Improved sensitivity of trypan blue dye exclusion assay with Ni2+ or Co2+ salts

A modified trypan blue dye exclusion assay was developed usingNi²⁺ or Co²⁺ salts to determine the viability ofprimary and transformed cells. When the cells were preincubatedwith NiSO₄ or CoCl₂ followed by trypan blue assay, thecontrast between stained and unstained cells was significantlyincreased as compared to the conventional trypan blue dyeexclusion assay.     

Benzidine-based dyes: effects of industrial practices,regulations, and world trade on the biological stains market

One of the most sweeping changes in the dye industry since the advent of synthetic dyes grew out of the health risks associated with benzidine. Dyes made from benzidine and its derivatives were used around the world until adverse health effects become incontrovertible. Workers and family members of workers involved in production and use of benzidine-based dyes had a high incidence of bladder cancer. Following publication of several reports documenting this health hazard, dye makers in the USA, Europe, and Japan phased these dyes out of production in the 1970s. Government regulations lent legal support for these voluntary initiatives. Two strategies subsequently evolved to compensate: developed nations brought alternative substances to market while emerging countries increased production of carcinogenic dyes and sold them at discount prices around the world. Nearly all dye manufacturing now has moved away from nations whose costs of production and compliance rendered them unable to compete. The purpose of this brief review is to publicize the health risks associated with dyes made from benzidine and its congeners, and to alert all companies and end users handling these dyes for biomedical applications that composition of the product and lot-to-lot variability may be problematic because of the manufacturing and distribution practices of the countries where they are produced.     

Quirks of dye nomenclature. 3. Trypan blue

Trypan blue is colorant from the 19^th century that has an association with Africa as a chemotherapeutic agent against protozoan (Trypanosomal) infections, which cause sleeping sickness. The dye still is used for staining biopsies, living cells and organisms, and it also has been used as a colorant for textiles.     

Re-evaluating the role of strongly charged sequences in amphipathic cell-penetrating peptides: a fluorescence study using Pep-1

Cell-penetrating peptides (CPPs) are able to translocate across biological membranes and deliver bioactive proteins. Cellular uptake and intracellular distribution of CPPs is commonly evaluated with fluorescent labels, which can alter peptide properties. The effect of carboxyfluorescein label in the Lys-rich domain of the amphipathic CPP pep-1, was evaluated and compared with non-labelled pep-1 in vitro and in vivo. A reduced membrane affinity and an endosomal-dependent translocation mechanism, at variance with non-labelled pep-1, were detected. Therefore, the charged domain is not a mere enabler of peptide adsorption but has a crucial role in the translocation pathway of non-labelled pep-1.     

Trichoplaxin — A new membrane-active antimicrobial peptide from placozoan cDNA

A method based on the use of signal peptide sequences from antimicrobial peptide (AMP) precursors was used to mine a placozoa expressed sequence tag database and identified a potential antimicrobial peptide from Trichoplax adhaerens. This peptide, with predicted sequence FFGRLKSVWSAVKHGWKAAKSR is the first AMP from a placozoan species, and was named trichoplaxin. It was chemically synthesized and its structural properties, biological activities and membrane selectivity were investigated. It adopts an α-helical structure in contact with membrane-like environments and is active against both Gram-negative and Gram-positive bacterial species (including MRSA), as well as yeasts from the Candida genus. The cytotoxic activity, as assessed by the haemolytic activity against rat erythrocytes, U937 cell permeabilization to propidium iodide and MCF7 cell mitochondrial activity, is significantly lower than the antimicrobial activity. In tests with membrane models, trichoplaxin shows high affinity for anionic prokaryote-like membranes with good fit in kinetic studies. Conversely, there is a low affinity for neutral eukaryote-like membranes and absence of a dose dependent response. With high selectivity for bacterial cells and no homologous sequence in the UniProt, trichoplaxin is a new potential lead compound for development of broad-spectrum antibacterial drugs.     

Trypan blue as a fluorochrome for confocal laser scanning microscopy of arbuscular mycorrhizae in three mangroves

Roots of three mangroves, Acanthus ilicifolius, Ceriops tagal and Excoecaria agallocha, collected from forests of the Sundarbans of India were stained with trypan blue to observe arbuscular mycorrhizal colonization. Spores of arbuscular mycorrhizal fungi isolated from rhizospheric soil, collected together with the root samples, also were stained for testing the suitability of the dye as a fluorochrome. Confocal laser scanning microscopy images were constructed. A. ilicifolius and E. agallocha exhibited “Arum” type colonization with highly branched arbuscules, whereas C. tagal showed “Paris” type association with clumped and collapsed arbuscules. We demonstrated that trypan blue is a suitable fluorochrome for staining arbuscular mycorrhizal fungal spores, fungal hyphae, arbuscules and vesicles, which presumably have a considerable amount of surface chitin. It appears that as the integration of chitin into the fungal cell wall changes, its accessibility to trypan blue dye also changes.     

Book Review

A DEPARTMENT DEVOTED TO ABSTRACTS OF BOOKS AND PAPERS FROM OTHER JOURNALS DEALING WITH STAINS AND MICROSCOPIC TECHNIC IN GENERAL

RILEY, JAMES F. The Mast Cells. 182 pp., 65 figures. Size 81/2 by 10 inches. Price $6.75. E. and S. Livingstone Ltd., Edinburgh and London. 1959. Agent in the U. S. A., The Williams & Wilkins Co., Baltimore 2, Md.

MICROSCOPE AND OTHER APPARATUS GARIN, A., and THELLIER, M. Méthode de microdétermination et de microdosage de Bore dans une solution ou un tissu végétal par activation aux neutrons et examen microscopique des autoradiographies. Bull. d'Micr. Appl., Ser. 2, 8, 129-48. 1958.

KUHL, W., and FISCHER, H. Vertikal-, Horizontal-, und Umkehrmikroskop, in Verbindung mit einer Filmkamera, mit schnellem Funktionswechsel und geringem Gewicht. Zschr. wiss. Mikr. 64, 73-83. 1959.

MAI, G., and HEINE, U. Beschreibung einer Mikrofilmeinrichtung mit Zeitraffung. Zschr. wiss. Mikr. 64, 65-72. 1959.

TERTIAN, ROBERT, and TRILLAT, JEAN-JACQUES. Les applications du titane en microscopie électronique. Bull. d'Micr. Appl., Ser. 2, 8, 1-6. 1958.

MICROTECHNIC IN GENERAL HIRSCH, TH. v., and BOELLAARD, J. W. Methacrylsäureester als Einbettungsmittel in der Histologie. Zschr. wiss. Mikr. 64, 24-9. 1958.

PIEKARSKI, G. Über mikroskopisch erfassbare Rückstände in einigen sog. flüchtigen organischen Fixierungsmitteln. Zschr. wiss. Mikr. 63, 499-502. 1958.

WALTER, FRIEDRICH. Die Wirkungsweise der Fliessbewegung von Polymethacrylate bei der Herstellung von Ultradünnschnitten. Zschr. wiss. Mikr. 64, 106-10. 1959.

ARVY, LUCIE. Mise en évidence simultanée des lipides figués et des substances métachromatique. Bull. Micr. Appl., Ser. 2, 8, 120-124. 1958.

DE GROODT, M., LAGASSE, A., and SEBRUYNS, M. Vesikulationsvorgänge der Blut-Luftschranke der Lunge. Zschr. wiss. Mikr. 64, 90-4. 1959.

JUSTER, M. Contribution a l'étude du tissue osseux non dimeneralisé. Bull. Micr. Appl., Ser. 2, 9, 34-36. 1959.

PONLOT, ROBERT. L'intérět du noir Soudan B en histologie des os. Bull. Micr. Appl., Ser. 2, 8, 125-126. 1958.

WUNDERLICH, H. Blutzählkammern mit fluoreszierender Netzteilung für Fluoreszenzuntersuchungen. Zschr. wiss. Mikr. 64, 47-9. 1958.

PLANT MICROTECHNIC HEIMBURGER, M. Cytotaxonomic studies in the genus Anemone. Canad. J. Bot. 37, 587-612. 1959.

MORRISON, J. W. Cytogenetic studies in the genus Hordeum. I. Chromosome morphology. Canad. J. Bot. 37, 527-38. 1959.

MICROORGANISMS BORZANI, W., and VAIRO, M. L. R. Quantitative adsorption of crystal violet by dead microorganisms. J. Bact., 77, 757-9. 1959.     

Rapid chromatography for evaluating adsorption characteristics of cellulase binding domain mimetics

The cost of cellulolytic enzymes is one barrier to the economic production of fermentable sugars from lignocellulosic biomass for the production of fuels and chemicals. One functional characteristic of cellulolytic enzymes that improves reaction kinetics over mineral acids is a cellulose binding domain that concentrates the catalytic domain to the substrate surface. We have identified maleic acid as an attractive catalytic domain with pK(a) and dicarboxylic acid structure properties that hydrolyze cellulose while producing minimal degradation of the glucose formed. In this study we report results of a rapid chromatographic method to assess the binding characteristics of potential cellulose binding domains for the construction of a synthetic cellulase over a wide range of temperatures (20 degrees to 120 degrees C). Aromatic, planar chemical structures appear to be key indicators of cellulose adsorption. Indole, the side-chain of the amino acid tryptophan, has been shown to reversibly adsorb to cellulose at temperatures between 30 degrees and 120 degrees C. Trypan blue, a polyaromatic, planar molecule, was shown to be irreversibly adsorbed to cotton cellulose at temperatures of <120 degrees C on the time scale of the experiments. These results confirm the importance of hydrophobic cellulose and the cellulose-binding component of cellulolytic enzymes and cellulolytic enzyme mimetics.     

Fluorescein Diacetate Uptake to Determine the Viability of Human Fetal Cerebral Cortical Cells

We investigated the accuracy of fluorescein diacetate uptake as an indicator of the viability of human fetal cerebral cortical cells. Cortical cells from 16-26-week-old normal fetuses were studied. The cortices were dissociated mechanically with normal saline to make a suspension. Fluorescein diacetate uptake and trypan blue exclusion were compared as methods for examining cell viability. Our results show that fluorescein diacetate uptake is a simple and sensitive method for examining human fetal cortical cell viability.     

A simple and rapid method for evaluating the survival of xeroderma pigmentosum lymphoid lines after irradiation with ultraviolet light

Summary A simple, rapid and reproducible test has been developed to measure the viability of cells after irradiation with ultraviolet light (UV). Epstein-Barr virus-transformed lymphoid lines, derived from patients with xeroderma pigmentosum (XP), were irradiated with UV, and the post-UV viability of the lymphoid lines was determined by the trypan blue dye exclusion method. The relative post-UV survival of the patients' lymphoid lines was similar to the relative post-UV survival of the patients' fibroblast strains.