The Toluidine Blue-Membrane Filter Method: Absorption Spectra of Toluidine Blue Stained Bacterial Cells and the Relationship Between Absorbance and Dry Mass of Bacteria

Propan-2-ol did not leach dye from toluidine blue stained bacteria on membrane filters but ethanol did. The absorption spectra of toluidine blue stained cells of two Gram-positive and two Gram-negative organisms differed with the latter organisms exhibiting metachromasia. The results suggest that toluidine blue stains the cell envelope. Linear regression equations were derived for each of four organisms, Streptococcus cremoris, Lactobacillus bulgaricus, Pseudomonas fluorescens and Escherichia coli, relating absorbance at the peak of the absorption spectra and the mass of cells on the filters. With these equations it should be possible to determine mass of cells with an error between 3% and 7.5% depending on the organism. Since the regression equations are similar, the amount of toluidine blue retained per milligram of cells may be constant under standard conditions, irrespective of species.     

A toluidine blue-membrane filter method for the quantitative staining of bacteria

A method for measuring the uptake of toluidine blue by bacteria on membrane filters was developed. Bacteria were filtered out of solution onto a cellulose acetate filter and stained on the filter at 50 C with toluidine blue in citrate-phosphate buffer, pH 4.0. The filter was destained in ethanol, placed on a glass slide and subsequently made transparent in a 1,4-dioxan and cyclohexanone mixture. The absorbance of the stained bacteria on the slide was measured in a spectrophotometer at 590 nm. The uptake of dye by cells of Streptococcus cremoris and Escherichia coli could be explained using the Freundlich adsorption isotherm. Cell concentrations of both these organisms can be determined with this technique.     

Acid polysaccharide content of frog rod outer segments determined by metachromatic toluidine blue staining

Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358 +/- 0.134 X 10(4) at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells.     

A Toluidine Blue-Membrane Filter Method for the Quantitative Staining of Bacteria

A method for measuring the uptake of toluidine blue by bacteria on membrane filters was developed. Bacteria were filtered out of solution onto a cellulose acetate filter and stained on the filter at 50 C with toluidine blue in citrate-phosphate buffer, pH 4.0. The filter was destained in ethanol, placed on a glass slide and subsequently made transparent in a 1,4-dioxan and cyclohexanone mixture. The absorbance of the stained bacteria on the slide was measured in a spectrophotometer at 590 nm. The uptake of dye by cells of Streptococcus cremoris and Escherichia coli could be explained using the Freundlich adsorption isotherm. Cell concentrations of both these organisms can be determined with this technique.     

Acid polysaccharide content of frog rod outer segments determined by metachromatic toluidine blue staining

Summary Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358±0.134×10⁴ at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells.Supported by NIH grants EY00012, EY07035 and EY01583     

Staining Mast Cells for Morphometric Evaluation on an Image Analysis System

Multiple skin sections from three nonhuman primates (Macaca mulatta) and three hairless guinea pigs (Cavia porcellus) were stained with 12 different histologic stains to determine whether mast cells could be selectively stained for morphometric analysis using an image analysis system (IAS). Sections were first evaluated with routine light microscopy for mast cell granule staining and the intensity of background staining. Methylene blue-basic fuchsin and Unna's method for mast cells (polychrome methylene blue with differentiation in glycerin-ether) stained mast cell granules more intensely than background in both species. Toluidine blue-stained sections in the guinea pig yielded similar results. Staining of the nuclei of dermal connective tissue was enhanced with the methylene blue-basic fuchsin and toluidine blue stains. These two stains, along with the Unna's stain, were further evaluated on an IAS with and without various interference filters (400.5-700.5 nm wavelengths). In both the methylene blue-basic fuchsin and toluidine blue stained sections, mast cell granules and other cell nuclei were detected together by the IAS. The use of interference filters with these two stains did not distinguish mast cell granules from stained nuclei. Unna's stain was the best of the 12 stains evaluated because mast cell granule staining was strong and background staining was faint. This contrast was further enhanced by interference filters (500.5-539.5 nm) and allowed morphometric measurements of mast cells to be taken on the IAS without background interference.     

Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.     

Differential use of Alcian blue and toluidine blue dyes for the quantification and isolation of anionic glycoconjugates from cell cultures: application to proteoglycans and a high-molecular-weight glycoprotein synthesized by articular chondrocytes

Alcian blue and toluidine blue dyes form complexes with anionic glycoconjugates (AG) such as proteoglycans (PG) and glycosaminoglycans (GAG). However, the Alcian blue-AG complexes do not readily dissociate, while the toluidine blue-AG complexes do so in salt solutions. This differential dissociation of the dye-AG complexes has been utilized in the analysis and isolation of radiolabeled AG elaborated by articular chondrocyte cultures incubated with the radiolabeled precursors of AG. For the rapid quantification of newly synthesized (35)S-labeled PG, small replicate aliquots of the radiolabeled culture media were applied directly to cellulose acetate strips, stained with Alcian blue and the stained immobilized radiolabeled PG was quantified by liquid scintillation counting. Comparison of anionic glycoconjugates quantified in the culture media employing toluidine blue and Alcian blue staining on cellulose acetate trips gave similar results. Staining on cellulose acetate strips using these two dyes is particularly suited for the simultaneous processing of large numbers of samples, as illustrated by the screening of the effects of biological materials and drugs on AG synthesis, in cultures labeled with [(35)S]-sulfate and [(3)H]-glucosamine. The Alcian blue and toluidine blue precipitation methods yielded similar results for the total AG recovered from the media of TGF-beta-stimulated chondrocytes. Electrophoretic analysis of toluidine blue- and Alcian blue-precipitated AG followed by autoradiography and Alcian blue staining in combination with silver nitrate demonstrated that both dyes yielded similar pattern of bands on gels. However, some AG from Alcian blue precipitate did not enter the gel, suggesting incomplete dissociation of Alcian blue-AG complex. The application of the toluidine blue precipitation method, in combination with enzymatic digestion of the GAG chains of the PGs, is illustrated by the isolation of a non-PG high-molecular-weight AG, as well as the PGs from the media of chondrocyte cultures stimulated by TGF-beta.     

Staining of fresh epiphyseal cartilage with toluidine blue

Summary Staining of fresh, unfrozen sections of rat tibia in 0.01% toluidine blue at pH 4.5 revealed the presence of large -metachromatic granules in the cells of the proliferative zone, small -metachromatic granules in the cells of the hypertrophic zone, and both types in cells of intermediate size in the maturing zone. The matrix of the reserve zone stained with toluidine blue; the matrix of the proliferative and upper hypertrophic zone was not stained, but staining appeared again in the lower hypertrophic (calcifying) zone and in the cartilage cores of the metaphyseal trabeculae. Similar but more erratic results were obtained with frozen sections of Carnoy-fixed, gelatin-embedded specimens. These results could not be obtained with simple frozen section techniques or ordinary histological processing, which disrupt the intracellular granules and alter the state of combination of the protein-polysaccharides of the matrix. The results support the concept that a change in the nature of the mucopolysaccharide-containing components is one of the factors involved in the change from non-calcifiable to calcifiable cartilage matrix.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82--for euchromatic arms--and 0.85--for centromeric heterochromatin--were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

Kinetic study of cooperative binding reaction of toluidine blue to poly (alpha,L-glutamic acid) by means of the electric-field pulse method.

Interaction of toluidine blue with helical and randomly coiled poly(α,L -glutamic acid) was studied with absorption spectra, titration, and electric-field pulse measurements. The obtained values of various parameters for the helical form of poly(α,L -glutamic acid)differed from those for its coiled form. The difference of parameters in these two forms of poly(α,L -glutamic acid) was attributed to differences of the binding mechanism in both forms. Furthermore, the binding of toluidine blue to poly(α,L -glutamic acid) was considered to be due to hydrogen binding in the helical conformation and ionic interaction in the coiled conformation of the polymer.     

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin

Summary The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes showed higher electron density than control chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82 — for euchromatic arms — and 0.85 — for centromeric heterochromatin — were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.     

A comparison of modified methenamine silver and toluidine blue stains for the detection of Pneumocystis carinii in bronchoalveolar lavage specimens from immunosuppressed patients.

Pneumonia due to Pneumocystis carinii is an increasingly frequent occurrence; a prompt, accurate diagnosis is important to successfully manage this infection. Methenamine silver and toluidine blue stainings of material recovered by bronchoalveolar lavage (BAL) have been the most widely employed techniques for detecting Pneumocystis organisms. The value of these two staining techniques for the detection of Pneumocystis organisms was prospectively compared in 220 BAL specimens obtained from 186 immunosuppressed individuals. The patients included those with disease-related and therapy-related immunosuppression; half of the BAL specimens came from organ transplant recipients. The results indicate that neither method is superior to the other and that both will correctly identify almost all Pneumocystis infections if a sufficient aliquot of BAL material is examined systematically by trained individuals. The toluidine blue method is somewhat simpler to perform, however. The reasons for discrepant results between the two staining methods were analyzed.     

Gene responsible for protecting Escherichia coli from sodium dodecyl sulfate and toluidine blue plus light.

Escherichia coli cells were killed by visible light irradiation in the presence of the photosensitizing dye, toluidine blue. Two uvrB mutant strains of E. coli K-12 (AB1885 and N3-1) were much more sensitive than the isogenic uvrA and uvrC strains to treatment with toluidine blue plus light, suggesting that the uvrB+ gene product was involved in repair of DNA damage induced by the treatment. The uvrB+ gene cloned in a high- or low-copy-number plasmid was transformed into the uvrB strain (AB1885). Although all the transformants showed the same resistance as its wild-type strain (AB1157) to UV irradiation, they were as sensitive as AB1885 was to treatment with toluidine blue plus light. The two uvrB strains were more sensitive to sodium dodecyl sulfate than the other strains, suggesting that these strains had a defect in the cell surface. A sodium dodecyl sulfate-resistant revertant obtained from AB1885 was more resistant than AB1885 was to treatment with toluidine blue plus light. The two uvrB strains (AB1885 and N3-1) appear to have a defective gene (tentatively called dvl) different from uvrB. Its map position was around 7 min on the E. coli map.     

Comparison between the toluidine blue stain and the Feulgen reaction for evaluation of rabbit sperm chromatin condensation and their relationship with sperm morphology

Sperm chromatin alteration is an important feature that can affect fertility of the male rabbit. This study compared toluidine blue staining with Feulgen reaction (as methods for evaluating chromatin alteration) and investigated the relationship between sperm morphology and chromatin alteration. Seven hundred rabbit ejaculates of animals with unknown fertility were used. Primary and secondary morphological sperm abnormalities were evaluated in semen smears with phase-contrast microscopy. Chromatin alterations were evaluated in semen smears stained with toluidine blue (pH 4.0 and 5.0) and with the Feulgen reaction. While the three methods were equally efficacious for identification of chromatin alterations, toluidine blue staining was more appropriate to characterize the intensity of chromatin alterations. The correlation between primary sperm defects and chromatin alteration was high and positive, suggesting that sperm chromatin structure affected sperm head morphology. The correlation between secondary sperm defects and chromatin alteration was also positive, but lower. The final chromatin compaction occurs in the epididymus, where secondary sperm defects originate. Therefore, the causes of secondary sperm defects could also intervene with final chromatin compaction. In summary, the toluidine blue stain was an effective means of evaluating the sperm chromatin alteration in rabbit spermatozoa.     

Anisotropic staining of apurinic acid with toluidine blue

Summary Using normal rat liver imprints, studies were carried out on the effects of histone extraction and the formation of aldehyde groups from deoxyribose on anisotropic toluidine blue staining of depurinized DNA after sodium bisulfite treatment. The anisotropic effect of bisulfite was found to be determined by binding of bisulfite ions to the aldehyde groups of apurinic acid which, together with free phosphate groups of DNA ensure coparallel attachment of the dye molecules. It was also shown that at pH 5.0 toluidine blue binds with both the phosphate and aldehyde groups of apurinic acid, to give anisotropic staining.     

Difference in localization of eosinophils and mast cells in the bovine ovary

The bovine ovary contains a considerable number of leucocytes which can be located with an antibody against the CD18 molecule. In the present study, subtyping and cell counting were carried out on histological sections stained with Sirius red for eosinophils and with toluidine blue for mast cells. The CD18(+) cells were identified immunohistologically. Eosinophils and mast cells contributed considerably to the CD18(+) pool. The number of eosinophils in the corpus luteum increased rapidly in early development to approximately 90% of the CD18(+) cells, and decreased to 30% during secretion and to 10% during regression. Mast cells were not detectable in the follicles, the corpus luteum and the periphery of the cortex, but were observed in the interstitial cortical stroma and the medulla. The number of mast cells in these regions, which corresponded to 60-76% of the CD18(+) cells, did not change significantly throughout the oestrous cycle. It is concluded that eosinophils are selectively recruited at the periovulatory period and that mast cells are unevenly distributed.     

Mutagenic potential of toluidine blue evaluated in the Ames test.

Toluidine blue is a vital, metachromatic thiazine dye which is used as an adjunct in clinical examination for the early detection of asymptomatic recurrent or secondary primary carcinoma in individuals who are at high risk for developing oral cancer. Because available data on the mutagenicity of toluidine blue was limited and contradictory, this study was conducted to evaluate the mutagenic potential of toluidine blue in the in vitro Ames Salmonella test. Tester strains TA97a, TA98, TA100 and TA102 were used. Toluidine blue was tested at concentrations of 0.1, 1.0, 10, 50, 100, 250 and 500 micrograms/plate, with and without S9 microsomal activation, and positive and negative controls were included. Results from tests without S9 showed a significant increase (p less than 0.05) in number of revertants in TA102 and in TA97a with 50 and 100 micrograms toluidine blue/plate, respectively. In tests with S9 activation, doses of toluidine blue ranging from 10 to 250 micrograms/plate induced dose-related increases in the number of revertants in all 4 strains. The results of this study indicate that toluidine blue has a mutagenic effect in the Ames test.     

Utility of toluidine blue staining and brush biopsy in precancerous and cancerous oral lesions

OBJECTIVE: To evaluate the usefulness of toluidine blue and brush biopsy in precancerous oral lesions and squamous cell carcinoma. STUDY DESIGN: The study was conducted at Moti Lal Nehru Medical College, Allahabad, India. Ninety-six patients with suspicious oral lesions who attended the outpatient clinics of otorhinolaryngology were screened with in vivo toluidine blue staining and oral brush biopsy. RESULTS: Oral brush biopsy showed high specificity and sensitivity. Toluidine blue staining was highly sensitive and moderately specific for malignant lesions but less sensitive for premalignant lesions. CONCLUSIONS: Early detection of oral carcinoma is possible even at the precancerous stages by using noninvasive, painless and outpatient procedures, such as in vivo toluidine blue staining and brush biopsy.     

A Full Color System for Quantitative Assessment of Histochemical and Immunohistochemical Staining Patterns

Quantitative measures of staining distributions are important to compare the presence and patterns of cells or macromolecules. Typically, achromatic thresholding systems are used to compare staining distributions. Achromatic video signals, however, lack sufficient resolution to identify and compare chromatic changes. The purpose of this study is to describe a full color system for analysis of chromatic staining distributions. The hardware system includes a Leitz Diaplan microscope, video camera, GVP videoboard and Amiga 3000 computer. Software was developed in “C” to partition the video signal into hue (H), saturation (S) and value (V). Also, percentage of stained area was determined. Kodak color filters were used to assess the accuracy and precision of the system. Craniofacial tissues were stained with varying concentrations of toluidine blue and primary anti-Brdll antibodies. HSV and the percentage of stained areas were determined and displayed low coefficients of error. HSV values also performed as expected for standard filters as well as cellular staining concentrations. This system is easily implemented and should be useful for comparing chromatic changes with any color resulting from histochemical or immunohistochemical procedures.