β-mercaptoethanol (BME) used in conjunction with the permanent mountant DPX (DPX-BME) retarded fluorescent fading of mithramycin, acridine orange and Hoechst 33258 stained chicken erythrocytes, each to a varying degree. The initial fluorescence of all dyes examined was more intense with DPX-BME than with DPX alone. Specimens mounted in DPX-BME showed strong fluorescence and excellent morphology; if kept in the dark, they could be stored indefinitely without deterioration. Retarding fading of fluorescence with DPX-BME faciliated quantitation of DNA using fluorescence cytophotometery.     

Four fluorochromes for the demonstration and microfluorometric estimation of RNA

Summary Microfluorometric estimates of total RNA were made in selected test material stained with berberine sulfate, acridine orange, and Hoechst 33258. These measurements were compared with those obtained with propidium iodide, which is known to interact only with double-stranded nucleic acids. It was observed that all of the fluorochromes, including propidium iodide, yielded very similar patterns of fluorescence in the various types of material tested. In isolated thymocyte and hepatocyte nuclei stained with either propidium iodide or Hoechst 33258 at pH 2, it was evident that RNA could be estimated only indirectly by measuring the amount of fluorescence before and after extraction with RNase. It was apparent that the total fluorescence of small thymocyte nuclei was affected much less by RNase extraction than that of 2c hepatocyte nuclei. Attempts to obtain direct estimates of RNA by exposing the preparations to DNase were not successful: the fluorescence of thymocyte nuclei dropped almost to zero, and hepatocyte nuclei could no longer be assigned to distinct ploidy classes. In addition, since the highly condensed chromatin of thymocyte nuclei was stained much more prominently than the looser chromatin of hepatocyte nuclei with Hoechst 33258, it was apparent that this fluorochrome — when used at pH 2 — has potential usefulness as a probe of organizational differences in chromatin.     

Fluorometric determination of the DNA concentration in municipal drinking water

DNA concentrations in municipal drinking water samples were measured by fluorometry, using Hoechst 33258 fluorochrome. The concentration, extraction, and detection methods used were adapted from existing techniques. The method is reproducible, fast, accurate, and simple. The amounts of DNA per cell for five different bacterial isolates obtained from drinking water samples were determined by measuring DNA concentration and total cell concentration (acridine orange epifluorescence direct cell counting) in stationary pure cultures. The relationship between DNA concentration and epifluorescence total direct cell concentration in 11 different drinking water samples was linear and positive; the amounts of DNA per cell in these samples did not differ significantly from the amounts in pure culture isolates. We found significant linear correlations between DNA concentration and colony-forming unit concentration, as well as between epifluorescence direct cell counts and colony-forming unit concentration. DNA concentration measurements of municipal drinking water samples appear to monitor changes in bacteriological quality at least as well as total heterotrophic plate counting and epifluorescence direct cell counting.     

Fluorometric determination of the DNA concentration in municipal drinking water.

DNA concentrations in municipal drinking water samples were measured by fluorometry, using Hoechst 33258 fluorochrome. The concentration, extraction, and detection methods used were adapted from existing techniques. The method is reproducible, fast, accurate, and simple. The amounts of DNA per cell for five different bacterial isolates obtained from drinking water samples were determined by measuring DNA concentration and total cell concentration (acridine orange epifluorescence direct cell counting) in stationary pure cultures. The relationship between DNA concentration and epifluorescence total direct cell concentration in 11 different drinking water samples was linear and positive; the amounts of DNA per cell in these samples did not differ significantly from the amounts in pure culture isolates. We found significant linear correlations between DNA concentration and colony-forming unit concentration, as well as between epifluorescence direct cell counts and colony-forming unit concentration. DNA concentration measurements of municipal drinking water samples appear to monitor changes in bacteriological quality at least as well as total heterotrophic plate counting and epifluorescence direct cell counting.     

Use of Electric Linear Dichroism and Competition Experiments with Intercalating Drugs to Investigate the Mode of Binding of Hoechst 33258, Berenil and DAPI to GC Sequences

Abstract

The drugs Hoechst 33258, berenil and DAPI bind preferentially to the minor groove of AT sequences in DNA Despite a strong selectivity for AT sites, they can interact with GC sequences by a mechanism which remains so far controversial. The 2-amino group of guanosine represents a steric hindrance to the entry of the drugs in the minor groove of GC sequences. Intercalation and major groove binding to GC sites of GC-rich DNA and polynucleotides have been proposed for these drugs. To investigate further the mode of binding of Hoechst 33258, berenil and DAPI to GC sequences, we studied by electric linear dichroism the mutual interference in the DNA binding reaction between these compounds and a classical intercalator, proflavine, or a DNA-threading intercalating drug, the amsacrine-4-carboxamide derivative SN16713. The results of the competition experiments show that the two acridine intercalators markedly affect the binding of Hoechst 33258, berenil and DAPI to GC polynucleotides but not to DNA containing AT/GC mixed sequences such as calf thymus DNA Proflavine and SN16713 exert dissimilar effects on the binding of Hoechst 33258, berenil and DAPI to GC sites. The structural changes in DNA induced upon intercalation of the acridine drugs into GC sites are not identically perceived by the test compounds. The electric linear dichroism data support the hypothesis that Hoechst 33258, berenil and DAPI interact with GC sites via a non-classical intercalation process.     

Chromosome banding homologies in three species of Aedes (Stegomyia)

The chromosome complements of the mosquitoes Aedes aegypti, Aedes mascarensis, and Aedes albopictus, belonging to the subgenus Stegomyia, gave a uniform response to the Q-, H-, and R-banding techniques. Of the three homomorphic chromosome pairs, only the shortest or sex pair (I) showed a consistent banding pattern. In the three species, a bright yellow intercalary band was present on one arm of both chromosomes of the sex pair after heat treatment and staining with acridine orange. The rest of the chromosome and the other two pairs fluoresced orange-red. The same intercalary region appeared completely dark with the fluorochromes quinacrine and Hoechst 33258, while the rest of the chromosomes fluoresced dull. The same banding pattern was present in males and females. Size variations of the Q- and H-negative and R-positive intercalary bands were observed within each species. The results are interpreted in terms of structural homology of the sex-determining chromosomes, which is retained within the subgenus.     

Further studies on the mechanism involved in differential staining of BUdR-substituted Vicia faba chromosomes

In a preceding publication we reported that photolysis of BUdR-substituted Vicia faba chromatids occurs during observation with a fluorescence microscope when chromosomes were mounted in a solution containing trypsin and a photosensitive dye (Hoechst 33258 or acridine orange). The present investigations support the hypothesis that the rapid dissolving of the double BUdR-substituted (BB) chromatids observed with our method is due to single-strand breaks induced by a photosensitive dye-visible light system. The agents cysteamine and potassium iodide which reduce BUdR radicals and in this way may inhibit single-strand breaks modify the rate of chromosomes showing differential staining. It was totally suppressed by high cysteamine concentrations and markedly reduced by potassium iodide. Several acridine dyes were tested concerning their ability to induce differential staining. Some of them, e.g. aurophosphine and coriphosphine O, yield good results, others, e.g. acriflavine and acridine yellow, give poor differential staining. In an experiment in which the trypsin concentration was varied to induce approximately optimum and non-optimum digestion conditions the necessity of trypsin treatment in our method was confirmed.