Combined stain for fish pituitary.

A new combined stain is described for the study of cell types in the fish pituitary. Tissues are prepared by fixing in formol-sublimate and then embedded in paraffin wax. Tissue is sectioned at 5 micron and than stained sequentially with performic acid-alcian blue, periodic acid-Schiff, orange G, and acid fuchsin. As a result of this procedure acidophils stain as follows: lactotropes, red; corticotropes, light pink; melanotropes, bright pink; and somatotropes, orange. Cyanophils stain either magenta red (gonadotropes) or blue (thyrotropes). Neurosecretory material and the fibers of the pars nervosa which penetrate the pars intermedia stain light blue.     

A Tetrachrome Stain for Fresh, Mineralized Bone Sections, Useful in the Diagnosis of Bone Diseases

Fresh, unprocessed bone is ground to sections 75-100 μ thick, stained in an aqueous solution composed of fast green FCF, 0.1 gm; orange G, 2.0 gm; distilled water, 100.0 ml; and adjusted to pH 6.65, then in a mixture of 1 part alcoholic solution of 0.25% celestine blue B and 9 parts of alcoholic solution of 0.1% basic fuchsin. Surface stain is removed by grinding sections to 50 μ and washing them in 1% invert soap (Zephiran) to remove adherent debris. (Commercial detergents and alkaline soaps may interfere with chromophore groups of the dyes.) Wash in tap water; rinse in distilled water and differentiate in 1% acetic alcohol. Dehydrate in ascending alcohols, clear in xylene and mount permanently in a neutral, synthetic resin. Active osteoid seams stain dark to light green; resting osteoid seams, red to bright orange red; transitional osteoid seams, geenish-yellow, orange red to red; older, partly mineralized matrix, orange; new, partly mineralized matrix, red; osteocyte nuclei, red; osteoblasts and osteoclasts, greenish-blue to dark purple nuclei and green or light green cytoplasm. Hyper-trophic and differentiating cartilage cells are stained light pink and dark red respectively. The staining reactions are consistent; the solutions are stable.     

Staining of Bacterial Colonies on the Millipore Filter to Improve Contrast

About 5 ml of 1% blue tetrazolium in 70% ethyl alcohol were poured over mature colonies of Pasteurella pestis and Malleomyces pseudomallei on Millipore filters (MF), contained in the filter holder apparatus, and allowed to drain through with the suction applied. The MF was washed with water and then covered with about 10 ml of 0.001% aqueous trypan blue and drained. This technique provided vivid white colonies sharply defined against a blue background.

Another method utilized 0.1% quinacrine-HCl (Atabrine) to stain colonies yellow and 0.05% vital red to stain the MF pink to light red.     

Haematoxylin-Phloxine-Alcian Blue-Orange G Differential Staining of Prekeratin, Keratin and Mucin

This is a modification of Kreyberg's stain with Alcian blue 8GS used to stain acid much while phloxine B and orange G stain keratin and prekeratin. Procedure: Dewax formalin-fixed paraffin sections in xylene and hydrate through alcohol. Stain in Mayer's haemalum, 10 min; blue in tap water; wash in distilled water; stain in 1% phloxine, 3 min; wash in running water, 1 min; wash in distilled water; stain in 0.5% aqueous Alcian blue in 0.5 acetic acid, 5 min; wash in distilled water; stain in 0.5% orange G dissolved in 2.0% phosphotungstic acid, 13 min; dehydrate quickly in 2 changes of 95% alcohol and 2 changes of absolute alcohol; clear in several changes of xylene; mount in a synthetic resin. Acid mucopolysaccharides are stained turquois blue; prekeratin and keratin are orange to red orange.     

Some fluorescent counterstains for neuroanatomical studies

Methods for counterstaining neural tissue that contains fluorescent markers have been developed. Acridine orange is useful for localizing cells that are retrogradely labelled with the fluorescent tracers true blue, bisbenzimide, and nuclear yellow because at low concentrations it yields a green Nissl stain when excited with blue, but not with ultraviolet, light; since the tracers fluoresce only when exposed to ultraviolet light, they are not masked by the counterstain. In addition, counterstaining at pH 2 increases bisbenzimide fluorescence considerably. Ethidium bromide is useful for immunohistochemistry (IHC) because it yields a bright red Nissl counterstain when excited by green light, and is only faintly visible when the fluorescein marker is excited with blue light, or when ultraviolet excitation is used. Ethidium bromide is therefore a good counterstain for fluorescent retrograde tracer and for combined IHC-retrograde tracer studies as well. Certain dyes are also useful for studies of the normal morphology of neural tissue. For example, bisbenzimide and nuclear yellow at low concentrations produce a brilliant Nissl stain at pH 2, and stain only nuclei at pH 7.2. The latter procedure may be particularly useful for cell counts. Finally, neutral red, astrazone red, and safranin-O differentially stain cells amd myelinated fibers, producing fluorescence analogs of the Klüver-Barrera stain.     

Direct Staining of Reticular Fibers with Gold Chloride

Reticular fibers are selectively stained in paraffin sections of formalin-fixed or Bouin's-fixed tissue as follows: 1% aqueous solution of gold chloride for 20 min, followed by a 10 min immersion in an aqueous solution containing 5% Na₂CO₃ and 0.5% KOH. The sections then are placed in a 5% aqueous solution of KI for 2 min. Counterstaining with a 0.25% aqueous solution of methylene blue chloride is optional. The reticular fibers stain dark pink; the collagen bundles are a light pink to straw color without the counterstain, or a light blue color when the methylene blue is used.     

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.     

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 fuchsin, 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 30 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.     

Viability and Acrosome Staining of Bull, Boar and Rabbit Spermatozoa

A practical and reliable staining procedure was developed to distinguish the viability and acrosomal status of bull, boar and rabbit spermatozoa. The first stain with trypan blue or Congo red is rapid and avoids artifacts. This stain is precipitated by neutral red during the 2 min required for fixation. The precipitate gives a high contrast black color, resistant to the subsequent rinsings and persists during the time required for staining the acrosome with Giemsa. Ten classes of spermatozoa are distinguished (live or dead with intact acrosomes, loose acrosomes, damaged acrosomes, no acrosome, or with no acrosome and no postacrosomal ring). The intact acrosomes are purple, the loose acrosomes are dark lavender and the damaged acrosomes are pale lavender. The anterior part of the head of live spermatozoa with no acrosome is white or light pink and the same area of dead spermatozoa is white or pale gray. The postacrosomal ring is red. The postacrosomal area of the head of live spermatozoa is white or light pink and the same part of dead spermatozoa is black, dark violet or gray. The procedure did not give satisfactory results for stallion spermatozoa.     

Suitability of different fluorescent powders for mass-marking the Chrysomelid, Diabrotica virgifera virgifera LeConte

Abstract:  The Western Corn Rootworm, Diabrotica virgifera virgifera LeConte (Col., Chrysomelidae), is an invasive alien pest of maize, Zea mays , in Europe. The suitability of 14 fluorescent powders for mass-marking the adults was studied in laboratory and in field cages. The visual discrimination between remaining spots of each colour on the beetles was investigated under ultraviolet (UV) light, as well as their retention time and the influences of those colours on the beetle survival and flight take-off response. The two best recognizable orange colours (i.e. of Radiant Colour and of Fiesta Colours Swada) were proposed for field experiments in first priority, followed by an orange and a yellow (both Magruder Colour), another yellow (Fiesta) and a pink (Radiant), as all did not affect beetle survival and flight take-off response and were recognizable under UV light for at least 10 days in the field. In contrast, the colours yellow and green (Radiant), red and blue (Magruder), yellow (Ciba Geigy) and pink (Fiesta) were unsuitable, because they either quickly disappeared from the beetles or adversely affected beetle survival or flight take-off response. For mass releases with differently marked beetles, only the use of a single orange colour together with a single yellow colour or the use of a pink colour together with a yellow colour can be used since few spots can clearly be discriminated from each other under UV light.     

Blue light reduces photosynthetic efficiency of cyanobacteria through an imbalance between photosystems I and II

Several studies have described that cyanobacteria use blue light less efficiently for photosynthesis than most eukaryotic phototrophs, but comprehensive studies of this phenomenon are lacking. Here, we study the effect of blue (450 nm), orange (625 nm), and red (660 nm) light on growth of the model cyanobacterium Synechocystis sp. PCC 6803, the green alga Chlorella sorokiniana and other cyanobacteria containing phycocyanin or phycoerythrin. Our results demonstrate that specific growth rates of the cyanobacteria were similar in orange and red light, but much lower in blue light. Conversely, specific growth rates of the green alga C. sorokiniana were similar in blue and red light, but lower in orange light. Oxygen production rates of Synechocystis sp. PCC 6803 were five-fold lower in blue than in orange and red light at low light intensities but approached the same saturation level in all three colors at high light intensities. Measurements of 77 K fluorescence emission demonstrated a lower ratio of photosystem I to photosystem II (PSI:PSII ratio) and relatively more phycobilisomes associated with PSII (state 1) in blue light than in orange and red light. These results support the hypothesis that blue light, which is not absorbed by phycobilisomes, creates an imbalance between the two photosystems of cyanobacteria with an energy excess at PSI and a deficiency at the PSII-side of the photosynthetic electron transfer chain. Our results help to explain why phycobilisome-containing cyanobacteria use blue light less efficiently than species with chlorophyll-based light-harvesting antennae such as Prochlorococcus, green algae and terrestrial plants.     

Identification of lymphocyte subpopulations with a polymethine dye

Using the polymethine dye p-ethoxyphenyl-p-aminostyryl-1,3,3-trimethyl-3H-indolium chloride as an aqueous stain applied to specimens of peripheral blood or buffy coat fixed in FAA fixative, differential coloration of leukocytes was achieved using darkfield illumination. Neutrophils stained dark maroon and contained green granules, eosinophils contained bright blue granules, basophils revealed yellow and pink granules, and monocytes stained green with green and yellow vacuoles. In studies of purified lymphocyte subpopulations obtained in a cell sorter, T-helper cells stained red, T-suppressor cells were yellow orange, B-cells appeared yellow and often contained yellow annular structures in the cytoplasm, and natural killer (NK) cells stained green and contained large green granules. As a rapid screening technique for identification of T-helper and T-suppressor cells and their ratios in health and disease, the new polymethine stain may complement the more complex monoclonal antibody techniques for identification of these cells.     

Sequential Use of Wright's and Ziehl-Neelsen's Stains for Demonstrating Phagocytosis of Bacterial Spores

Nongerminating spores, germinating spores, and vegetative cells of Clostridium botulinum type A were observed during phagocytosis in the peritoneal fluid of white mice. Since phagocytes are easily ruptured by heat, the method described avoids heating, as this has been employed in conventional spore staining methods. A thin smear of the fluid is air dried on the slide for 2 hr, and stained by Wright's method: stain, 2 min; dilution water, 2 min; and rinsed; then in 0.005% methylene blue for 30 sec, and rinsed. This is followed by Ziehl-Neelsen's stain for 3-4 min and destained with 1: acetone-95% ethanol for 10 sec. The slide is rinsed, and Wright's staining repeated: stain 1 min, dilution 2-3 min; and thereafter washed about 5 ml of Wright's buffer. Blotting and air drying completes the staining. Non-germinating spores stain light red with a red spore wall, germinating spores are deep red throughout, vegetative cells are blue, and leucocytes show a dark purple nucleus and light blue cytoplasm.     

Suggested Counterstains for Davenport Reduced Silver Preparations of Peripheral Nerves

—Peripheral nerves which have been fixed in a mixture of formaldehyde and acetic acid and stained according to the method of Davenport can be successfully counterstained for demonstration of myelin sheaths and stroma. After mounted sections have been silvered, reduced and toned, the coating of nitrocellulose is removed by passing thru two changes of acetone. Following brief washes in 100,95,85 and 75% alcohols they are stained in an acidified aqueous solution of azo carmine for 30 to 60 minutes. Excess azo carmine is extracted with anilin alcohol followed by acetic alcohol after which the sections are mordanted for 15 to 60 minutes in a 5% aqueous solution of phosphotungstic acid. Without washing they are transferred to a stain mixture of either anilin blue and orange G (acidified) or light green and orange G (acidified) where they remain from 1 to 5 hours. After destaining in 95% alcohol and dehydration in absolute alcohol the sections are mounted in dammar. Result: axons stain black; sheath and fibroblast nuclei, red; myelin sheaths, orange; and connective tissue, blue or green. When the counterstains are applied to ganglia, cytological details of individual cells are demonstrated.     

A Differential Staining Method for Elastic Fibers, Collagenic Fibers, and Keratin

A method allowing for the differential presentation of elastic fibers, other connective tissue fibers, epithelial and other types of cytoplasm, and keratin is described. The procedure is based on the affinity of orcein for elastic fibers, of anilin blue for collagenic material, and of orange G for keratin. Bouin-fixed, tissue-mat embedded sections are stained in Pinkus' acid orcein for 1 1/2 hours and rinsed in distilled water. The sections are differentiated in 50% alcohol containing 1% hydrochloric acid, washed in tap and then in distilled water. The sections are next transferred for I to 2 minutes to the anilin blue, orange G, phosphomolybdic acid combination known as solution No. 2 of Mallory's connective tissue stain, diluted 1:1 with distilled water. They are then rinsed in distilled water, quickly passed into 95% alcohol, and dehydrated in absolute alcohol containing some orange G, after which they are cleared and mounted. Within less than two hours sections may be stained and mounted with the following results: elastic fibers — red; collagenic fibers — blue; muscle fibers — yellow; keratin — orange.     

Stain Technology a Bone Stain for Osteoid Seams in Fresh, Unembedded, Mineralized Bone

Fresh, ground, mineralized bone sections 75-100 μ thick are stained 90 minutes or 48 hours in the Bone Stain, a preparation containing fast green FCF, orange G, basic fuchsin, and azure II. Surface stain is then removed by grinding under running water. Sections are washed in 0.1% zephiran chloride (benzalkonium chloride) or in 0.01% mild soap and again washed in tap water, followed with distilled water. Sections are next differentiated in 0.01% acetic acid in 95% methanol, dehydrated in 95% ethanol and 100% ethanol, cleared in alcohol:xylene 1:1, 1:4, 1:9 and 2 changes of xylol, and then mounted permanently in Eukitt's mounting media.

Osteoid seams stain either green to jade green or red to dark red, incompletely mineralized bone red or orange yellow, and the zone of demarcation light green. The walls of lacunae, canaliculae, feathered bone, procedural artifacts and periosteocyte lacunar low-density versions stain red.

The method helps in the differential diagnosis of certain metabolic bone diseases in human biopsy and autopsy material.     

Photosensitivity of pigmented and nonpigmented strains ofUstilago violacea

White, pink, pumpkin, orange, and yellow strains ofUstilago violacea containing high and low levels of cytochrome c and various carotenes were subjected to high light intensities in the presence and absence of the endogenous photosensitizer toluidine blue (TB). The white strain 15.10 and the cytochrome-c-accumulating pink strain 1.C429 were completely lacking carotenes and were the most sensitive to visible light under all conditions. The phytoene-containing white strain 2.C419, the carotene-containing pink strains AB278a-1, 1.C421, and 2.C425, and pumpkin strain 2.C415 were more resistant to photodestruction in the presence and absence of TB than strains 15.10 and 1.C429. Consistently, the most light resistant strains were the high carotene, low cytochrome-c-accumulating orange, and yellow strains. The photosensitivity of strains ofU. violacea was related to the level of carotene, cytochrome c, the presence or absence of TB, and the age of the culture.     

The Pink Membrane: The Stable Photoproduct of Deionized Purple Membrane

When cations are removed from the purple membrane of Halobacterium halobium it turns blue (λ_max = 603 nm); continuous irradiation with intense red light (λ's ≥ 630 nm) converts this deionized blue membrane into a pink membrane (λ_max ≈ 491 nm). The rate and extent of the transformation from the blue to the pink membrane is facilitated by the removal of the last twenty COOH-terminal amino acids of bacteriorhodopsin. While the chromophore of the blue membrane is a 32:68 mixture of the 13-cis and all-trans isomers of retinal, the chromophore of the pink membrane is 9-cis rectinal. The quantum efficiency of the pink to blue membrane photoconversion is relatively high compared with that of the blue to pink membrane photoconversion. Proton release is observed when the pink membrane is converted to the blue form, and proton uptake occurs during the reverse transition. Unlike the blue membrane, the absorbance maximum of the pink membrane is only slightly affected by cation addition at low pH and ionic strength.     

颜色对梨小食心虫产卵选择性的影响

梨小食心虫(Grapholitha molesta Busck)是一种重要的果树害虫,早春产卵喜在桃嫩梢叶上,为探明寄主颜色在其产卵选择中的作用,利用彩色卡纸模拟寄主颜色,室内比较了红、粉红、浅粉、橙黄、深黄、浅黄、青绿、深绿、浅绿、蓝、紫、褐色等12种不同颜色基质对其成虫产卵选择性的影响。结果发现:基质颜色对梨小食心虫的产卵选择性有显著影响,其产卵偏嗜浅黄和浅绿色,白色和黑色参比时其产卵选择率分别依次为68.9%、63.8%和64.1%、65.5%,蓝和浅粉色则表现一定的拒避作用,白色和黑色参比时其产卵选择率分别依次为47.7%、40.4%和47.2%、42.7%,且参比色对其产卵选择性影响差异显著。基质颜色对其产卵量亦有显著性影响,无论黑或白色参比,黄、绿颜色上的产卵量均较多,尤其是深黄、深绿和青绿色。基质颜色对1—7日龄梨小食心虫成虫的产卵选择性均有显著影响。白色参比时,2、3日龄蛾对浅绿和浅黄色的产卵选择率显著高于其他颜色;黑色参比时,2日龄时明显偏嗜浅绿色(79.7%),6日龄时明显偏嗜浅黄色(74.8%)。表明寄主颜色在梨小食心虫产卵场所选择中具有重要作用,为其卵期监测和防控中颜色应用乃至进一步揭示其产卵寄主选择机理提供了依据和参考。     

The Demonstration of Beryllium Oxide in Paraffin Sections with Chromoxane Stains

Aqueous solutions of the arylmethane dyes Chromoxane pure blue BLD (C.I. No. 43825) and Chromoxane pure blue B (C.I. No. 43830) will stain beryllium oxide. In the presence of EDTA the staining of other metals is masked. As a specific stain for BeO, formol saline fixed paraffin sections are hydrated and stained for 1 hr with either 0.1 gm of pure blue BLD in 100 ml of pH 4.0 Na-acetate buffer or with 0.1 gm of pure blue B in 1 N NaOH adjusted to pH 9.0 with HCl. To mask interference from other metal ions, 9 gm of Na₂-EDTA is added to 100 ml of the stain solution. BeO is stained blue, organic tissue components are either unstained or pink. Results of tests against other materials show that a high degree of specificity may be expected from these dyes. A 1% aqueous solution of neutral red may be used as a counterstain.