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Phenothiazine dyes, methylene blue, new methylene blue, azure A, and azure B, photosensitized the oxidation of nicotinamide adenine dinucleotide (NADH), an important coenzyme in the living cells, through electron transfer. The reduced forms of these phenothiazine dyes, which were produced through electron extraction from NADH, underwent reoxidation to the original cationic forms, leading to the construction of a photoredox cycle. This reoxidation process was the rate-determining step in the photoredox cycle. The electron extraction from NADH using phenothiazine dyes can trigger the chain reaction of the NADH oxidation. Copper ions enhanced the photoredox cycle through reoxidation of the reduced forms of phenothiazine dyes. New methylene blue demonstrated the highest photooxidative activity in this experiment due to the fast reoxidation process. Electron-transfer-mediated oxidation and the role of endogenous metal ions may be important elements in the photosterilization mechanism.

A modified electrode was developed by immobilizing poly(azure A) (pAA) onto the surface of a glassy carbon electrode via the electropolymerization of azure A (AA). The pAA immobilized on the electrode exhibited redox response during cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The redox reaction obeyed the Nernst equation because of the involvement of H+ ions. In addition, the peak potential was shifted according to the solution pH. The shifts of the oxidation peak potential could be more easily observed using DPV than when using CV, indicating that the developed electrode could be useful as a pH sensor. This pH measurement method can be successfully applied in the pH range of 1 to 10 and can be successfully repeated more than 50 times.

Methylene blue (MB) is commonly used in aquaculture as a fungicide and antidotes. This study was designed to explore the pharmacokinetics of MB in Japanese eel (Anguilla Japonica) immersed in 10 mg L−1 and 20 mg L−1 MB for 0.5 h. The concentrations of MB and its derivatives in the blood, liver, kidney, skin and muscle were determined by HPLC after immersion. The results showed that the changes of drug concentrations in Japanese eel were basically the same in different dose groups, showing a general trend of increasing at first and then decreasing, but the peak time was slightly different. The peak concentration of the drug was positively correlated with the dose level. The peak concentrations of MB in MB (20 mg L−1) group, MB, azure A and azure B in the tissues of Japanese eel were significantly higher than those in MB (10 mg L−1) group. Moreover, MB, azure A and azure B remained for a long time and could still be detected at 64 days, and azure C was not detected in tissues.

A sensitive and reliable method was developed to determine methylene blue (MB) and its metabolite residues, including azure A (AZA), azure B (AZB), and azure C (AZC) in aquatic products by HPLC–MS/MS. The samples were extracted by acetonitrile and cleaned up by alumina-neutral (ALN) cartridges. The analytes were separated on a Sunfire C18 column (150 mm × 2.1 mm, 5 µm). The method was validated according to the European criteria of Commission Decision 2002/657/CE. Good linearity between 1–500 µg/L was obtained with correlation coefficients (R2) greater than 0.99. The limit of quantification (LOQ) was 1.0 µg/kg. The average recoveries at three levels of each compound (1, 5, and 10 µg/kg) were demonstrated to be in the range of 71.8–97.5%, with relative standard deviations (RSDs) from 1.05% to 8.63%. This method was suitable for the detection of methylene blue and its metabolite residues in aquatic products.

Life manifestation is mainly based on biopolymer-ligand molecular recognition; therefore, the elucidation of energy and speed associated with protein-ligand binding is strategic in understanding and modulating biological systems. In this study, the interactions between methylene blue (MB) or azure A (AZA) dyes and bovine lactoferrin (BLF) were investigated by surface plasmon resonance, fluorescence spectroscopy, and isothermal titration microcalorimetry. Despite the molecular similarities between the dyes, the BLF-AZA binding thermodynamic parameters (ΔGAZAo = −30.50 and ΔHAZAo = 10.8 (kJ·mol−1)) were higher in magnitude than those of the BLF-MB systems (ΔGMBo = −27.3 and ΔHMBo = 5.72 (kJ·mol−1)). To increase the systems' entropy (TΔSAZAo = 41.3 and TΔSMBo = 33.0 (kJ·mol−1)), the hydrophobic interactions must outweigh the electrostatic repulsion, thereby promoting BLF-dye binding. The activation complex formation (Eac, aMB = 33, Eac, aAZA = 32, ∆Ha, MB‡ = 31, ∆Ha, AZA‡ = 30, ∆Ga, MB‡ = 51.84, ∆Ga, AZA‡ = 50.7, T∆Sa, MB‡ = −21, T∆Sa, AZA‡ = −21 (kJ·mol−1)), owing to free BLF and MB (or AZA) associations, was not affected by the dye chemical structure, while for the thermodynamically stable BLF-dye complex dissociation, the same energetic parameters (Eac, dMB = 16, Eac, dAZA = 6.4, ∆Hd, MB‡ = 14, ∆Hd, AZA‡ = 3.9, ∆Gd, MB‡ = 81.4, ∆Gd, AZA‡ = 74.93, T∆Sd, MB‡ = −68, T∆Sd, AZA‡ = −71.0 (kJ·mol−1)) were considerably affected by the number of methyl groups. Our results may be very useful to determine binding processes controlled by kinetic parameters, as well as to optimize the application of these photosensitive dyes in biological systems.

© 2017 Informa UK Limited, trading as Taylor & Francis Group Earlier studies have clarified that NADH and NADPH, re-energized repeatedly by red blood cell (RBC) glycolysis, can be used in extracellular chemical reactions, where electron energies are extracted by electron mediators, such as methylene blue (MB). The electron mediators, which are reduced by NAD(P)H, permeate both the membranes of RBC and phospholipid bilayer of liposomes encapsulating haemoglobin (Hb-vesicles, HbV) and reduce autoxidized ferric methemoglobin (metHb) in HbV to ferrous Hb. Moreover, in vitro screening study clarified some other potential electron mediators with comparable capacity to reduce metHb. Given this background, eight of these compounds: MB, 1,9-dimethyl MB, azure A, azure B (AB), azure, toluidine blue, brilliant cresyl blue and toluylene blue, were evaluated in both in vitro and in vivo studies in this work. Compared with MB as a reference, in vitro experiments demonstrated that most compounds caused effective metHb reduction of HbV in the presence of RBC. However, in vivo experiments of bolus injection of autoxidized HbV to rats (10 mL HbV/kg body weight) followed by injection of the dye (1.53 mL/kg body weight, 2.6 mM) led to some differences from in vitro results. Effective metHb reduction was found for the combination of AB. To evaluate AB effectiveness further, a haemorrhagic shock and resuscitation model was used, where the rats were resuscitated with HbV. When the level of metHb increased to 50%, a dye solution was injected. Again, AB caused sufficient reduction of metHb. Through these in vivo experiments, this study clarified that AB is a suitable electron mediator to prolong the functional lifetime of HbV. (Less)

The biotransformation and biodecolorization of methylene blue (MB) dye using the bacterium Ralstonia pickettii was investigated. This experiment was conducted in a nutrient broth (NB) medium after adding MB at 100 mg L–1 concentration. Approximately 98.11% of MB was decolorized after 18 h of incubation. In addition, the metabolic products detected by LC-TOF/MS were Azure A (AA), thionine, leuco-MB, and glucose-MB, which indicated the MB degradation through a reductase that attacked the heterocyclic central chromophore group present in the structure. Moreover, azure A and thionine fragments resulted from the N-demethylase enzyme that attacked the auxochrome group. Thus, this research was assumed to be the first scientific report suggesting the potential to use R. pickettii in the biodecolorization and biotransformation of dye waste, particularly MB.

This paper describes the role of a structure breaking additive (thiourea) on the photophysical and molecular association of a series of phenothiazine dyes in aqueous media using the absorption and fluorescence spectroscopic methods for the first time. The studied dyes were thionine, azure A, azure B, toluidine blue, and methylene blue. The spectral data were analyzed using DECOM program. Relevant spectral parameters in the dye solutions were estimated and discussed based on the chemical structure of the additive and excitonic treatment. The observation of spectral changes in the spectral data of the (water-additive-dye) system indicates the possible structure formation between the dye and additive molecules. However, it is found that in the high concentrations of thiourea the dimer geometries (H- and J-type dimers) are influenced by the dye-additive interactions. As a result, a competition between the dye-additive and dye-dye interactions was also observed.

Grape stalk (GS) and pomegranate peel (PP) were chemically modified with hydrochloric acid and applied as potential adsorbents for the removal of Azure A, one of the products of oxidized methylene blue, from aqueous solutions. The surface chemistry and morphology of the modified grape stalk (H-GS) and pomegranate peel (H-PP) were characterized by FTIR and SEM. The results showed that the adsorption capacity of H-PP was higher than PP, GS and H-GS. The experimental data indicated that PP, H-PP and H-GS displayed Freundlich adsorption properties while GS displayed both the Langmuir and Freundlich adsorption properties. The adsorption kinetic of Azure A for all adsorbents obeys the pseudo-second-order kinetic model. Moreover, the thermodynamic parameters were also determined.

This study focuses on the understanding of the interaction of phenothiazinium dyes methylene blue (MB), new methylene blue (NMB), azure A (AZA) and azure B (AZB) with tRNAPhe with particular emphasis on deciphering the mode and energetics of the binding. Strong intercalative binding to tRNAPhe was observed for MB, NMB and AZB, bound by a partial intercalative mode. AZA has shown groove binding characteristics. From spectroscopic studies binding affinity values of the order of 105 M-1 were deduced for these dyes; the trend varied as MB > NMB > AZB > AZA. The binding was characterized by an increase of thermal melting temperatures and perturbation in the circular dichroism spectrum of tRNA. All the dyes acquired optical activity upon binding to tRNA. The binding was predominantly entropy driven with a favorable enthalpy term that increased with temperature in all the cases. Dissection of the Gibbs energy to polyelectrolytic and non-polyelectrolytic terms revealed a major role of the non-electrostatic forces in the binding. The small but significant heat capacity changes and the observed enthalpy-entropy compensation phenomenon confirmed the involvement of multiple weak non-covalent forces driving the interaction. The mode of binding was confirmed from quenching, viscosity and cyclic voltammetric results. Using density functional theory, ground state optimized structures of the dyes were calculated to provide insight into theoretical docking studies to correlate the experimental approaches. The modeling results verified the binding location as well as the binding energy of complexation. The results may provide new insights into the structure-activity relationship useful in the design of effective RNA targeted therapeutic agents.

The evaluation of the IC50 values of phenothiazinium dyes in neoplastic immortalized cells (HeLa) in the dark and under irradiation showed that the effectiveness of the photosensitizers (PSs) increased in the order of Azure A (AA) < Azure B (AB) < Methylene Blue (MB). Singlet oxygen production was similar in all of the compounds and the cell uptake of MB was lower than those of AA and AB. The evaluation of the dimer to monomer ratio showed that aggregation increased in the order of MB < AB < AA. Thus, the photodynamic efficiency of these PSs was dependent on aggregation. In order to improve the effectiveness of photodynamic therapy (PDT), it is important to develop phenothiazinium dyes with structural modifications and/or to design formulations that could reduce aggregation.

Background Photodynamic antimicrobial chemotherapy (PACT) is an important therapeutic platform for antimicrobial activity, especially due to the resistant strains, however, the physical-chemical properties of the photosensitizers may affect the final outcome. Recently, this research group showed that the control of aggregation potentiates the PACT action of Methylene Blue. In this study, the researchers investigated the PACT action of other phenothiazinium dyes (PDs), such as Azure A, Azure B (AB), and 1,9-dimethyl methylene blue (DMMB), and the effects of different mediums which modulate PD aggregation were studied. Methods The C. albicans planktonic culture was treated with PDs at different concentrations (0–100.00 mg/L), both in the dark (5 min) and irradiated (640 ± 12 nm LED during 30 min, 2.60 mW/cm2). After the treatments, the diluted samples were grown on Sabouraud Dextrose Agar and maintained at 37 °C for 24 h. Later, one single concentration was defined and the inoculum was treated with the compounds in different media (water, phosphate buffer saline – PBS, physiological solution – 0.90 % NaCl, urea 1.00 mol/L, and sodium dodecyl sulfate 0.25 % - SDS), at 20.00 mg/L for AA and AB, and at 0.50 mg/L for DMMB. The cell uptake and the dimer to the monomer ratios were determined by spectrophotometry. Results PACT with the PDs showed a reduction in the CFU/mL, with DMMB being the most effective due to the higher cell uptake within the series. When PACT was applied in the different mediums, the inactivation response was influenced by the medium. In water, the physiological solution, PBS, and urea showed no significant differences in relation to the control group. On the other hand, SDS potentiated the PACT action, and the inactivation of the microorganism was achieved with AB and DMMB. Conclusions In water, DMMB presented the highest PACT action within the PDs being studied, reaching a microorganism inactivation with a 1.00 mg/L solution, mainly due to its high cell uptake. SDS 0.25 % increased the PACT action of the PDs studied, mainly with DMMB and AB, and this may be used in formulations to develop antimicrobial treatments to be used in clinics.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2021-10-25T23:01:00Z No. of bitstreams: 1 José Marcelo Lacerda Alves Gonçalves.pdf: 2026250 bytes, checksum: e8546c4944f3e52c577de28d25f2d289 (MD5) Made available in DSpace on 2021-10-25T23:01:00Z (GMT). No. of bitstreams: 1 José Marcelo Lacerda Alves Gonçalves.pdf: 2026250 bytes, checksum: e8546c4944f3e52c577de28d25f2d289 (MD5) Previous issue date: 2019-02-04 Photodynamic therapy is a promising treatment option that can be used in several areas of health. The technique is based on the combination of a photosensitizing agent and light. The photosensitizer after exposure to visible radiation, generates reactive oxygen species that lead to cell death. Phenothiazinium dyes are a class of molecules that have properties suitable for Photodynamic Therapy, with only Methylene Blue and Toluidine Blue being extensively studied. Recently other phenothiazinium dyes, such as Azure A and Azure B, have started to be studied and it has been reported that their efficacy is dependent on aggregation. In this context, it is not yet known whether aggregation influences the mechanism of photoinduced cell death. The objective of this work was to evaluate the mechanisms of cell death induced by photodynamic therapy with the photosensitizers Azure A, Azure B and Methylene Blue, relating to the production data of reactive oxygen species. Tests were performed to quantify the production of reactive oxygen and nitrogen species, and evaluation of mechanisms of cell death using the IC50 concentrations of photosensitizers, at different times after exposure to light. The results showed that Methylene Blue was the photosensitizer that presented the highest production of reactive species (oxygen and nitrogen), followed by Azure B, whereas the production of these species photoinduced by Azure A is very low, with no difference in relation to the control group. Methylene blue is the most effective in causing cell death among the compounds tested and the photosensitizer that causes greater loss of mitochondrial membrane potential. All SFs lead to some labeling with propidium iodide 3h after irradiation, but only AA continues to present high PI + marking at 24 and 48h after treatment. Other assays are still needed to clarify the mechanisms of photodamaged cell death after photodynamic Therapy, but there are indications that Azure A causes necrosis to a greater extent due to loss of cell membrane integrity at different times after treatment, whereas Methylene Blue and Azure B would cause necrosis to some extent, but apoptosis would be the mechanism most involved. A terapia fotodinâmica é uma promissora opção de tratamento que pode ser utilizada em diversas áreas da saúde. A técnica é baseada na combinação de um agente fotossensibilizador (FS) e luz. O FS após exposição à radiação visível, gera espécies reativas de oxigênio que levam à morte celular. As fenotiazinas são uma classe de moléculas que apresentam propriedades adequadas para a Terapia Fotodinâmica, sendo que somente o Azul de Metileno (AM) e Azul de Toluidina têm sido amplamente estudados. Recentemente outras fenotiazinas, como o Azure A (AA) e Azure B (AB), começaram a ser estudadas e foi relatado que a eficácia das fenotiazinas é dependente da agregação. Neste contexto, ainda não se sabe se a agregação influencia no mecanismo de morte celular fotoinduzido. O objetivo deste trabalho foi avaliar os mecanismos de morte celular induzidos por terapia fotodinâmica com os FS AA, AB e AM. Foram realizados ensaios para quantificação da produção de espécies reativas de oxigênio e nitrogênio, e avaliação de mecanismos de morte celular utilizando as concentrações de IC50 dos fotossensibilizadores, em diferentes tempos após a exposição à luz. Os resultados mostraram que AM foi o FS que apresenta maior produção de espécies reativas (de oxigênio e de nitrogênio), seguido por AB, enquanto a produção destas espécies fotoinduzida por AA é muito baixa, sem diferença em relação ao grupo controle. O FS AM é o mais efetivo em causar morte celular dentre os FS testados e também o FS que causa maior perda de potencial de membrana mictocondrial. Todos os FS levam à alguma marcação com iodeto de propídeo 3h após a irradiação, porém somente AA continua apresentando elevada marcação PI+ em 24 e 48h após o tratamento. Outros ensaios ainda são necessários para esclarecer os mecanismos de morte celular fotoinduzida após TFD, porém existem indícios que AA causa necrose em maior extensão devido à perda de integridade de membrana celular dos diferentes tempos após tratamento, enquanto AM e AB causaria necrose em certa extensão, porém apoptose seria o mecanismo majoritariamente envolvido.

Silicon based materials on 2D landscape are gaining significant interest due to their exotic properties along with well-developed processing technologies. We report use of 2D siloxene nanosheets as a potential photocatalyst for remediation of water polluted from cationic dyes. Degradation of methylene blue (MB) is used as a representative case study. Systematic studies were performed by varying reaction time, pH, material mass concentration, and dye concentration. Results suggest that adsorption mechanism is prominent over the basic pH range. Adsorption isotherms reveal that the process can be described by Langmuir model, suggesting monolayer adsorption of MB on siloxene nanosheets and pseudo second order kinetics is obeyed. Samples exhibited adsorption capacity of 8.93 mg/g of MB within equilibrium time of ∼ 10 min. These are also found to be effective in removal of other cationic dyes such as Azure A, Azure B and Malachite Green. Siloxene nanosheets are expected to open a new avenue for efficient remediation for treatment of textile effluents by adsorption under diffuse light.

Molecular clip 1 remains monomeric in water and engages in host-guest recognition processes with suitable guests. We report the K-a values for 32 1.guest complexes measured by H-1 NMR, UV/Vis, and fluorescence titrations. The cavity of 1 is shaped by aromatic surfaces of negative electrostatic potential and therefore displays high affinity and selectivity for planar and cationic aromatic guests that distinguishes it from CB[n] receptors that prefer aliphatic over aromatic guests. Electrostatic effects play a dominant role in the recognition process whereby ion-dipole interactions may occur between ammonium ions and the C=O groups of 1, between the SO3- groups of 1 and pendant cationic groups on the guest, and within the cavity of 1 by cation-pi interactions. Host 1 displays a high affinity toward dicationic guests with large planar aromatic surfaces (e.g. naphthalene diimide NDI + and perylene diimide PDI +) and cationic dyes derived from acridine (e.g. methylene blue and azure A). The critical importance of cation-pi interactions was ascertained by a comparison of analogous neutral and cationic guests (e.g. methylene violet vs. methylene blue; quinoline vs. N-methylquinolinium; acridine vs. N-methylacridinium; neutral red vs. neutral red H+) the affinities of which differ by up to 380-fold. We demonstrate that the high affinity of 1 toward methylene blue (K-a = 3.92 x 10(7) M-1; K-d = 25 nm) allows for the selective sequestration and destaining of U87 cells stained with methylene blue.

Five selected amine-derivatives of phenothiazine were electropolymerized on an ITO/glass substrate and then used in the daylight-activated process to produce in situ singlet oxygen which degrades phenol in a solution. The phenothiazines were immobilized in a simple electrochemical procedure in an acidic solution which led to the formation of an ultrathin transparent polymeric film. All films obtained on the ITO substrate including azure A (AA), azure C (AC), methylene blue (MB), toluidine blue (TBO), and thionine (Th) had a comparable surface coverage at the level of picomoles/cm 2 . The activity of these materials was then compared and presented in terms of an efficiency of the phenol degradation process in an aqueous solution by photogenerated singlet oxygen. That efficiency was determined by the UV–vis spectroscopy employing a phenol/4-aminoantipyrine complex. All the phenothiazine ultrathin polymeric films were capable of generating the singlet oxygen in the aqueous solution under daylight activation, which was used in the consecutive process of phenol degradation. The highest efficiency at a level of 51.4% and 45.4% was found for the AC/ITO and MB/ITO layers, respectively.

Antimicrobial photodynamic inactivation (APDI) using phenothiazinium dyes is mediated by reactive oxygen species consisting of a combination of singlet oxygen (quenched by azide), hydroxyl radicals and other reactive oxygen species. We recently showed that addition of sodium azide paradoxically potentiated APDI of Gram-positive and Gram-negative bacteria using methylene blue as the photosensitizer, and this was due to electron transfer to the dye triplet state from azide anion, producing azidyl radical. Here we compare this effect using six different homologous phenothiazinium dyes: methylene blue, toluidine blue O, new methylene blue, dimethylmethylene blue, azure A, and azure B. We found both significant potentiation (up to 2 logs) and also significant inhibition (>3 logs) of killing by adding 10 mM azide depending on Gram classification, washing the dye from the cells, and dye structure. Killing of E. coli was potentiated with all 6 dyes after a wash, while S. aureus killing was only potentiated by MB and TBO with a wash and DMMB with no wash. More lipophilic dyes (higher log P value, such as DMMB) were more likely to show potentiation. We conclude that the Type I photochemical mechanism (potentiation with azide) likely depends on the microenvironment, i.e. higher binding of dye to bacteria. Bacterial dye-binding is thought to be higher with Gram-negative compared to Gram-positive bacteria, when unbound dye has been washed away, and with more lipophilic dyes.

Visible absorption spectra of thionine (Th), azure A (AA), and methylene blue (MB) in aqueous and aqueous additive solutions (0.1–3 M) were studied as a function of the dye concentration. The added additives were urea and aminoethyl ethanolamine (AEEA). Due to the strong structural similarity of the investigated dyes, their spectral parameters were compared at room temperature. The spectral data for each dye were analyzed using DECOM Program. The dimerization behavior of the investigated dyes in water with or without urea was analyzed in terms of monomer–dimer equilibria. The dimerization constant, K d , values of Th, AA, and MB in aqueous solutions were calculated to be (5.6 ± 0.4) × 10 2 M − 1 , (2.5 ± 0.1) × 10 3 M − 1 , and (6.5 ± 0.3) × 10 3 M − 1 , respectively. However, a reduction of the dimerization constant for the dyes in aqueous urea solutions was observed. The dimer structure and the nature of the interacting pairs in these dyes were discussed using the exciton theory. Finally, due to the unique properties of AEEA, different absorption characteristics were observed for these dyes in pure AEEA and its aqueous solutions.

This study examined the reaction of methylene blue (MB) with tunneled manganese oxide pyrolusite regarding pH and reaction time. MB was cleaved through N-demethylation, in which reaction azure B (AB), azure A (AA), azure C (AC), and thionin (TH) were stepwise generated at all tested pH. Pyrolusite predominantly serves as the oxidant in the oxidative degradation of MB at a pH under the pHiep of pyrolusite (4.70) while playing the role of the catalyst at pH higher than pHiep. Among all oxidative products and original MB molecule, TH is the alone compound adsorbed onto the pyrolusite surface at all tested pH. However, the quantity of adsorbed TH increases with pH because of the stronger affinity between the cationic TH molecule and the more negatively charged surface of pyrolusite with pH increasing. Because the lattice oxygen and surface hydroxyl groups form excited oxygen firstly to cause the oxidation of MB, the tunneled pyrolusite with less constrained corner and edge oxygen catalytically promote the oxidation reaction at pH beyond pHiep. The vacancy of the consumed lattice oxygen forms the active sites for the other oxidation and could be replenished by molecular oxygen to complete a catalytic cycle.

This work describes solid-phase extraction-ultra-performance liquid chromatography with electrospray ionization tandem spectrometry for determination of malachite green and metabolite leucomalachite green, crystal violet and metabolite leucocrystal violet, methylene blue and metabolites including azure A, azure B and azure C in aquatic products. Samples were extracted with acetonitrile and ammonium acetate buffer and purified by liquid extraction with dichloromethane, and then on MCAX solid-phase extraction cartridges. Then the extract was evaporated at 45°C by nitrogen blow. The residue was dissolved and separated by an Acquity BEH C18 column. The mobile phase was acetonitrile (A) and 5 mmol/L of ammonium acetate containing 0.1% formic acid (B). Analytes were confirmed and quantified using a tandem mass spectrometry system in multiple reaction mode with triple quadrupole analyzer using positive polarity mode. The limits of detection of malachite green, leucomalachite green, crystal violet and leucocrystal violet were 0.15 µg/kg, the limits of quantification were 0.50 µg/kg, and the average recoveries were more than 75% with spiked residues from 0.5 to 10 µg/kg. The relative standard deviations were less than 13%. The limits of detection of methylene blue, azure A, azure B and azure C were 0.3 µg/kg, the limits of quantification were 1.0 µg/kg, the average recoveries were more than 70% with spiked residues from 1.0 to 10 µg/kg and the relative standard deviations were less than 15%. The method has the merits of simplicity, sensitivity and rapidity, and can be used for simultaneous determination of the analytes in aquatic products.

This paper analyzes the experimental evaluation of methylene blue concentration applied during photocatalytic experiments. In most studies a satisfactory evaluation of the concentration of methylene blue by means of visible spectroscopy is masked by the presence of intermediates during the reaction, resulting in an overestimation of the amount of methylene blue present. The deconvolution technique described in the present study solves this problem by estimating the contribution by intermediates to the visible spectrum. In the photodegradation of methylene blue by Degussa P25 TiO 2 particles the main intermediate detected in the visible spectral region was azure A, produced from the gradual demethylation of azure B. The methylene blue concentrations obtained if the artefact is not taken into account give lower values for the kinetic rate constants of photocatalytic degradation than those provided by this novel deconvolution technique.

A method for the determination of methylene blue (MB) and its metabolites (azure A, azure B and azure C) in rat blood by CE-electrospray ionization mass spectrometry (CE-ESI-MS) was developed in this paper. Different analytical parameters were investigated in detail such as pH and concentration of separation buffer, and ESI-MS instrumental parameters. Under the optimum conditions, MB and its metabolites were separated and detected in 27.3 min. LODs (defined as S/N=3) of this method were 0.22, 0.25, 0.10 and 0.30 μg/mL for MB, azure A, azure B and azure C, respectively. To get a satisfactory extraction efficiency of MB and its metabolites in rat blood, different extraction solutions were studied. By using this method, MB and its metabolites (azure A, azure B and azure C) were successfully analyzed in rat blood samples.

This paper describes the successful use of thin layers of birnessite to degrade methylene blue (MB) contained in aqueous solutions. This dye is a representative of a class of dyestuffs resistant to biodegradation. The thin layers of birnessite, electrodeposited onto a cheap transparent semiconductor substrate (SnO 2 ), in neutral aerated sulphate solutions at room temperature, are very homogeneous, adherent and well crystallised. We showed that these thin layers can discolor the MB solutions totally at room temperature after only 2 h. Total organic carbon (TOC) analyses report the absence of organic compounds in solutions after treatment, in the best conditions. Mass spectrometry reveals the presence of intermediate reaction products (azure A, azure B, azure C, and thionin) during interaction confirming the real degradation of dye. Nitrate and ammonium ions were quantified showing that thin layer of birnessite can mineralise MB. To understand mechanism, interaction experiments were made with each intermediary compound. The electron transfers lead to a rapid N-demethylation oxidative reaction to form colored intermediary compounds. In all cases, thionin was identified as the last adsorbed organic compound onto the birnessite external surface, which is then mineralised slowly into nitrate and ammonium ions. These results suggest that birnessite thin layers may be envisaged as a new non-toxic material for treatment of diluted colored wastewater.

Cyclic voltammetric investigations have been performed on the clean glassy carbon electrode (GCE) at (25 ± 0.1) °C on five progressively alkylated thiazine dyes, namely, thionine (Th), methyl thionine (Azure C), dimethyl thionine (Azure A), trimethyl thionine (Azure B), and tetramethyl thionine (methylene blue) in water, water−ethanol, and water−Triton X-100 media. Diffusion coefficient values of the dyes, measured from the electrochemical data in different media, are clearly influenced by the effect of progressive alkylation to the thionine moiety along with the solvent property. In aqueous media the values of diffusion coefficient fall in the range of (2.66·106 to 5.70·106) cm2·s−1, whereas in presence of ethanol and Triton X-100 the rather lower ranges of (1.16·106 to 3.70·106) cm2·s−1 and (0.76·106 to 1.80·106) cm2·s−1 are observed, respectively. At higher pH’s the voltammograms display cathodic and anodic post/prepeaks which are the characteristics of the strong adsorption of reactants and/or product...

A new method for flow-injection analysis (FIA) for the determination of penicillins based on the extraction and spectrophotometric determination of ion associates with selected thiazine dyes (methylene blue, azure A, and azure B) is proposed. The reaction conditions (cdye = 2 × 10−4 mol l−1, cKCl = 1 mol l−1, pH ≅ 6, λ = 635 nm) were found. The factorial design has been carried out to determine the optimum flow conditions. A wide linear dynamic range of calibration curves (5.1–700 µg ml−1 for penicillin V with all dyes, R = 0.9985) and good repeatability (e.g., relative standard deviation [RSD] = 4.6–0.6% in this concentration range for the reaction with azure B) were found. The detection limit for penicillin V is 1.5 µg ml−1, and the determination limit is 5.1 µg ml−1. The maximum analysis rate is 35 samples per h. The practical samples of pharmaceutics were tested. There are no interferences from the additives in pharmaceutics.

This paper details the comparison of different electropolymerized thiazine electrocatalysts for NADH oxidation. Electropolymerized thiazines have been shown to be electrocatalysts for NADH, but no comprehensive comparison of their properties in the same environment has been performed. The electropolymerization and electrocatalysis is very dependent on chemical and electrochemical environment, so the thiazines (methylene green, methylene blue, toluidine blue, azure a, azure b, and azure c) were all electropolymerized in the same chemical and electrochemical environment and tested for NADH electrocatalysis. All of the thiazines can be electropolymerized to form stable polymer modified electrodes on glassy carbon electrodes and all shown electrocatalytic activity toward NADH. However, each polymer has different properties and therefore would be employed in different applications, depending on whether open circuit potential, current density, or lifetime is the most important condition of the biofuel cell. This paper further compares NAD-dependent glucose dehydrogenase bioelectrocatalysis with poly(methylene green) and poly(methylene blue) electrocatalysts in terms of sensitivity to glucose and biofuel cell performance.

This paper reviews the nature of Romanowsky staining and the relationship between Romanowsky dyes and the Romanowsky-Giemsa effect (RGE). On blood and bone marrow smears the RGE is characterized by a purple colouration of nuclei and neutrophil granules. The nuclear purple contrasts strongly with the blue cytoplasmic staining of cells rich in RNA. Requirement for the occurrence of RGE are: I A cationic dye: The best dye is azure B and, though azure A gives the nuclear purple colour, the cytoplasmic blue is inferior. No other cationic dye such as methylene blue is suitable. 2 An anionic dye: Most commonly eosin Y is used, but it can be replaced by the erythrosins. Full halogenation of the fluorescein (four atoms of bromine or iodine) is not necessary. Phloxine and rose bengal are unsuitable. 3 An appropriate substrate: These are proteins with acidic side groups or proteins bound to a polyanion. For the interaction with the dyes substrates must provide a suitable three-dimensional network which is why the RGE is not obtained in solutions. A tentative theory of RGE is advanced and briefly discussed.

The inclusion complexation of p-sulfonatocalix[6]arene (Calix-S6) with three kinds of phenothiazine dyes was studied spectrophotometrically in a mixture of a room-temperature ionic liquid [bmim]BF4 (1-butyl-3-methylimidazolium tetrafluoroborate) and ethanol. We have determined the association constants of Calix-S6 with phenothiazine dyes under external static pressure up to 767 bar in the [bmim]BF4-ethanol and alcohol-water mixtures. With increasing external pressure, the inclusion equilibrium in the alcohol-water mixtures was shifted to the dissociation side. Conversely, the inclusion equilibrium of methylene blue (MB) and azure A (AA) in the ionic liquid mixture was shifted to the association side. From the analysis of the pressure effects, the reaction volumes ΔV for inclusion complexation were estimated as −7 to 9 cm3 mol−1 in the [bmim]BF4-ethanol mixture and 20–32 cm3 mol−1 in the alcohol-water mixtures. Based on the results, we have suggested that there is a competing complexation between the included dye and [bmim]BF4 molecules in the ionic liquid.

Micellar preconcentration has been proposed to improve the procedure of spectrophotometric determination of sodium dodecyl sulfate (SDS). It involves quantitative extraction by reversed micelles of Triton N-42 in n-decane and the subsequent formation of an ion associate with methylene blue and azure A upon destruction of the micellar solution by diluting it with a mixture of chloroform and n-decane in the presence of small concentrations of a dye solution. The absence of losses of 10−7−10−5 M SDS upon from 5-to 50-fold preconcentration is confirmed by the standard addition method (RSD = 4–5%); the determination limit of SDS equals 5 × 10−8 M.

Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases.

Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6) and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions and (3) predispose chronically treated individuals to cancer via the inhibition of caspases.

Developing a functional blood substitute as an alternative to donated blood for clinical use is believed to relieve present and future blood shortages, and to reduce the risks of infection and blood type mismatching. Hemoglobin vesicle (HbV) encapsulates a purified and concentrated human-derived Hb solution in a phospholipid vesicle (liposome). The in vivo safety and efficacy of HbV as a transfusion alternative have been clarified. Auto-oxidation of ferrous Hb in HbV gradually increases the level of ferric methemoglobin (metHb) and impairs the oxygen transport capabilities. The extension of the functional half-life of HbV has recently been proposed using an electron mediator, methylene blue (MB), which acts as a shuttle between red blood cells (RBC) and HbV. MB transfers electron energies of NAD(P)H, produced by RBC glycolysis, to metHb in HbV. Work presented here focuses on screening of 15 potential electron mediators, with appropriate redox potential and water solubility, for electron transfer from RBC to HbV. The results are assessed with regard to the chemical properties of the candidates. The compounds examined in this study were dimethyl methylene blue (DMB), methylene green, azure A, azure B, azure C, toluidine blue (TDB), thionin acetate, phenazine methosulfate, brilliant cresyl blue, cresyl violet, gallocyanine, toluylene blue, indigo carmine, indigotetrasulfonate, and MB. Six candidates were found to be unsuitable because of their insufficient diffusion across membranes, or overly high or nonexistent reactivity with relevant biomolecules. However, 9 displayed favorable metHb reduction. Among the suitable candidates, phenothiazines DMB and TDB exhibited effectiveness like MB did. In comparison to MB, they showed faster reduction by electron-donating NAD(P)H, coupled with showing a lower rate of reoxidation in the presence of molecular oxygen. Ascertaining the best electron mediator can provide a pathway for extending the lifetime and efficiency of potential blood substitutes.

A new electrocatalytic scheme for the detection of DNA is described. This scheme relies on the different interactions of single- and double-stranded oligonucleotides with the organic dyes methylene blue, thionin, azure A, azure B, toluidine blue O, brilliant cresyl blue and neutral red, which in addition can act as NADH catalysts. This catalytic activity can be used for amplifying differentiation between duplex and single-stranded DNA. Pyrolytic graphite electrodes modified with adsorbed dsDNA–organic dye complexes gave higher electrocatalytic NADH oxidation currents than those measured with electrodes modified with adsorbed ssDNA–organic dye. This difference is maximum for brilliant cresyl blue. These results suggest that the proposed scheme may be useful for the detection of DNA hybridisation and damage. Keywords: DNA hybridisation, Catalytic NADH oxidation, Phenoxazines, Phenothiazines, Phenazines

Visual oxygen indicators can give information on the quality and safety of packaged food in an economic and simple manner by changing color based on the amount of oxygen in the packaging, which is related to food spoilage. In particular, ultraviolet (UV)-activated oxygen indicators have the advantages of in-pack activation and irreversibility; however, these dye-based oxygen indicator films suffer from dye leaching upon contact with water. In this work, we introduce carrageenans, which are natural sulfated polysaccharides, to develop UV-activated colorimetric oxygen indicator films that are resistant to dye leakage. Carrageenan-based indicator films were fabricated using redox dyes [methylene blue (MB), azure A, and thionine], a sacrificial electron donor (glycerol), an UV-absorbing photocatalyst (TiO2), and an encapsulation polymer (carrageenan). They showed even lower dye leakage in water than conventional oxygen indicator films, owing to the electrostatic interaction of anionic carrageenan with cationic dyes. The MB/TiO2/glycerol/carrageenan oxygen indicator film was successfully bleached upon UV irradiation, and it regained color very rapidly in the presence of oxygen compared to the other waterproof oxygen indicator films.

The use of magnetic micro- and nanoparticles for the removal of pollutants from wastewater is gaining increasing attention. Here, amine-functionalized magnetic microparticles (AFMMs) and carboxylic-functionalized magnetic microparticles (CFMMs) were synthesized by modifying the surface of Fe3O4 with amino and carboxyl groups for fast and efficient removal of textile dyes from aqueous solution. The functionalized magnetic microparticles were characterized by TEM, SEM, FTIR, and VSM. The adsorption experiments were carried out by varying the regulating parameters like solution pH and adsorbent dosage and analyzed in terms of kinetic and isotherm models. It was demonstrated that simple electrostatic interactions between functionalized magnetic microparticles and adsorbates played a dominating role in the adsorption of textile dyes. The positively charged AFMMs adsorbed the negatively charged dyes vat blue (VB) and direct violet (DV) at pH 6 with the maximum removal percentages of 95.72 and 97.29 %, respectively. The maximum removal percentages of cationic dyes methylene blue (MB) and azure A chloride (AA) on the negatively charged CFMMs were 92.28 and 92.22 % at pH 11, respectively. Moreover, the adsorbed dyes could be desorbed completely from the surface of CFMMs at a lower pH, and AFMMs also allowed rapid removal of VB and DV in different water samples. All the results in the present work demonstrated that the functionalized magnetic microparticles as efficient, magnetically separable adsorbents are attractive for the removal of dye pollutants.

Phanerochaete chrysosporium lignin peroxidase (LiP) can degrade synthetic dyes such as heterocyclic, azo, and triphenylmethane on its activation by H2O2. Analysis of the reaction products indicated that N-demethylation reactions are involved in the degradation of crystal violet and methylene blue (MB). We studied LiP oxidation of methylene blue and azure B (AB) in reaction mixtures containing different dye:H2O2 stoichiometric relations aiming at the selective formation of N-demethylated derivatives. High yields, about 70%, of the mono- and didemethylated derivatives, azure B and azure A, were obtained with the use of 1:1 and 1:2 MB:H2O2, respectively. Using azure B as substrate in reaction mixtures containing 1:1 AB:H2O2, a yield of 70% was also observed in azure A. Reaction mixtures containing 1:3 MB:H2O2 and 1:2 AB:H2O2, originated several oxidation products in similar proportions. These results indicated that the process of enzymatic degradation of methylene blue and azure B initiates via N(CH3)2 oxidation. According to the yields that were obtained for azure B and azure A, this enzymatic route can be used for the synthesis of these dyes since these data compare favorably to the chemical route that has a yield of 35%. The use of a dye:H2O2 relation of 1:10 resulted in a decoloration level of about 85%, showing the usefulness of this procedure for wastewater treatment. The reaction products were followed by spectrophotometric analysis within the wavelength of 500-700 nm. The product identifications were performed using a reverse-phase high-performance liquid chromatography (HPLC) C-18 column and thin-layer chromatography.

b-Cyclodextrin forms 1:1 inclusion complexes with methylene blue, azure A, toluidine blue, resorcinol blue, neutral red, safranine T, indigo carmine and acridine orange in aqueous media. The formation constants are determined by differential pulse polarography and spectrophotometry. The supramolecular interaction in the inclusion complexes can be employed to immobilize dyes on an electrode. This gives high sensitivity and stable electrochemical behavior for H2O2 detection at the mmol l ˇ1 level by means of the supramolecular interaction between b-CD and dye molecules. # 1999 Elsevier Science B.V.

The dimerization and inclusion complexation equilibria of six phenothiazine (PN) dyes with cyclodextrins (α-, β-, and γ-CDs) in aqueous media have been studied using absorption and fluorescence spectroscopy. The PN dyes used in this study are thionine (TH), azure A (AZA), methylene blue (MB), toluidine blue (TB), new methylene blue (NMB), and 1,9-dimethylmethylene blue (DMMB). The dimerization constants (KD) of the dyes having two methyl substituents at the phenothiazine ring (NMB and DMMB) are much greater than those of other dyes having unsubstituted rings, and the presence of methyl groups on the amine groups affects little the KD values. The positions of the monomer/dimer equilibria do not change with the presence of α-CD, while the addition of β-CD suppresses and γ-CD enhances the dimerization of the dyes except DMMB. The equilibrium constants for the inclusion complexation of the dye monomers and dimers with CDs are determined from the analysis of the dependence of the absorption spectra of the dye ...

Methylene blue (MB) and its derivatives azure A, B, C and thionine are photoactive and, in principle, are suitable for photodynamic virus inactivation of blood and blood products, such as therapeutic plasma. Methylene blue was selected for plasma decontamination because it is being clinically used and because of its known toxicological and other properties. The standard procedure for photodynamic treatment of single units of fresh plasma involves illumination with visible light at an MB concentration of 1 microM. Polymerase chain reaction analysis revealed that, in addition to model viruses, the bloodborne viruses hepatitis B virus, hepatitis C virus, human immune deficiency virus-1 and probably also the nonenveloped parvovirus B19 are sensitive to MB/light treatment. The procedure is further improved when the fluorescent tubes routinely used for illumination are replaced by more intense light sources, e.g. light-emitting diodes or low-pressure sodium lamps. Surprisingly, the improved virus kill is accompanied by reduced damage to plasma proteins.

The electropolymerization of thionine in an aqueous solution has been carried out by using both cycling voltammetry and chronoamperometry experiments. Electropolymerization has been applied at various pHs by using different working electrodes. The characterization of poly(thionine) thin films is performed by using cyclic voltammetry, current–time transients, atomic force microscopy, scanning tunnelling microscopy, ATR-FTIR, and UV–vis absorption spectroscopy techniques. It is deduced from current–time transients and morphological data that poly(thionine) film forms with random adsorption process and exhibits an irregular polymeric film. Poly(thionine) film formation was compared with that of previously published results of phenothiazine derivatives of poly(azure A) and poly(methylene blue). It has been concluded that type of amine functional group of phenothiazine dyes is very effective on the formation of polymeric film structure. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 39686.

In this report we describe a simple and rapid staining technique for cartilage and bone embedded in Araldite. Semithin sections of embryonic vertebrae obtained from 15 to 17 day mouse fetuses were stained using an aqueous solution 0.25% with respect to methylene blue, 0.25% with respect to azure A, and 0.5% with respect to Na2 CO3, then counterstained with 1% aqueous pararosaniline chloride (MAP). Results were compared with toluidine blue stained sections. MAP permitted good discrimination of developmental stages of both cells and extracellular matrix within vertebral ossification centers during endochondral ossification. The technique is simple, rapid and applicable to plastic embedded sections, and can be used prior to ultrastructural examination.

Decomposition mechanism of methylene blue (MB) caused by a mixture of metallic iron-maghemite (Fe0-γFe2O3) was investigated by means of 57Fe-Mossbauer spectroscopy, X-ray diffractometry (XRD), Ultraviolet-Visible Absorption Spectroscopy (UV-vis) and electrospray-ionization mass spectroscopy (ESI-MS). Ten day-leaching test of 10 μmol L − 1 MB aqueous solution and Fe0-γFe2O3 mixture (mass ratio 3:7) showed a decrease in the concentration from 10.5 to 4.45 μmol L − 1 with first-order rate constant (k) of $1.5_{7} \times 10^{-1}$ day − 1. An ESI-MS study of Fe0-γFe2O3 mixture (3:7) after the leaching test revealed new peaks at m/z of 100, 110 and 137 due to fragmentation of MB, in addition to those observed at m/z of 284, 270 and 256 which were ascribed to MB, Azure B and Azure A, respectively. 57Fe-Mossbauer spectra of Fe0-γFe2O3 mixture (3:7) resulted in a decrease in absorption area (A) for Fe0 (δ = 0.00±0.01 mm s − 1, H int = 33.0±0.1 T) from 37.5 to 21.3, 9.7, 7.9, 7.0 and 4.5±0.5 %, together with an increase in A from 0.0 to 5.0, 13.8, 17.2, 21.0 and 22.4±0.5 % for octahedral (O h) iron (FeII + FeIII) of Fe3O4. XRD study of these samples indicated that the peak intensity at 2Θ of 44.7° being ascribed to Fe0 was decreased, while that of 35.6° due to Fe3O4 was almost constant; relative peak ratio of Fe $^{0}/\mathrm{Fe}_{3}$ O4 was decreased from 26.3 to 2.76 after the leaching, indicating that Fe0 in the Fe0-γFe2O3 mixture was oxidized to Fe3O4. These experimental results suggest that Fe0-γFe2O3 mixture could be utilized for the cleaning or decomposition of toxic organic compounds like trichloroethylene.

A simple room-temperature photochemically induced fluorescence (RTPF) method was developed for the determination of several biomedically important phenothiazines including azure A, methylene blue, thionine, promethazine and unsubstitued phenothiazine in aqueous solutions. These compounds are weakly or non-fluorescent under these conditions but their fluorescence can be induced on UV irradiation. The influence of pH and irradiation time on their excitation and emission fluorescence wavelengths and on their fluorescence intensity was investigated. It was found that the optimum pH values ranged from 2.0 for unsubstituted phenothiazine to 7.0 for promethazine and 10–13 for the other compounds, the selected irradiation times being between 5 s and 17 min, depending on the compound. Linear calibration graphs were obtained over about two orders of magnitude, with relative standard deviations in the range 3.9–6.9%. RTPF limits of detection were between 12 and 70 ng ml−1, according to the compound. RTPF was applied to the determination of phenothiazines in urine samples. The mean recoveries ranged from 87 to 116%.

We tested various cationic dyes chemically related to ruthenium hexaammine trichloride (RHT) [i.e., the RHT-cyclohexanedione complex (RHT-CC), pentaamine ruthenium N-dimethylphenylenediimine trichloride (PRT), tris-(bipyridyl)ruthenium (II) chloride (TRC), tris (bipyridyl) iron (II) chloride (TIC), and cobalt hexaammine trichloride (CHT)] for their effectiveness in precipitating cartilage matrix proteoglycans in situ. Dyes were introduced into media at the onset of processing and were present throughout both aldehyde fixation and osmium tetroxide post-fixation. Contrary to expectation, most of the dye-proteoglycan complexes generated and stable under aldehyde fixation conditions were found to be unstable during post-fixation despite the continuing presence of the dye. A similar phenomenon was also found for the cationic dyes commonly used for precipitation of proteoglycans in cartilage tissue sections (such as Acridine Orange, Alcian Blue, Azure A, Methylene Blue, and Ruthenium Red). Only two dyes, i.e., RHT and the newly tested RHT-CC, formed proteoglycan precipitates sufficiently stable to resist disruption and extraction during osmium tetroxide post-fixation. The latter may be particularly useful in semiquantitative analyses of proteoglycan content in unstained tissue sections owing to its intense brown-black color. For applications in which the osmium tetroxide post-fixation step may be omitted, TRC and PRT may also be valuable for semiquantitative histochemistry by virtue of their stable fluorescence and intense violet color signals, respectively.

Aqueous solutions of a number of biological stains were completely decontaminated to the limit of detection using Amberlite resins. Amberlite XAD-16 was the most generally applicable resin but Amberlite XAD-2, Amberlite XAD-4, and Amberlite XAD-7 could be used to decontaminate some solutions. Solutions of acridine orange, alcian blue 8GX, alizarin red S, azure A, azure B, Congo red, cresyl violet acetate, crystal violet, eosin B, erythrosin B, ethidium bromide, Janus green B, methylene blue, neutral red, nigrosin, orcein, propidium iodide, rose Bengal, safranine O, toluidine blue O, and trypan blue could be completely decontaminated to the limit of detection and solutions of eosin Y and Giemsa stain were decontaminated to very low levels (less than 0.02 ppm) using Amberlite XAD-16. Reaction times varied from 10 min to 18 hr. Up to 500 ml of a 100 micrograms/ml solution could be decontaminated per gram of Amberlite XAD-16. Fourteen of the 23 stains tested were found to be mutagenic to Salmonella typhimurium. None of the completely decontaminated solutions were found to be mutagenic.