The suitability of further thiazine and quinoline dyes for topo-optical reactions on the plasmalemma (author's transl)]

The present studies prove that the thiazin dyes, azure B, azure C and thionin, and the quinolin dyes pinacyanol and its hydrochloride, are suitable for topo-optical staining of the plasmalemma. On the membrane surface the orientated bound dye molecules become stabilized, and with subsequent precipitation the anisotropic effect is reinforced. On optical analysis, the thiazin dye molecules (azure B, AZURE C and Thionin) are bound radially on the membrane. The molecules of the previously studied quinolin dye, N,N'-diethylpseudoisocyanide chloride are bound parallel to the membrane, while pinacyanol and its hydrochloride, like the thiazin dyes, are bound in the radial position.     

Topo-optische Reaktionen an der Membran von menschlichen Erythrocytenschatten

Zusammenfassung Frühere Untersuchungen erwiesen, daß die Thiazinfarbstoffe Toluidinblau, Azur A, Azur B, 1:9-Dimethyl-Methylenblau, sowie die Chinolinfarbstoffe N,N-Diäthylpseudoisocyaninchlorid und N,N-6,6-Dichlorpseudoisocyaninchlorid für die topo-optische Reaktion an der menschlichen Erythrocytenmembran geeignet sind. In dieser Studie wird die Verwendbarkeit der Thiazin- und Chinolinfarbstoffe an den menschlichen Erythrocytenschatten gezeigt. Auf Grund der optischen Analyse sind die Thiazinfarbstoffmoleküle radiär zur Membran ausgerichtet, während sich die Chinolinfarbstoffmoleküle membranparallel anlagern.

---

Topo-optical reactions on the membrane of the human red cell ghost

Summary Previous studies have proved that the thiazin dyes toluidine blue, azure A, azure B, 1.9-dimethyl methylene blue and the quinolin dyes N,N-diethylpseudoisocyanine chloride, N,N-6,6-dichlorpseudoisocyanine chloride are suitable for topo-optical reaction on the membrane of the red blood cells. In the present study the applicability of the thiazin and quinolin dyes on the membrane of the human red cell ghost was examined. Optical analysis revealed that the thiazin dyes are bound in radial position to the membrane, while the quinolin dyes are bound parallel to the membrane's plane.

     

Zur Eignung weiterer Thiazin- bzw. Chinolinfarbstoffe für die topo-optische Reaktion am Plasmalemm

Zusammenfassung Die hier durchgeführten Untersuchungen erwiesen, daß die Thiazinfarbstoffe Azur B, Azur C und Thionin sowie der Chinolinfarbstoff Pinacyanol bzw. sein Hydrochlorid für die topooptische Reaktion am Plasmalemm geeignent sind.-Die an der Membranoberfläche orientiert gebundenen Farbstoffmoleküle werden durch eine nachträgliche Präzipitation stabilisiert und gleichzeitig wird der anisotrope Effekt verstärkt.-Die Thiazinfarbstoffmoleküle (Azur B, Azur C, Thionin) sind nach der optischen Analyse radiär zur Membran ausgerichtet.-Gegenüber dem früher untersuchten Chinolinfarbstoff N,N-Diäthylpseudoisocyaninchlorid, dessen Farbstoffmoleküle parallel zur Membran lagen, sind das Pinacyanol bzw. sein Hydrochlorid in gleicher Weise, wie die Thiazinfarbstoffe radiär zur Membran ausgerichtet.

---

The suitability of further thiazin and quinolin dyes for topo-optical reactions on the plasmalemma

Summary The present studies prove that the thiazin dyes, azure B, azure C and thionin, and the quinolin dyes pinacyanol and its hydrochloride, are suitable for topo-optical staining of the plasmalemma. On the membrane surface the orientated bound dye molecules become stabilized, and with subsequent precipitation the anisotropic effect is reinforced. On optical analysis, the thiazin dye molecules (azure B, azure C and Thionin) are bound radially on the membrane. The molecules of the previously studied quinolin dye, N,N-diethylpseudoisocyanide chloride are bound parallel to the membrane, while pinacyanol and its hydrochloride, like the thiazin dyes, are bound in the radial position.

     

Methods for the Standardization of Biological Stains Part V

In this paper are given the methods for determining the suitability of certain dyes of the pyronin, thiazin, oxazin, azin and natural dye groups for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Pyronin G, pyronin B, neutral red, safranin, nigrosin water-soluble, brilliant cresyl blue, cresyl violet, Nile blue A, thionin, methylene blue, methylene azure (azure A), azure C, toluidine blue O, indigo carmin (indigotine) and carmin. For each of these dyes methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Methods for the Standardization of Biological Stains Part V

In this paper are given the methods for determining the suitability of certain dyes of the pyronin, thiazin, oxazin, azin and natural dye groups for certification by the Commission on Standardization of Biological Stains. These methods have been developed by the Commission in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Department of Agriculture. The dyes for which the methods are given in the present paper are: Pyronin G, pyronin B, neutral red, safranin, nigrosin water-soluble, brilliant cresyl blue, cresyl violet, Nile blue A, thionin, methylene blue, methylene azure (azure A), azure C, toluidine blue O, indigo carmin (indigotine) and carmin. For each of these dyes methods are discussed under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Mediation of 8-hydroxy-guanine formation in DNA by thiazin dyes plus light

We have discovered that methylene blue plus light mediates the formation of 8-OHdG in DNA. Methylene blue is one of several thiazin dyes and we report here that the other thiazin dyes tested, in combination with white light, are effective in mediating 8-OHdG formation in DNA. The effectiveness of light plus the thiazin dyes in forming 8-OHdG in DNA were as follows: methylene blue greater than azure B greater than azure A greater than toluidine blue greater than thionin. Two other compounds tested; riboflavin and fuschin acid, in combination with light, caused formation of very little, if any, 8-OHdG in DNA. Thiazin dye mediated formation of 8-OHdG in DNA was not inhibited by the spin trap alpha-phenyl-t-butyl nitrone, which supports our previous observations that oxygen free radical scavengers did not inhibit methylene blue plus light mediated 8-OHdG formation in DNA. Ascorbate addition to methylene blue plus DNA, in the absence of light, was ineffective in mediating 8-OHdG formation in DNA.     

Topo-optische Färbung der Erythrocytenmembran mit Thiazin- und Chinolinfarbstoffen

Zusammenfassung Die Untersuchungen erwiesen, daß die Farbstoffe 1:9-Dimethyl-Methylenblau, Azur A und N,N-Diäthylpseudoisozyaninchlorid für topo-optische Reaktionen an der Membran von Erythrocyten geeignet sind. Die Farbstoffmoleküle werden an der Membran orientiert gebunden. Ihre Bindung kann durch Behandlung mit Präzipitationslösungen stabilisiert werden, und zugleich wird die Anisotropie verstärkt. Die optische Analyse ergab, daß 1:9-Dimethyl-Methylenblau und Azur A radiär zur Membran ausgerichtet sind, während sich N,N-Diäthylpseudoisozyaninchlorid membranparallel anlagert.

---

Tope-optical staining with thiazin and quinolin dyestuffs of the erythrocyte membrane

Summary The present studies prove the dyestuffs 1.9-dimethyl methyleneblue, azure A and N,N-diethylpseudoisocyanine chloride suitable for topo-optical reactions with the membrane of the red blood cell. The dye molecules are bound in orientated fashion. Treatment with precipitants stabilizes the binding of dye molecules and, in addition, it enhances the birefringence. Optical analysis revealed 1.9-dimethyl methyleneblue and azure A bound in radial position, however, N,N-diethylpseudoisocyanine chloride was bound parallel to the membrane's plane.

     

Purification of Thionin, Azure A, Azure B and Methylene Blue

Column and paper chromatography of four thiazin dyes revealed both inorganic and organic impurities. In thionin, azure A, azure B and methylene blue, sodium and other metal cations were found as inorganic impurities. The analysis for organic impurities revealed that the dyes were mixtures; specifically each dye contained one or more of the other dyes as impurities. Inorganic impurities were detected by ashing the dyes in the presence of H₂SO₄ and chromatographing the sulfate salts on paper. They were removed by filtration through ion exchange resins. Organic impurities were detected by paper chromatography and removed by column chromatography on Woelm's neutral alumina.     

Mechanisms of chromosome banding

A thorough understanding of the mechanisms of R-, C-and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral adsorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. — Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not a necessary component for banding. — The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their adsorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.     

The purification of methylene blue and azure B by solvent extraction and crystallization.

Detailed schemes are described for the preparation of purified methylene blue and azure B from commercial samples of methylene blue. Purified methylene blue is obtained by extracting a solution of the commercial product in an aqueous buffer (pH 9.5) with carbon tetrachloride. Methylene blue remains in the aqueous layer but contaminating dyes pass into the carbon tetrachloride. Metal salt contaminants are removed when the dye is crystallized by the addition of hydrochloric acid at a final concentration of 0.25 N. Purified azure B is obtained by extracting a solution of commercial methylene blue in dilute aqueous sodium hydroxide (pH 11-11.5) with carbon tetrachloride. In this pH range, methylene blue is unstable and yields azure B. The latter passes into the carbon tetrachloride layer as it is formed. Metal salt contaminants remain in the aqueous layer. A concentrated solution oa azure B is obtained by extracting the carbon tetrachloride layer with 4.5 X 10(-4)N hydrobromic acid. The dye is then crystallized by increasing the hydrobromic acid concentration to 0.23 N. Thin-layer chromatography of the purified dyes shows that contamination with related thiazine dyes is absent or negligible. Ash analyses reveal that metal salt contamination is also negligible (sulphated ash less than 0.2%).     

Lower azure B methylene blue ratios in Giemsa type blood and malaria stains

Starting from ancient reports that rare samples of methylene blue were apparently sufficiently contaminated with azures to give red plasmodial and red purple nuclear chromatin in Chenzinsky type methylene blue eosin stains, it was decided to determine how little azure B would suffice for such staining in methylene blue eosin stains. The traditional 1902 Giemsa had an azure : methylene blue : eosin ratio of about 6 : 3 : 6.3 : 10; Lillie's 1943 formula had a 5 : 7 : 10 ratio. In the current series of tests 5 : 7 : 10 (I), 4 : 8 : 10 (II), 3 : 9 : 10 (III), 2 : 10 : 10 (IV), 1 : 11 : 10 (V), and 0 : 12 : 10 (VI) were used. Malaria and blood stains were better than the standard 5 : 7 : 10 (I) in III, IV and II in that order. Normal and leukemic human blood, mouse blood with Plasmodium berghei, and monkey blood with the CDC strain of Pl. falciparum were used as test materials. The staining mixtures were made from highly purified samples of azure B and methylene blue. Staining mixtures contained 12 ml 0.1% thiazin dye, 10 ml 0.1% eosin, 2 ml each of glycerol, methanol and 0.1 M phosphate buffer pH 6.5, 3 ml acetone as accelerator, and distilled water to make 40 ml; staining times of 10--30 min were used.     

The Azure Dyes their Purification and Physicochemical properties. II. Purification of Azure B

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or plychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polyineric adsorbent (XAD-2) the acetate-formate anions are exchanged for chloride. Regeneration of both columns is possible: KMnO₄, Na₂S₂O₄ and water are run through column A, 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

Zinc Chloride Methylene Blue. I. Biological Stain History, Physical Characteristics and Approximation of Azure B Content of Commercial Samples

Zinc chloride methylene blue appeared on the market almost contemporaneously with the zinc-free medicinal form. The former has rarely been reported as being used in blood stains. Recent suspension of manufacture of medicinal methylene blue by it. principal American producer has excited interest in the use of the zinc chloride form for the preparation of blood stains. According to Lillie (1944a,b) the azure B content of zinc chloride methylene blue may have varied from 5 to 30% in the samples studied. Taking the Merck Index (1968, 1976) figures for the spectroscopic absorption maximum (λmax) of 667.8 and 668 nm as standard, recent samples of zinc chloride methylene blue are calculated to contain 6-8% azure B. These figures are baaed on 1) the shift of λmax after exhaustive pH 9.5 chloroform extraction, 2) evaluation of the actual ratio of the observed TiCl₂ dye content to the theoretical for pure zinc chloride methylene blue, 3) comparison of spectroscopic and staining effects of graded hot dichromate oxidation products with those of highly purified azure B-methylene blue mixtures of known proportions.

As far as can be found, medicinal methylene blue is almost the exclusive source of cosin polychrome methylene blue blood stains. Lillie (1944c) included a short series comparing 5 zinc chloride methylene blues with a dozen medicinal methylene blue samples; all were oxidized with hot dichromate to produce successful Wright stains. No effort was made to remove the zinc Exhaustive pH 9.5 chloroform extraction of zinc chloride methylene blue (lot MCB 12-H-29) yielded a small amount of red dye which when extracted into 0.1 N HCI gave λmax = 650. The extraction moved the absorption peak of the zinc chloride methylene blue from 667 to 668 nm and the midpoint of the 90% maximum absorption band, 18 nm wide, from 666.5 to 667.5 nm.     

The azure dyes: their purification and physicochemical properties. II. Purification of azure B.

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or polychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polymeric adsorbent (XAD-2) the acetate-formate anions are exchanged in chloride. Regeneration of both columns is possible: KMnO4, Na2S2O4 and water are run through column A; 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

Lower Azure B Methylene Blue Ratios in Giemsa Type Blood and Malaria Stains

Starting from ancient reports that rare samples of methylene blue were apparently sufficiently contaminated with azures to give red plasmodial and red purple nuclear chromatin in Chenzinsky type methylene blue eosin stains, it was decided to determine how little azure B would suffice for such staining in methylene blue eosin stains. The traditional 1902 Giemsa had an azure:methylene blue: eosin ratio of about 6:3:6.3:10; Lillie's 1943 formula had a 5:7:10 ratio. In the current series of tests 5:7:10 (I), 4:8:10 (II), 3:9:10 (III), 2:10:10 (IV), 1:11:10 (V), and 0:12:10 (VI) were used. Malaria and blood stains were better than the standard 5:7:10 (I) in III, IV and II in that order. Normal and leukemic human blood, mouse blood with Plasmodium berghei, and monkey blood with the CDC strain of Pl. falciparum were used as test materials. The staining mixtures were made from highly purified samples of azure B and methylene blue. Staining mixtures contained 12 ml 0.1% thiazin dye, 10 ml 0.1% eosin, 2 ml each of glycerol, methanol and 0.1 M phosphate buffer pH 6.5, 3 ml acetone as accelerator, and distilled water to make 40 ml; staining times of 10-30 min were used.     

Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions.

Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur. One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation. The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components. Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark. Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue. Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage. In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing. Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue. Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased. Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution. Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage. Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.     

Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions

Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur. One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation. The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components. Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark. Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue. Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage. In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing. Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue. Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased. Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution. Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage. Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.     

Investigation of photosensitizing dyes for pathogen reduction in red cell suspensions

Despite recent advances in blood safety by careful donor selection and implementation of infectious disease testing, transmission of viruses, bacteria and parasites by transfusion can still rarely occur. One approach to reduce the residual risk from currently tested pathogens and to protect against the emergence of new ones is to investigate methods for pathogen inactivation. The use of photosensitizing dyes for pathogen inactivation has been studied in both red cell and platelet blood components. Optimal properties of sensitizing dyes for use in red cell suspensions include selection of dyes that traverse cell and viral membranes, bind to nucleic acids, absorb light in the red region of the spectrum, inactivate a wide range of pathogens, produce little red cell photodamage from dye not bound to nucleic acid and do not hemolyze red cells in the dark. Early research at the American Red Cross focused on the use of a class of dyes with rigid structures, such as the phenothiazine dyes, beginning with the prototypical sensitizer methylene blue. Results revealed that methylene blue phototreatment could inactivate extracellular virus, but resulted in undesirable defects in the red cell membrane that resulted in enhanced hemolysis that became evident during extended refrigerated blood storage. In addition, methylene blue phototreatment could neither inactivate intracellular viruses nor appreciably inactivate bacteria under conditions of extracellualar viral killing. Attempts to improve intracellular viral inactivation led to the investigations of more hydrophobic phenothiazines, such as methylene violet or dimethylmethylene blue. Although these dyes could inactivate intracellular virus, problems with increased red cell membrane damage and hemolysis persisted or increased. Further studies using red cell additive storage solutions containing high levels of the impermeable ion, citrate, to protect against colloidal osmotic hemolysis as well as competitive inhibitors to limit sensitizer binding to red cell membranes revealed that photoinduced hemolysis stemmed from dye bound to the red cell membrane as well as dye free in solution. Use of red cell additive solutions to prevent colloidal-osmotic hemolysis and use of novel flexible dyes that only act as sensitizers when bound to their targets are two techniques that currently are under investigation for reducing red cell damage. Ultimately, the decision to implement a photodynamic method for pathogen reduction will be determined by weighing the risks of unintended adverse consequences of the procedure itself, such as the potential for genotoxicity and allergic reactions, against the cost and benefits of its implementation.     

Investigation of Thiazin Dyes as Biological Stains I. The Staining Properties of Thionin and its Derivatives as Compared with Their Chemical Formulae

An investigation has been made of the staining properties of eight dyes of the thionin group. The dyes studied are as follows: tetra-ethyl thionin, asymmetrical di-ethyl thionin, tetra-methyl thionin (methylene blue), tri-methyl thionin (azure B), asymmetrical di-methyl thionin (azure A), symmetrical di-methyl thionin, mono-methyl thionin (azure C), and unsubstituted thionin. The staining properties were tested on sections of paraffin embedded material following five different methods of fixation. No counterstain was employed. It was shown that there was a general correlation between the extent of ethylation or methylation of the dyes and their staining properties. As one passes from tetra-ethyl thionin down the series to thionin itself, there is a progressive decrease in the amount of green showing in the preparations, and an increase in the amount of red present, also an increase in the metachromatic effects, and in the intensity of nuclear staining. There seems, also, to be a similar relation between staining qualities on the one hand and the color and solubility of the dye base on the other.     

Investigation of Thiazin Dyes as Biological Stains I. The Staining Properties of Thionin and its Derivatives as Compared with Their Chemical Formulae

An investigation has been made of the staining properties of eight dyes of the thionin group. The dyes studied are as follows: tetra-ethyl thionin, asymmetrical di-ethyl thionin, tetra-methyl thionin (methylene blue), tri-methyl thionin (azure B), asymmetrical di-methyl thionin (azure A), symmetrical di-methyl thionin, mono-methyl thionin (azure C), and unsubstituted thionin. The staining properties were tested on sections of paraffin embedded material following five different methods of fixation. No counterstain was employed. It was shown that there was a general correlation between the extent of ethylation or methylation of the dyes and their staining properties. As one passes from tetra-ethyl thionin down the series to thionin itself, there is a progressive decrease in the amount of green showing in the preparations, and an increase in the amount of red present, also an increase in the metachromatic effects, and in the intensity of nuclear staining. There seems, also, to be a similar relation between staining qualities on the one hand and the color and solubility of the dye base on the other.