A Nile Blue Staining Technic for the Differentiation of Melanin and Lipofuscins

When paraffin sections are stained in 0.05-.01% Nile blue in 1 % sulfuric acid, washed thoroughly in water and mounted in aqueous media, lipofuscins color deep blue, melanins dark green, myelin and red cells lighter greens and background pale green. If, immediately after staining, the preparations are at once extracted with acetone with or without a 1% sulfuric acid rinse, melanins remain dark green, mast cells color purple, lipofuscins and background decolorize and nuclei may stain light green.     

Structure and function of the conidiospore pigments of Penicillium cyclopium.

The cell wall of mature, green condiospores of Penicillium cyclopium Westling contains at least two pigments: A green chromoprotein which is extractable by means of formic acid or liquified phenol and a black insoluble pigment. Both fractions after long term treatment with boiling conc. HCl leave black amorphous residues which, due to their chemical and physico-chemical properties, belong to the group of melanins. The chemical structure of these melanins is still unidentified. No degradation products typical for indol-type or catechol-type melanins have so far been detected. During spore maturation parallel to an increase of pigmentation (determined by remission), the melanin residue left after acid hydrolysis of spores increases. The mature, dark green spores of the wild type strain contain about 40% melanin, the yellow-green spores of the mutant aux-glu 1 about 36%. The unpigmented spores of mutant res-eth 1 possess a melanin content of only about 5%. This value is nearly the same as that found in hyphae, which in all strains are yellowish-brown. The heavily pigmented condia of the wild type strain are about 100-times less sensitive to UV-radiation compared with the unpigmented spores of the mutant res-eth 1. The reduced sensitivity indicates that, as with other microorganisms, the conidia pigments of P. cyclopium are protective components of the spores.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

Luxol Fast Blue Mbs and Phloxine; a Stain for Mitochondria

The applicability of Luxol fast blue MBS as a 0.1% solution in 0.05% acetic acid to the staining of mitochondria, first recognized in rat kidney by Shanklin and Nassar (Stain Techn., 34: 257-60. 1959), was confirmed in various organs (formalin-Zenker and Regaud's fixations; paraffin embedding) of the mouse and bullfrog. In liver cells and in the epithelium of renal tubules, mitochondria were stained green, selectively and clearly. The dark cells of the renal tubules and the middle piece of sperms in both animals were conspicuously demonstrated by their dense assemblages of green granules. The periodic acid-Schiff procedure proposed by Shanklin and Nassar as a counterstain was replaced by staining in 0.5% aqueous phloxine, 2-3 min; differentiation in 5% phosphotungstic acid, 2 min; and washing in water, 5 min. This simplified and accelerated the techique, and gave a better color contrast. Advantages of Luxol fast blue MBS and phloxine staining over traditional methods for mitochondria in paraffin sections are: durability of the stain, high specificity, simplicity of procedure, and constant result.     

A differential staining technique for simultaneous visualization of mitotic spindle and chromosomes in mammalian cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanol:acetic acid solution containing 4 mM MgCl2 and 1.5 mM CaCl2 at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

A Differential Staining Technique for Simultaneous Visualization of Mitotic Spindle and Chromosomes in Mammalian Cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanobacetic acid solution containing 4 mM MgCl₂ and 1.5 mM CaCl₂ at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

Carotenoid and Pterin Pigment Localization in Fish Chromatophores

The classical sulfuric acid method for the histochemical detection of carotenoids has been adapted to give a reliable cytological localization of these compounds in fish chromatophores. This procedure consists mainly in fixing skin fragments in glutaraldehyde and dehydrating in a 50% solution of glycerin followed by exposure to air. It is essential that the preparations permit direct contact of the sulfuric acid with the pigment cells. Under these conditions, carotenoid containing cells stain green or blue. When associated with the extraction of the carotenoids by acetone, the procedure permits the distinction between pterin and carotenoid in fish chromatophores.     

Elevated levels of plasma lipofuscins in patients with chronic renal failure

The fluorescence excitation and emission spectra observed in plasma from patients with chronic renal failure were reproduced by the generation of soluble lipofuscins in normal plasma samples by incubation with mixtures of L-dopa, dopamine, L-norepinephrine, L-epinephrine, 3-hydroxy-DL-kynurenine and 3-hydroxy-anthranilic acid for 24 h at 37 degrees C. Relative fluorescence intensity measurements consistently showed elevated plasma levels of the soluble lipofuscins in chronic renal failure: the means (n = 27) were 73.9 +/- 33.4 (SD) and 71.1 +/- 14.8 at emissions 413 nm and 445 nm respectively, in contrast to those of normal plasma samples (n = 11), 18.2 +/- 5.3 and 23.1 +/- 5.6. The maximum or shoulder at approximately 413 nm represents soluble lipofuscin that can be generated from 3-hydroxyanthranilic acid and the maximum or shoulder at approximately 445 nm represents soluble lipofuscins derived from the precursors listed above and probably from other related precursors. Gravimetric measurements also showed elevated levels of melanins in the plasma samples of patients with chronic renal failure: 2.72 +/- 0.38 mg/ml (n = 16), as compared to normal values: 1.70 +/- 0.10 mg/ml (n = 6). In individual patients haemodialysis reduced the fluorescence intensities to a range of 65-99% and the melanin levels to a range of 86-99% of the pre-dialysis values.     

Brom Cresol Green and Brom Phenol Blue as Indicators of Respiration Deficiency in Yeast

Strains of Saccharomyces which contained cells of respirationally normal (wild-type) and of respiration-deficient (RD) mutants were grown on untrient agar plates containing 20-23 mg/liter of either brom cresol green (BCG) or brom phenol blue (BPB). Glucose content of the media was varied experimentally in the range of 1.5-6%. After 2-4 days of incubation, normal colonies were very palely stained whereas RD colonies were drak green with BCG and dark blue with BPB. Optimum glucose content in the medium was 2-3% for S. cerevisiae, S. carlbergensis and S. chevalieri, but Fleischmann's (baker's) yeast developed the best color contrast with 4-5% glucose.     

Male mate searching behavior in the black chafer Holotrichia loochooana loochooana (Sawada) (Coleoptera: Scarabaeidae): response to pheromone lures of various colors and intensities

In order to understand the visual orientation mechanisms of the male black chafer Holotrichia loochooana loochooana to females, we examined male behavioral response to colored objects (lures) when female sex pheromone was present. In the field, males landed more frequently on darker lures than on lighter ones of the same color. Among lures of different colors but with the same brightness, males preferred landing on black, blue, or red ones to green or grey ones. The reflectance intensities of preferred lures were weaker than those of green and grey in the range of 540–580 nm. This range is considered to affect choice by males. When females perch on the leaf edges of the food plant to adopt a calling posture, the dominant background color is that of the leaves (i.e., green). This means that the landing target forms a silhouette (dark spot) which is prominent against a background of green leaves, providing maximum visual acuity for mating.     

The effect of light color on the nucleocytoplasmic and chloroplast cycle of the green chlorococcal algaScenedesmus obliquus

The color of light (white, red, blue, and green) had a significant effect on the growth and reproductive processes (both in the nucleocytoplasmic and chloroplast compartment of the cells) in synchronous cultures of Scenedesmus obliquus. This effect decreased in the order red > white > blue > green. In the same order, the light phase of the cell cycle (time when first autospores started to be released) was prolonged. The length of dark phase (time when 100 % of daughters were allowed to release from mothers) was not influenced and was the same for all colors. Critical cell size for cell division in green light was shifted to a smaller size (compared with cells grown in other lights) and so was the size of released daughters. The nuclear cycle was slowed in blue and even in green light, contrary to cells grown in red and white light. At the beginning of the cell cycle, one-nucleus daughters possess approximately 10 nucleoids; during the cell cycle their number doubled in all variants before the division of nuclei. Both events were delayed in cultures grown more slowly most markedly in green light. Smaller daughters in the green variant possessed a lower number of nucleoids. Motile cells released in continuous green or blue lights but not in red one were rarely observed.     

Species‐dependent microarchitectural traits of iridescent scales in the triad taxa of Ornithoptera birdwing butterflies

Ornithoptera birdwing butterflies have blue, green, or orange iridescent scales in different species or subspecies. To understand the species‐ or subspecies‐dependent scale color differences, we performed comparative morphometric analyses of iridescent scales from three closely related taxa: O. priamus priamus (green), O. priamus urvillianus (blue), and O. croesus (orange). The three types of Ornithoptera wings exhibited reversible color changes to longer wavelengths with different kinetics upon immersion in methanol, suggesting that their color differences are at least partly based on differences in the size of air cavities made by nanostructures. Cover scales of all three color types were visually semi‐transparent glass scales that exhibited color when placed on a dark background. The dorsoventral differences in coloration were observed in single scales, suggesting the optical importance of scale surfaces. Scanning electron microscopy of cover scales in cross section revealed that all color types exhibited finely sculpted tapered ridges and thick, irregular basal multilayers containing tandemly clustered granular objects and air cavities. Scale thickness, ridge height, and multilayer thickness were significantly different among the three color types, and granular object size was significantly different between orange scales and blue and green scales. We conclude that each of the three taxa of Ornithoptera butterflies possesses unique quantitative size values on tapered ridges and irregular multilayers with granular objects and air cavities to express unique structural color. These species‐ or subspecies‐dependent structural colors might have evolved via quantitative shifts in these microarchitectural traits rather than via changes in the basic developmental or architectural plan for color expression.     

Chemical Characterization of Melanins in Sheep Wool and Human Hair

The color of hair and wool in mammals and feathers in birds is mostly determined by the quantity and quality of melanins that are synthesized in follicular melanocytes and transferred to keratinocytes. There are two chemically distinct types of melanin pigments: the black to brown eumelanins and the yellow to reddish pheomelanins. Melanins in sheep wool and human hair of various colors were characterized by HPLC methods to estimate 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-derived units in eumelanins and benzothiazine units in pheomelanins. Melanins were also characterized by spectrophotometric methods after differential solubilization in alkalies. It was demonstrated that 1) black wool in Asiatic sheep contains eumelanin with the DHICA content similar to black mouse melanin, while black to brown melanins from human hair contain much lower ratios of DHICA-derived units, comparable to the slaty mutation in mice, 2) dark brown to brown hair in human contains eumelanin whose chemical properties are indistinguishable from those of black hair, 3) dark red wool and red human hair contain pheomelanic pigments whose chemical properties are rather different from those of yellow pheomelanins in mice, and 4) light brown, blonde, and red hairs in human can be differentiated from each other with this methodology.     

Ophthalmoscopic observations of ocular fundus in colony-born cynomolgus monkeys aged from 0 day to 90 days

Apparently healthy 242 colony-born cynomolgus monkeys (Macaca fascicularis) aged from 0 day to 90 days were examined for the findings of ocular fundus by using ophthalmoscope. One drop of mixed solution of 0.5% tropicamide and 0.5% phenylephrine hydrochloride was instilled into each eye of the monkey. Then, those monkeys were anesthetized with ketamine-HC1 at the dose level of 10mg/kg B. W. Regular and fluorescein photographs were taken with Kowa RC-II ophthalmoscope-camera by using daylight typed color film. Following findings were obtained in each age class: Retinal color was salmon pink with 0 to 3-days-old neonates, salmon pink and blue to green with 7 days to 14-days-old animals and blue to green with 60-days to 90-days-old monkeys. As regards optic disc, 0- to 14-days-old animals were observed to be light orange in color, and the infant aged more than 28-days showed orange color. Retinal arteries and veins were lightly reddish in color with every age class. Macular color was salmon pink in 0-day-old cases, slightly dark in 3-days-old neonates and very dark after 14-days of age. Lightly retinal reflex was noted in 0- and 7-days-old animals. The reflex was observable in 14-days-old animals without any case of exception. Retinal hemorrhages were recorded in 22 (67%) of 36 neonates born in natural condition and 10 (33%) of 30 neonates born by cesarean section. These findings will be useful as the criteria for ophthalmoscopic observations of the cynomolgus monkeys as laboratory use.     

Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry

Three metallic ions, NiCl2, CoCl2, and CuSO4, were found to modify the color of the normally brown diaminobenzidine (DAB) reaction. The colors ranged from purplish blue (NiCl2), dark blue/bluish black (CoCl2), to greyish blue (CuSO4). We have found that the CoCl2 + DAB is the ion of choice because: 1) it yields a distinct dark blue color that is easily distinguishable from brown DAB; 2) the blue reaction product is very stable throughout the entire staining procedure; and 3) background staining is minimal. These findings can be applied to the double staining technique of two different antigens in the same section. Among three staining procedures discussed, the avidin-biotin peroxidase complex (Co-DAB)-peroxidase-antiperoxidase (DAB) technique produced the best results because: 1) no antibody elution was needed following the avidin-biotin-peroxidase complex procedure when the CoCl2-DAB modification was used; and 2) no background staining occurred.     

A Cresyl Fast Violet Stain for Bacteria and Fungi in Tissue

The cresyl fast violet staining method was modified to eliminate differentiation. Paraffin sections from tissues fixed in Zenker-formol were stained in a 1% aqueous solution of cresyl fast violet (Chroma), adjusted to pH 3.7 with acetic acid, washed in running tap water, dehydrated and covered. Because basophilia increases with time of fixation or storage in formalin or Kaiserling's fluid, dilution of the dye solution to 0.5-0.1% is recommended for such material. Bacteria, nuclei, Nissl substance, and lipofuscin were colored dark blue; fungi, blue to purple; and cytoplasm and muscle fibers, light blue. Collagen and reticulum fibers were only faintly stained. Thus, microorganisms were easily visible against the lightly colored background. In formalin-fixed material, bile pigment was colored olive green. Because this method does not require differentiation, it gave uniform results even in the hands of different users. Little or no fading was observed in sections stored for more than 2 yr.     

Growth and cell cycle of Ulva compressa (Ulvophyceae) under LED illumination

The cell‐cycle progression of Ulva compressa is diurnally gated at the G₁ phase in accordance with light–dark cycles. The present study was designed to examine the spectral sensitivity of the G₁ gating system. When blue, red, and green light‐emitting diodes (LEDs) were used for illumination either alone or in combination, the cells divided under all illumination conditions, suggesting that all colors of light were able to open the G₁ gate. Although blue light was most effective to open the G₁ gate, red light alone or green light alone was also able to open the G₁ gate even at irradiance levels lower than the light compensation point of each color. Occurrence of a period of no cell division in the course of a day suggested that the G₁ gating system normally functioned as under ordinary illumination by cool‐white fluorescent lamps. The rise of the proportion of blue light to green light resulted in increased growth rate. On the other hand, the growth rates did not vary regardless of the proportion of blue light to red light. These results indicate that the difference in growth rate due to light color resulted from the difference in photosynthetic efficiency of the colors of light. However, the growth rates significantly decreased under conditions without blue light. This result suggests that blue light mediates cell elongation and because the spectral sensitivity of the cell elongation regulating system was different from that of the G₁ gating system, distinct photoreceptors are likely to mediate the two systems.     

What's in a band? The function of the color and banding pattern of the Banded Swallowtail

Butterflies have evolved a diversity of color patterns, but the ecological functions for most of these patterns are still poorly understood. The Banded Swallowtail butterfly, Papilio demolion demolion, is a mostly black butterfly with a greenish‐blue band that traverses the wings. The function of this wing pattern remains unknown. Here, we examined the morphology of black and green‐blue colored scales, and how the color and banding pattern affects predation risk in the wild. The protective benefits of the transversal band and of its green‐blue color were tested via the use of paper model replicas of the Banded Swallowtail with variations in band shape and band color in a full factorial design. A variant model where the continuous transversal green‐blue band was shifted and made discontinuous tested the protective benefit of the transversal band, while grayscale variants of the wildtype and distorted band models assessed the protective benefit of the green‐blue color. Paper models of the variants and the wildtype were placed simultaneously in the field with live baits. Wildtype models were the least preyed upon compared with all other variants, while gray models with distorted bands suffered the greatest predation. The color and the continuous band of the Banded Swallowtail hence confer antipredator qualities. We propose that the shape of the band hinders detection of the butterfly's true shape through coincident disruptive coloration; while the green color of the band prevents detection of the butterfly from its background via differential blending. Differential blending is aided by the green‐blue color being due to pigments rather than via structural coloration. Both green and black scales have identical structures, and the scales follow the Bauplan of pigmented scales documented in other Papilio butterflies.     

A Method for Staining Pollen Tubes in Pistil

A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsia, 6 ml; 1% aqueous aniline blue, 4 ml; 1 % orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45±2 C They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45±2 C, hydrolyzed in the clearing and softening fluid at 58±1 C for SO min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.