The mechanism of action of “mordant” dyes — a study using preformed metal complexes

Summary For small basic dyes blocking or selective extraction of tissue sections can reduce staining (methylation, or trichloracetio acid extraction) or increase staining (acetylation or nitrous acid deamination). This is as predicted by a simple electrostatic model of dye binding. In contrast, the staining with both large basic dyes and with metal complex dyes was only slightly affected by these treatments. This is in keeping with the probable role of van der Waals attractions and hydrophobic bonding in the binding of large dyes.Since MCDs are all large, there is no need to invoke the traditional, special mechanism of mordanting (i.e. tissue-metal ion-dye covalencies) to explain the peculiarities of these MCDs. An apparent exception to this was the resistance of MCDs, unlike either small or large basic dyes, to the action of processing fluids. However, this was due to the insolubility of MCDs in alcoho, lsin contrast to the solubility of most basic dyes, irrespective of size, in these media.     

Side effects of reaction media in histochemical blocking procedures

Synopsis 0.2 N NaOH, the reaction medium for 1,2-cyclohexanedione, a specific reagent for arginyl residues in proteins, was found to intensify, at some sites in rat abdominal skin and human gingiva, the Sakaguchi reaction, staining with Pauly's reagent, and anionic dye binding at pH 6.4; at other sites these reactions were reduced, presumably due to extraction of material from sections. 0.2 N NaOH slightly reduced staining after the ninhydrin-Schiff procedure at all sites in rat skin. The interpretation of this finding is obscure, because some sites giving a positive Sakaguchi reaction and staining with anionic dyes failed to stain after the ninhydrin-Schiff procedure. There were also alterations in staining, with the cationic dyes Alcian Blue and Alcian Yellow. It is suggested that 0.2 N NaOH ruptures linkages between polycationic residues of proteins and polanions, demonstrable by Alican Blue. The blockade produced by acetic anhydride-pyridine (4060 v/v) mixtures was stable, in the alkaline conditions required for staining with Pauly's reagent. Pretreatment with pyridine alone reduced tissue binding of anionic dyes.     

A convenient buffer system for controlled staining with basic dyes

Summary A new buffer system is described for use in histochemical staining with basic dyes. The buffer is made up by mixing solutions of formic acid and sodium acetate. Tables giving the proportions for closely-spaced pH values in the range 3.0–5.6 are presented. A table for an acid phosphate series down to a pH of 2.0 is also included. The value of these buffer mixtures in histological as well as histochemical staining with basic dyes is stressed.With 1 Figure in the Text     

Quantitative microspectrophotometry of RNA in plant tissue

Synopsis Chemical estimation of nucleic acid, essentially RNA, in fixed tissue from Jerusalem artichoke tubers, coupled with an examination of the types of RNA in the fixed tissue by gel electrophoresis, demonstrates that ribosomal and soluble RNA are preserved in this tissue after various fixation procedures including methanol, ethanol-acetic acid and aqueous formaldehyde. Tissue fixed in ethanol-acetic acid or formaldehyde is resistant to loss of nucleic acid by aqueous extraction but tissue fixed in all three standard fixatives loses nucleic acid in citrate buffer under conditions used for Azure B staining. The presence of Azure B in the buffer does not wholly prevent this loss. Tissue fixed in formaldehyde or mixed fixatives containing formaldehyde is resistant to loss of nucleic acid during treatment with EDTA to obtain cell suspensions.Preliminary experiments with Azure B, Gallocyanin chrome-alum and Methylene Blue showed that the Gallocyanin technique is the most practicable for demonstrating RNA cytochemically. Its specificity was confirmed by ribonuclease extraction of the tissue. Optimum staining conditions, requiring treatment of the tissue in Gallocyanin chrome-alum solution overnight at 40°C, are established for the artichoke tissue and for paraffin sections of pea shoot apices.     

Understanding Romanowsky staining

Summary Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of toxic granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).To whom offprint should be sent     

The action of chromium(III) in fixation of animal tissues

Summary Chromic salts have been studied as fixatives of mammalian tissues for light microscopy, and the binding of the metal has been examined histochemically. Tissues bind chromium(III) from aqueous solutions less acid than pH 2.5; the metal attaches mainly to collagen and basement membranes. Solutions containing chromium(III) as the only active ingredient cannot be used as fixatives because they destroy cytoplasm and cause great structural distortion. When mixed with other fixative agents, however, chromic salts can bring about considerable improvement in structural preservation. In aqueous mixtures more acid than pH 2, and in aqueous-methanolic solutions in the pH range 4.0–5.3, a chromic salt provides only a nonspecific osmotic effect: little or no metal is bound to the tissue, and an aluminium or a sodium salt can be effectively substituted. In less acid (pH 2.3–3.2) aqueous mixtures, the beneficial action of chromium(III) cannot be imitated by aluminium or sodium ions.Chromium(III) forms coordinate bonds that cross-link ionized carboxyl groups of macromolecules. The reaction occurs so slowly that such cross-links can internally strengthen a tissue only after the structure has been stabilized by rapidly acting fixative agents. Thus, a valuable future use of chromic salts may be in a post-fixation treatment to protect specimens against the adverse effects of embedding in paraffin wax. Chromium(III) might also be useful for enhancing the opacity of collagen fibrils in electron microscopy.Address until 30 June 1986: Department of Anatomy and Cell Biology, University of Sheffield, Sheffield, S10 2TN, UK     

The interrelation of the size and substantivity of dyes; the role of van der Waals attractions and hydrophobic bonding in biological staining

Summary Using a pH signature criterion, it was found that whereas electrostatic attractions and repulsions were paramount in the binding of low molecular weight acid and basic dyes to tissue sections, high molecular weight dyes were also bound non-electrostatically.By studying the effects on staining of adding to aqueous dyebaths agents destroying the iceberg structure of water, the importance of hydrophobic bonding was established. It was noticed that the hydrophobic elastic fibres were stained by large dyes from dyebaths inhibitory both to electrostatic attractions and hydrophobic bonding (i.e. using acid dyes from alkaline aqueous-ethanol or aqueous-dimethylformamide dyebaths). This indicated that strong van der Waals attractions occurred, at least with some substrates. Supporting this idea was the observation that in tissue sections benzoylated before staining (i.e. made less acidophilic but more hydrophobic) additional structures were stained when using large acid dyes from alkaline aqueous-ethanol or aqueous-dimethylformamide dyebaths.Applications of the size-substantivity relationship were suggested, e.g. commenting on a standard stain for basic proteins; explaining the modes of action of traditional stains for elastic fibres and amyloid; rationalising the varied substantivities of tetrazolium salts; and finally suggesting guide lines for use in the design of new staining methods.     

Does progressive nuclear staining with hemalum (alum hematoxylin) involve DNA,and what is the nature of the dye-chromatin complex?

Previous investigators have disagreed about whether hemalum stains DNA or its associated nucleoproteins. I review here the literature and describe new experiments in an attempt to resolve the controversy. Hemalum solutions, which contain aluminum ions and hematein, are routinely used to stain nuclei. A solution containing 16 Al³⁺ ions for each hematein molecule, at pH 2.0–2.5, provides selective progressive staining of chromatin without cytoplasmic or extracellular “background color.” Such solutions contain a red cationic dye-metal complex and an excess of Al³⁺ ions. The red complex is converted to an insoluble blue compound, assumed to be polymeric, but of undetermined composition, when stained sections are blued in water at pH 5.5–8.5. Staining experiments with DNA, histone and DNA + histone mixtures support the theory that DNA, not histone, is progressively colored by hemalum. Extraction of nucleic acids, by either a strong acid or nucleases at near neutral pH, prevented chromatin staining by a simple cationic dye, thionine, pH 4, and by hemalum, with pH adjustments in the range, 2.0–3.5. Staining by hemalum at pH 2.0–3.5 was not inhibited by methylation, which completely prevented staining by thionine at pH 4. Staining by hemalum and other dye-metal complexes at pH ≤ 2 may be due to the high acidity of DNA-phosphodiester (pK_a ~ 1). This argument does not explain the requirement for a much higher pH to stain DNA with those dyes and fluorochromes not used as dye-metal complexes. Sequential treatment of sections with Al₂(SO₄)₃ followed by hematein provides nuclear staining that is weaker than that attainable with hemalum. Stronger staining is seen if the pH is raised to 3.0–3.5, but there is also coloration of cytoplasm and other materials. These observations do not support the theory that Al³⁺ forms bridges between chromatin and hematein. When staining with hematein is followed by an Al₂(SO₄)₃ solution, there is no significant staining. Taken together, the results of my study indicate that the red hemalum cation is electrostatically attracted to the phosphate anion of DNA. The bulky complex cation is too large to intercalate between base pairs of DNA and is unlikely to fit into the minor groove. The short range van der Waals forces that bind planar dye cations to DNA probably do not contribute to the stability of progressive hemalum staining. The red cation is precipitated in situ as a blue compound, insoluble in water, ethanol and water-ethanol mixtures, when a stained preparation is blued at pH > 5.5.     

Enzymatic and nonenzymatic degradation of myelin basic protein

A procedure for large scale isolation of myelin basic protein (BP) has been modified to insure BP preparations free of neutral proteinase activity. Fractions were monitored by electrophoretic analysis of BP solutions incubated under various conditions of temperature and pH. Maximum degradation of human BP prepared by the old batch procedure occurs at pH 7, 47°C. BP preparations obtained by the new procedure, as well as BP preparations purified by CM-cellulose chromatography, are stable under these conditions. The latter, however, do undergo significant breakdown at pH 9, 100°C. The results suggest that the degradation observed under these conditions is non-enzymatic in nature.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.     

Effect of water soluble non-membrane proteins on the phospholipid structure

The interaction of water-soluble nonmembraneous proteins (trypsin and the basic pancreatic trypsin inhibitor (BPTI)) with soybean phospholipids was studied using multilamellar vesicles. Multilamellar vesicles were obtained from soybean lipid extracts and mixtures of individual phospholipids based on phosphatidylcholine. These mixtures contain different phospholipids: "bilayer", "nonbilayer", and negatively charged. It was shown that the content of both proteins in the complex depends on pH and the presence of negatively charged components. On the basis of this finding, the conclusion about the electrostatic nature of lipid-protein interaction was made. The structural organization of soybean phospholipids in multilamellar vesicles was studied in the presence and absence of the proteins using broad-line 31P-NMR spectroscopy. It was found that, in mixtures of phospholipids of complex composition, different types of phases coexist, and phospholipids of different classes can compensate the effects of each other. Trypsin and BPTI affect the structure of phospholipids in a similar way, inducing considerable structural changes in multilamellar vesicles of preparations containing negatively charged components in whose structure there coexisted primordially the bilayer and isotropic phases.     

The influence of inorganic salts when staining with preformed metal complex dyes

Summary The influence of various inorganic salts on the staining of tissue sections by a variety of preformed metal complex dyes (MCDs) has been studied. The staining patterns resulting were found to be extremely complex, though certain generalisations are possible.Usually the staining of basophilic tissue components (e. g. cartilage matrix and cell nuclei) was reduced as the amount of salt in the dyebath was increased. On the other hand the staining of some acidophilic tissue components (especially elastic fibres) was increased as the salt concentration rose. Both these effects can be rationalised by use of the Donnan equilibrium.The limited occurrence of salt-induced increases in staining intensity may be attributed in part to the extremely low rates of staining observed with acidophilic substrates.The decreases and increases in staining seen were to some extent dependant on the particular salt used, and presented an extremely complex picture. One general effect seen was that salts of Al³⁺, Cr³⁺, and Mg²⁺ had a greater inhibitory effect on staining than salts of K⁺ and Na⁺. This is explicable in terms of coordinative binding of the polyvalent ions by tissue substrates.On the basis of the information obtained comments were possible on various topics of biological staining interest. Thus it was concluded that of the MCDs studied only the aluminon-chromium complex showed evidence of binding by a mordanting mechanism. Further, it was found that the Critical Electrolyte Concentration theory was commonly inapplicable when staining tissue sections with MCDs.     

Gallocyanin chromalum as a nuclear stain in cytology. I. A cytophotometric comparison of the Husain-Watts Gallocyanin chromalum staining protocol with the Feulgen procedure

Summary In the present study, the staining characteristics of the Gallocyanin chromalum technique devised by Husain and Watts are compared with the Feulgen reaction. Liver imprints, blood smears, and cervical smears were fixed in ethanol and stained with either the Husain and Watts Gallocyanin chromalum reagent or the Feulgen-Schiff reagent. The slides were then post-treated with 70% ethanol-HCl pH 1.0, or with phosphotungstic acid for 0.5–30 min. The integrated optical density of cell nuclei was measured with a VIDAS image analyzer. In the material stained with the Husain and Watts procedure, some Gallocyanin chromalum was removed from the nuclei in the early phase (5 min) of all the post-treatment steps, followed by a plateau phase where the integrated optical density remained constant for 30 min. In this phase, the nuclear absorbance was highly reproducible and of the same size regardless of the post-treatment. Both the Husain and Watts procedure and the Feulgen-reaction gave quantitative staining of DNA. The Gallocyanin chromalum stain after Husain and Watts is a quick staining procedure for quantitative evaluation of DNA in cytological material. Proper rinsing of the slides is necessary for a good reproducibility of results.     

Exploration of dye chemistry in Taenzer Unna Orcein type elastin staining

Summary Musso's demonstration of the amino- and hydroxyphenoxazone structure of synthetic orcein suggested trial of simpler mixtures and isolated pure dyes of the phenoxazine phenoxazone series. It was further thought that explorations of this sort might reveal which of the hitherto demonstrated 14 chromatographic components of orcein might take part in or even be chiefly responsible for the elective staining of elastin.Accordingly trials were made in a hydrochloric acid (0.12 N) 70% alcohol technic based on Taenzer's original method of a number of commercially available dyestuffs and indicators and a number of products were synthesized by variants of Musso's technic for resorcin blue: air oxidation of resorcinol in the presence of ammonia. Those tested are the following: Azolitmin, Lacmoid, Resorufin, Resorcin Blue (MLB), C.I. No. 51020, Gallocyanin C.I. 51030, Brilliant Cresyl Blue C.I. 51010, Nile Blue C.I. 51180, a Nile red preparation, Bernthsen's Methylene Violet; Azure A, Toluidine Blue C.I. 52040, several laboratory synthetic lots of Musso's Resorcin Blue in which oxidation was done with H₂O₂ and varying amounts of ammoni were used, and 2 batches in which resorcinol was partly or completely replaced by m-aminophenol.Successful to excellent elastin stains were achieved with Lacmoid, Resorcin Blue MLB, part of the Musso Resorcin Blue products and the two m-aminophenol oxilation products Elastin purple FP and Elastin Videt PR.From the failure of azolitmin and resorufin, both 7-hydroxy-2-phenoxazones and the success of resorcin blue (MLB) 7-N,N-dimethylamino-2-phenoxazone, it appears suggested that the aminophenoxazone structure may be a determining characteristic. The success with m-aminophenol substitution in the Musso air oxidation NH₃ synthesis tends to support this view.While I have had some success with the Victoria blues first used by Lustgarten, using other technics, these dyes and some other triphenylmethanes do not successfully take the place of orcein in acid alcohol staining methods. Rosanilin and pararosanilin do stain rodent elastica from acid aqueous and alcoholic solutions but adult human elastica does not so stain. We suspect a diphenamine Schiff base condensation with the known free aldehyde of rodent elastica. This is confirmed by more or less complete blockade of pararosanilin acid alcohol staining with p-toluidine in glacial acetic acid, 1 hr, hydroxylamine: sodium acetate: H₂O 102040 3 hr and 5% phenylhydrazine HCl 3 hr on dog, rat and guinea pig arteries. The hydroxylamine gave complete blocking, the other two reagents partial. Altogether about 50 dye samples were tested.Presented before the Histochemische Gesellschaft September 28, 1968.Supported by National Cancer Institute Research Grant C-4816, National Institutes of Health.     

Factors affecting the formation of metallic silver and the binding of silver ions by tissue components

Summary The rate of formation of metallic silver has a maximum when plotted as a function of pH. The site of this maximum on a pH scale differs noticeably for various tissue elements. By contrast, the amount of silver ions bound to the tissue is a monotonously increasing function of the pH. A temperature rise decreases the length of the induction period and increases the gradient of the ascending section of the kinetic curve representing the formation of metallic silver. It also increases the maximum amount of silver ions bound to the tissue. An increase in the concentration (activity) of the silver ions in the impregnating bath has the same effect. Chemical composition and concentration of the complexing agent, as well as special ions in the impregnating bath to which earlier some definitive role has been attributed in the silver staining methods, proved to be ineffective when both pH and activity of silver ions were kept constant. Illumination of the reaction was also ineffective. The kinetic curves obtained in nonaqueous but polar media (e.g., acetone) exhibited the same qualitative characteristics as those obtained in aqueous solutions. No reaction between silver ions and tissue was observed in apolar solvents.     

The Effect of Preliminary Treatment (Fixing Fluids) on Staining Properties of the Tissues

As was reported in a previous paper,¹ staining properties depend on the chemical composition of the tissues and on the strength of the dyes themselves. Applying mixtures of basic and acid dye on tissues (methylene blue, eosin Y) at different pH-values, it is possible to find differences in the isoelectric points of the nuclei and cytoplasm of different tissues. For example, the nucleus of polymorphonuclear cells of the blood consists of the most acid protein, with an isoelectric point around pH 2.5, while the nucleus of lymphatic tissues has an isoelectric point of about pH 4.0, and that of connective tissue about pH 3.4.

With a knowledge of the above, a constant method of staining at various pH-values was used to study the effect of different fixing fluids on the staining properties of the tissues. In this way it was found that many fixing fluids gave very stable compounds with tissue proteins, and that they almost permanently change the chemical composition (i.e. the staining properties of the tissues). In some instances, these changes can be easily explained from the regular chemical standpoint. For example, formalin forms inert compounds with amino groups of the amino acids of proteins and in this way it makes the tissue proteins more acid, i.e. it moves the isoelectric point of the proteins toward a lower pH-value. The same is true in the case of the polivalent acids. The bivalent heavy metals such as mercury, on the contrary, it is assumed, combine with carboxyi groups of amino acids and in this way move the isoelectric point of the proteins toward a higher pH.     

Species specificity in the extrinsic absorption changes exhibited by some invertebrates stained with voltage-sensitive dyes

Summary The absorption changes of several invertebrate neuronal preparations stained by the potentiometric dyes (WW 375, WW 433, WW 401 and RGA 84) in response to electrical nerve stimulation were examined. The dyes did not penetrate the connective sheath of insect preparations, but stained it. Only a decremental spreading of optical signals was seen onPeriplaneta americana, Gryllus bimaculatus and GitGryllus campestris ganglia and nerves. In contrast to insect preparations, pond snail and leech neurons were well stained by these dyes. The dye WW 375 behaved somewhat distinctly on insect and pond snail preparations than had been previously reported on other invertebrates. Like the signals from vertebrate neurons, they usually had triphasic action spectra. Therefore, this kind of action spectrum is not found only in membranes of vertebrate neurons. The main conclusion of this work is that the species-specific effects of the dye on different invertebrate preparations have a common feature: the existence of three peaks in the change of absorption (at 575, 675 and 750 nm) in both kinds of WW 375 action spectra (monophasic or triphasic). The wavelength dependence of the change in absorption was not affected by concentration, staining time, pH, osmolarity or ionic composition of physiological saline.     

On the stainability of plant cell walls with alcian blue

This work is a continuation of a communication on the stainability of broad bean (Vicia faba L.) root tip cells with alcian blue, published some time ago. Following the standard method of staining with alcian blue, the cell walls are very strongly stained, the nuclei (except nucleolus) lightly, the nucleolus and cytoplasm are practically colourless. The weak dyeing of the nucleus is not equal throughout the whole section so that the comparison of stainability of cell walls and nuclei by itself cannot explain the staining with alcian blue. The results of this work on the staining of cell walls (if not including model experiments and experiments in vitro, which are not considered as decisive here) can be summarized as follows: the pH dependence of staining, the loss of stainability as a result of pectinase digestion, blocking of staining by methylation and regeneration of stainability by demethylation and, finally, the impossibility of staining in the presence of NaCl lead to the conclusion that the staining of the material studied in this work is primarily caused by the salt linkage of alcian blue with the free carboxyls of pectic substances. From the comparison of staining with alcian blue and with other basic dyes it follows that in the case of alcian blue some other factors may also take part and are the reason for the selectivity and firmness (fastness) of the staining of cell walls with this dye. Otherwise, the staining of plant cell walls with alcian blue corresponds quite well to the staining of carboxyls containing polysaccharides of animal tissues with this dye. By staining with alcian blue it was found impossible to distinguish between younger and older cell walls within the meristem. However, this staining is suitable for routine use when studying the meristematic tissue. It is often possible to use solutions of a higher pH than generally used.     

The use of heavy metals in mycoremediation of synthetic dyes

Synthetic dyes represent a major class of toxic pollutants that are resistant to biological degradation, and heavy metals play a special role in this process. In this study, two isolates of lignicolous macromycetes, Lenzites betulina as well as another species less frequently studied, Trametes gibbosa, were tested in order to assess their remediation potential against three different synthetic dyes under specific conditions. The effect of heavy metal ions in the discoloration of synthetic dyes process, and the optimal concentration of manganese ions necessary were evaluated. The dyes’ discoloration efficiency of the fresh isolates were compared to the isolates maintained by refrigeration, isolates that were repeatedly sub-cultivated and isolates that were previously grown on dye-supplemented media, and then were assessed. The discoloration process was evaluated in liquid nutrient media, 10 replicates were used for each working version. Evaluation of discoloration rate was obtained by using a UV-VIS spectrometer. The results were interpreted statistically by the Kruskal-Wallis test and by use of a new complex method, Multiple Factor Analysis. The fresh isolates showed the highest discoloration capacity while the isolates previously grown on the dye-supplemented media presented a low discoloration rate. Manganese ions gave a positive effect on enzyme induction while copper and cobalt ions inhibited the process.     

Characterization of a salt resistant bacterial strain <Emphasis Type="Italic">Proteus</Emphasis> sp. NA6 capable of decolorizing reactive dyes in presence of multi-metal stress

Microbial biotechnologies for the decolorization of textile wastewaters have attracted worldwide attention because of their economic suitability and easiness in handling. However, the presence of high amounts of salts and metal ions in textile wastewaters adversely affects the decolorization efficiency of the microbial bioresources. In this regard, the present study was conducted to isolate salt tolerant bacterial strains which might have the potential to decolorize azo dyes even in the presence of multi-metal ion mixtures. Out of the tested 48 bacteria that were isolated from an effluent drain, the strain NA6 was found relatively more efficient in decolorizing the reactive yellow-2 (RY2) dye in the presence of 50 g L^?1 NaCl. Based on the similarity of its 16S rRNA gene sequence and its position in a phylogenetic tree, this strain was designated as Proteus sp. NA6. The strain NA6 showed efficient decolorization (>90 %) of RY2 at pH 7.5 in the presence of 50 g L^?1 NaCl under static incubation at 30 °C. This strain also had the potential to efficiently decolorize other structurally related azo dyes in the presence of 50 g L^?1 NaCl. Moreover, Proteus sp. NA6 was found to resist the presence of different metal ions (Co⁺², Cr⁺⁶, Zn⁺², Pb⁺², Cu⁺², Cd⁺²) and was capable of decolorizing reactive dyes in the presence of different levels of the mixtures of these metal ions along with 50 g L^?1 NaCl. Based on the findings of this study, it can be suggested that Proteus sp. NA6 might serve as a potential bioresource for the biotechnologies involving bioremediation of textile wastewaters containing the metal ions and salts.     

Mechanisms of Giemsa banding

Summary We investigated the capability of individual thiazins in Giemsa mixtures (methylene blue and azures A, B, and C) and of two related dyes (toluidine blue and thionin) to produce G-banding. We further tested the effects of variations of buffer composition and concentration, dye concentration, and staining time.G-banding was produced by all of the dyes at low concentrations, although differences were noted. Overall, methylene blue and azure B produced the best banding, azures A, C, and toluidine blue produced moderately good banding, and thionin produced poor banding. This order did not appear to be altered essentially by different treatments. The optimal conditions for G-banding for all dyes and treatments included the use of (1) 0.025–0.05M phosphate buffer, (2) dye concentrations of 0.002%–0.005%, and (3) staining times of 6–15 min.