Photosensitizing dyes and fluorochromes as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) chromosome technique

Summary UsingAllium cepa chromosomes after 5-bromo, 2-deoxyuridine (BrdU) incorporation, we studied several acid and basic dyes and fluorochromes for their potential as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) technique. All of the dyes and fluorochromes investigated showed a photosensitizing capacity which was slightly lower than 33258 Hoechst in the cases of daunomycin, phloxin, fluorescein, thioflavine T and nuclear fast red, and somewhat higher in the case of eosin Y. Observation and cytophotometric analysis of differentially Giemsa-stained sister chromatids when eosin Y was used as the photosensitizing agent revealed the unsubstituted chromatid to be reddish violet in colour (absorption maximum, 550 nm), while the BrdU-substituted chromatid was blue or pale violet blue (absorption maximum, 580 nm). These results indicate that eosin Y is a useful photosensitizing dye which could be used as a substitute for 33258 Hocchst in the FPG staining technique     

Cytochemical characterization of the modified Guard procedure a regressive staining method for demonstrating chromosomal basic proteins

Summary A number of acidic dyes, including various fluorochromes, were substituted for biebrich scarlet in the modified Guard (1959) procedure, a regressive staining method which appears to demonstrate basic chromosomal proteins. These substitutions were made to test the possibility that dyes other than biebrich scarlet might provide advantages in sensitivity and/or contrast, or that more control could be exerted over the differentiation step in which solutions of phosphotungstic and phosphomolybdic acids (polyacids) are used.Of the dyes tested in this investigation, six were found to be especially suitable: procion brilliant blue, procion yellow, geranine G, brilliant sulfoflavine, eosin Y, and eosin B. While procion brilliant blue could be used as an absorption dye only, the other dyes were used more profitably as fluorochromes. The various dyes displayed considerable variability in the ease with which they could be displayed from substrates with polyacid solutions during the differentiation step. Procion brilliant blue, procion yellow, and geranine G were displaced gradually and thus resembled biebrich scarlet. In contrast, eosin B, eosin Y, and brilliant sulfoflavine were displaced more easily from all but the most highly condensed chromatin in substrates. Brilliant sulfoflavine yielded exceptionally bright and nearly selective fluorescence of consensed chromosomes in division, X chromosomes of grasshopper spermatocytes, and sperm heads.Weak, but selective fluorescence was observed when monazo sulfonated dyes, including ponta chrome violet SW, eriochrome black, diamond red, and ponta chrome blue black, were substituted in the modified Guard procedure. Similar results were obtained with solochrome cyanin R. As expected, these dyes seemed to function as weakly acidic dyes.     

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.     

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation

The fluorescent dye 4′-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.     

Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining

The essential steps of the 33258 Hoechst-Giemsa method for differential chromatid staining consist of (1) 33258 Hoechst treatment, (2) exposure to light, and (3) Giemsa staining. The staining was shown to be a function of the concentration of 33258 Hoechst and the light exposure. The dye was successfully replaced by various metachromatic dyes such as thionine. Two simple methods are proposed. Failure of the pale stained chromatids to restore Giemsa affinity with urea and trypsin and the diminished Feulgen reaction after light exposure suggest that not masking proteins but photolysis of the BrdU-incorporation chromatid components in the present of photosensitive dyes play a role in the differential staining.     

Harmine as a substitute for 33258 Hoechst in the FPG technique

We studied the effectiveness of harmine as a substitute for 33258 Hoechst in the fluorescence-plus-Giemsa technique, using Allium cepa chromosomes after 5-bromo-2'-deoxyuridine (BrdU) incorporation. Harmine showed a photosensitizing capacity which was somewhat higher than 33258 Hoechst and used half of the time established for the usual treatment.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-UV irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Summary Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-U.V irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.In honour of Prof. P. van Duijn     

Stain Solubilities Part 1. Data in the Literature

The rather meager data found in the literature concerning the solubilities of the dyes used as biological stains is reviewed. Solubility data have been found concerning the following dyes: picric acid, martius yellow, crystal ponceau, methyl orange, tropaeolin O, orange II, Bismarck brown, Congo red, auramine, malachite green, fuchsin, methyl violet, gentian violet, crystal violet, methyl green, diphenylamine blue, aurin, corallin, phenolphthalein, flluorescein, eosin Y, iodo-eosin, methylene blue, alizarin, indigo carmine, and carmine. Much of this information is of questionable reliability. The writer is investigating the matter and his original data are to appear in subsequent papers.     

Stain Solubilities Part 1. Data in the Literature

The rather meager data found in the literature concerning the solubilities of the dyes used as biological stains is reviewed. Solubility data have been found concerning the following dyes: picric acid, martius yellow, crystal ponceau, methyl orange, tropaeolin O, orange II, Bismarck brown, Congo red, auramine, malachite green, fuchsin, methyl violet, gentian violet, crystal violet, methyl green, diphenylamine blue, aurin, corallin, phenolphthalein, flluorescein, eosin Y, iodo-eosin, methylene blue, alizarin, indigo carmine, and carmine. Much of this information is of questionable reliability. The writer is investigating the matter and his original data are to appear in subsequent papers.     

Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)     

Use of Hoechst Dyes 33258 and 33342 for Enumeration of Attached and Planktonic Bacteria

The DNA-specific fluorochromes Hoechst 33258 and 33342 were used to enumerate aquatic bacteria by epifluorescent direct counts. Cultures of estuarine bacteria gave identical counts when stained with Hoechst 33258 or acridine orange, whereas natural populations of aquatic bacteria gave 92 to 98.5% of the acridine orange counts. The technique had distinct advantages over acridine orange when enumerating bacteria on surfaces which bind acridine orange, such as polystyrene.     

Harmine as a substitute for 33258 Hoechst in the FPG technique

Summary We studied the effectiveness of harmine as a substitute for 33258 Hoechst in the fluorescence-plus-Giemsa technique, using Allium cepa chromosomes after 5-bromo-2-deoxyuridine (BrdU) incorporation. Harmine showed a photosensitizing capacity which was somewhat higher than 33258 Hoechst and used half of the time established for the usual treatment.     

Selected dyes for residence time distribution evaluation in bioreactors

Summary A selection of dyes for tracer studies in bioreactors, specially for wastewater treatment, is presented. Substances that showed no adsorption on air or biomass, stability in time, good solubility and no color change between pH 6.5 to 8.5, were: bromocresol green, bromophenol blue, dextran blue, eosin Y and mordant violet. Consequently they seem to be adequate for common biochemical engineering processes. In addition, dyes that showed some limitations, but may be employed in special cases, were: bromophenol red and phenol red (color change between pH 5.0 to 6.8 and 6.8 to 8.4 respectively) and methylene violet Bernsthen (low spectrophotometric response).     

Binding of Hoechst 33258 to chromatin in situ

The binding of Hoechst 33258 to rat thymocytes, human lymphocytes, and NHIK 3025 tissue culture cells was studied by measuring the fluorescence and light scattering of the cells as functions of dye concentration using flow cytometry. The results indicated that there were two different modes of binding of Hoechst 33258 to chromatin in situ at physiological pH. Type 1 binding, which dominated at total dye/phosphate ratios below 0.1 (0.15, M), was characterized by a binding constant of the order 10(7) M-1 and fluorescence with high quantum yield. Further binding of the dye resulted in a reduced blue/green fluorescence ratio, indicating that secondary sites were occupied. Binding at secondary sites above a certain density (0.1 less than or equal to bound dye/phosphate less than or equal to 0.2) induced strong quenching of fluorescence and precipitation of chromatin. Precipitation was quantitated by measuring the large-angle (greater than or equal to 15 degrees) light scattering of the cells above 400 nm, i.e., outside the Hoechst 33258/DNA absorption spectrum, as a function of dye concentration. In contrast, the light scattering at 365 nm, i.e., within the absorption spectrum of Hoechst 33258/DNA, was independent of the total dye/phosphate ratio. The coefficient of variation of the light-scattering (greater than or equal to 400 nm) histograms decreased with Hoechst 33258 concentration. Type 2 binding to histone-depleted chromatin was cooperative (Hill-coefficient approximately 2) and the apparent binding constant was 2-3 X 10(5) M-1 as determined from quenching and precipitation data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability

This study was designed to assess whether nucleic acid stains such as propidium iodide and Hoechst 33258 and the cytosolic stain eosin identified equivalent proportions of non-viable cells. Sub-samples of boar spermatozoa stored for up to 72 h, and frozen bull spermatozoa stored in straws and thawed before staining, were exposed to either propidium iodide or Hoechst 33258 alone or in combination. Additional sub-samples were stained with eosin-nigrosin and subsequently with Giemsa. The proportion of non-viable cells identified by propidium iodide alone was equivalent to that observed when it was used in combination with the other fluorescent probe. Similar results were observed for Hoechst 33258. However, direct microscopic examination of sub-samples exposed to both stains revealed that a proportion of spermatozoa stained with propidium iodide did not incorporate Hoechst 33258. This was found consistently in boar and bull spermatozoa under the different experimental conditions used. Quantification showed that the proportion of propidium iodide-positive cells was significantly higher than Hoechst 33258-positive cells. Furthermore, the proportion of propidium iodide-positive cells was higher than cells stained with eosin, but no differences were found between the number of cells stained with Hoechst 33258 or eosin. The proportion of cells stained with propidium iodide was positively correlated with the proportion stained with either Hoechst 33258 or eosin, despite the observation that more cells incorporated propidium iodide. Taken together, these results indicate that there are differences in the ability of fluorescent probes to identify non-viable sperm cells and that this should be considered when staining protocols are used to analyse sperm viability, or when viability is used as a discriminating factor in functional studies, such as those related to acrosomal exocytosis.     

Additional Schiff-Type Reagents for Use in Cytochemistry

Twenty-four new Schiff-type reagents were discovered in a survey of 140 different dyes. These dyes include acid fuchsin, acridine yellow, acriflavine hydrochloride, azure C., Bismarck brown R, Bismarck brown Y, celestine blue B, chrysoidine 3R, chrysoidine Y extra, cresyl violet, crystal violet, gentian violet, methylene blue, neutral violet, phenosafranin, phosphine GN, proflavine, toluidine blue O, and toluylene blue. Positive results obtained with crystal violet and a few samples of methylene blue are considered due to impurities. Various chemical extractions, aldehyde blocking reagents, and enzymatic treatments were used to verify the aldehyde specificity of the above dye-SO₂, reagents as well as azure A, brilliant cresyl blue, neutral red, safranin O, and thionin which have been mentioned by other workers. These reagents were tested in the Feulgen reaction for DNA and the PAS reaction for polysaccharides. Absorption curves were obtained from individual nuclei stained for DNA. The absorption peaks ranged from 450 mμ, to 630 mμ. depending on the dye studied. The Feulgen reaction could be followed by the PAS reaction or vice versa in mouse intestine using reactive dyes of complementary colors. The evidence indicates that a potential Schiff-type reagent must have at least one free NH₂ group on the dye molecule.     

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.     

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.     

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.