Alkaline bismuth solution as an en bloc stain for formaldehyde-glutaraldehyde potassium permanganate fixed fungal spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.     

Localization of Plant Lipids for Light Microscopy Using P-Phenylenediamine in Tissues of Arachis Hypogaea L.

p-Phenylenediamine (pPD) can be used en bloc to preserve and differentiate cell lipids in aldehyde-fixed peanut plant tissues treated with osmium tetroxide during dehydration in 70% ethanol. Semithin plastic sections for light microscopy need o further staining and can be mounted in Histoclad after drying on a slide. Brown staining above background differentiates lipid-containing structures. Nonspecific staining can be distinguished in control preparations extracted en bloc with lipid solvents.     

Identification of Cells by Backscattered Electron Imaging of Silver Stained Bulk Tissues in Scanning Electron Microscopy

Anuran tadpole tail muscle was stained en bloc by a modified light microscope silver stain for light microscopy and freeze-fractured in liquid nitrogen after partial dehydration with ethanol. The fractured specimens were observed in both secondary electron and backscattered electron modes in a scanning electron microscope. Since the cell nuclei specifically stained with silver provided high contrast against the unstained background due to atomic number contrast of backscattered electron image, various cells were easily identified by a comparison of secondary electron images and compositional images of backscattered electron signals.     

A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

Preservation and Long-Term Storage of Feulgen-Stained Mouse Tissues for Subsequent Autoradiography

Tissue of the jejunal crypts of mouse intestine was fixed for 24-48 hr in acetic-ethanol, 1:3, and stained en bloc by the Feulgen reaction. The stained preparations were then stored 4 mo at -25 C in either 45% acetic acid alone or in dimethyl sulfoxide (DMSO) or glycerol to which 45% acetic acid had been added to make 15% of the total volume. Such storage preserved not only the stain but allowed autoradiographs to be made. No loss of silver grains or a decrease of labeling index was observed. The procedures are equally successful when used with double-labeling experiments. Solid, transplantable, experimental carcinomas can be preserved in a manner identical to that suggested for the epithelial cells of the crypts.     

Aldehyde Pararosanilin (True Aldehyde Fuchsin) Stains Purified Insulin, Oxidized or Not, Following Adequate Fixation with Either Formalin or Bouin'S Fluid

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from “basic fuchsin” whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC.

Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content.

We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam® sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.     

En Bloc Staining of Articular Cartilage and Bone

Blocks of canine and porcine articular cartilage were stained en bloc with Weigert's iron hematoxylin or Harris' hematoxylin with or without eosin Y counterstaining and cleared in methyl salicylate. The morphology and three-dimensional relationships of chondrocytes were best demonstrated with Weigert's iron hematoxylin. The morphology of the cartilage and chondrocytes was superior to that in sections of routine hematoxylin and eosin stained, paraffin processed samples. The three-dimensional localization of intracellular lipids in individual and clones of chondrocytes was observed when cartilage samples were stained with oil red O and mounted directly in a water-based medium. Blocks of decalcified bone were stained en bloc with Weigert's iron hematoxylin and cleared with methyl salicylate. The three-dimensional orientation of osteocytes around osteonal canals, in circumferential lamellae, and in interstitial lamellae was demonstrated. The morphology of “cutting cones” in cortical bone also was observed.     

A Hot Aldehyde-Peroxide Fixation Method for Electron Microscopy of the Free-Living Nematode Caenorhabditis Elegans

An improved method for the fixation of the third and fourth larval stages and adults of Caenorhabditis clegans has been developed. Worms are placed in a mixture of 1.5% paraformaldehyde and 1.0% glutaraldehyde at pH 7.0 and 70 C and the suspension promptly cooled in a water bath at 20 C for 1 hr. The fixed worms are then immersed in a mixture of 5% glutaraldehyde and hydrogen peroxide at 4 C for 1 hr, stained en bloc in uranyl acetate, and embedded in resin for electron microscopy. The procedure results in superior fixation, particularly of micro filaments and micro tubules. The high temperature of the initial fixation straightens the worms and thus facilitates serial sectioning.     

Aldehyde pararosanilin (true aldehyde fuchsin) stains purified insulin, oxidized or not, following adequate fixation with either formalin or Bouin's fluid

Gomori reported that aldehyde fuchsin stained the granules of pancreatic islet beta cells selectively and without need of permanganate pretreatment. Others adopted permanganate oxidation because it makes staining faster though much less selective. All aldehyde fuchsins are not equivalent, being made from "basic fuchsin" whose composition may vary from pure pararosanilin to one of its methylated homologs, rosanilin or a mixture. Mowry et al. have shown that only aldehyde fuchsin made from pararosanilin stained unoxidized pancreatic beta cells (PBC). Aldehyde fuchsins made from methylated homologs of pararosanilin stain PBC cells only after oxidation, which induces basophilia of other cells as well; these are less selective for PBC. Is the staining of PBC by aldehyde fuchsins due to insulin? Others have been unable to stain pure insulin with aldehyde fuchsins except in polyacrylamide gels and only after oxidation with permanganate. They have concluded that insulin contributed to the staining of oxidized but not of unoxidized PBC. This view denies any inherent validity of the more selective staining of unoxidized PBC cells as an indication of their insulin content. We describe here indisputable staining of unoxidized pure insulins by aldehyde fuchsin made with pararosanilin. Dried spots of insulin dissolved in the stain unless fixed beforehand. Spots of dried insulin solution made on various support media and fixed in warm formalin vapor were colored strongly by the stain. Insulin soaked Gelfoam sponges were dried, fixed in formalin vapor and processed into paraffin. In unoxidized paraffin sections, presumed insulin inside gel spaces was stained strongly by aldehyde pararosanilin. Finally, the renal tubules of unoxidized paraffin sections of kidneys from insulin-injected mice fixed in either Bouin's fluid or formalin were loaded with material stained deeply by aldehyde pararosanilin. This material was absent in renal tubules of mice receiving no insulin. The material in the spaces of insulin-soaked gels and in the renal tubules of insulin-injected mice was proven to be insulin by specific immunostaining of duplicate sections. The same material was also stained by aldehyde pararosanilin used after permanganate. So, this dye stains oxidized or unoxidized insulin if fixed adequately.     

Alkaline Bismuth Stain as A Tracer for Golgi Vesicles of Plant Cells

An alkaline solution of bismuth subnitrate reacts well with carbohydrate-rich components of Golgi bodies in sections prepared from plant leaves fixed with glutaraldehyde and osmium tetroxide and embedded in Epon. The metal deposits formed are so fine that the stain is appropriate to ultrastructural observation at high magnification. The Golgi vesicles show polarity with respect to the localization of the reactive deposits. Golgi vesicles that had migrated farther from the Golgi cisternae showed greater reactive deposits and higher membrane contrast than those close to the Golgi cisternae. These results indicate that the alkaline bismuth stain is an excellent tracer for Golgi bodies of plant cells.     

Staining of Fungi With Saccharated Iron Oxide

A 0.2% aqueous solution of saccharated iron oxide with a pH of 10.8 is shown to be suitable for supravital staining of fungi. Colonies or fresh tissue materials presumed to contain fungi are immersed in the solution for 1-24 hr, fixed in a 10% neutral solution of buffered formalin about 1 hr, washed, and treated with 10% potassium ferrocyanide in 0.5 N, HC1. Cell membranes of hyphae, perithekia, and walls of sporangia appear dark blue; cytoplasm of the hyphae, sporangiospores and spores stain greenish blue or yellowish green.     

Electron Dense Artefactual Deposits in Tissue Sections: The Role of Ethanol, Uranyl Acetate and Phosphate Buffer

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.     

Quantitative Determination of Murine Dermal Elastic Fibers by Color Image Analysis: Comparison of Three Staining Methods

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Klig-man's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.     

Conjugates of Chitinase with Fluorescein Isothiocyanate or Lissamine Rhodamine as Specific Stains for Chitin In Sztu

The fluorescent chitinase technique is based on the specific affinity of the enzyme for its substrate and applicable when an enzyme can be coupled with a fluorescent dye. Fluorescent chitinase specifically stained chitinous structures in several fungi and an insect, but failed to stain other polysaccharides in bacterial and algal cell walls. Freezing-microtome sections of Drosophila and fungal mycelia 6 μ thick were fixed in acetone for 5 min, then stained and mounted in fluorescent chitinase. Staining of smears of unsectioned fungal material required 5 min in absolute acetone, 5 min in 95% ethanol-1 N aqueous acetic acid (1:1), 10 min in 0.2 M phosphate buffer, PH 5.7, 1 sec in enzyme-dye conjugate, and 10 min in carbonate-bicarbonate buffer (0.2 M, pH 10.7, for chitinase-FITC; pH 7.6, for chitinase-LRBC). Preparations are viewed microscopically with ultraviolet light.     

The Use of Alizarin Red S To Detect and Localize Calcium in Gametophyte Cells of Ferns

An aqueous solution of alizarin red S containing chloral hydrate both clears intact chlorophyllous gemma cells of Vittaria graminifolia and stains for protoplasmic calcium. Verification that the stain was protoplasmic rather than in the cell wall was shown by a positive reaction in extruded protoplasm. Similar staining was found in extruded protoplasm of Onoclea sensibilis spores. Differentiating gemma cells show localized protoplasmic accumulations of Ca²⁺ at sites where asymmetric cell divisions initiate the formation of rhizoids, antheridia or vegetative cells. The staining properties of the dye depend on careful control of pH and the addition of appropriate amounts of KC1 to the mixture. Treatment of Onoclea spores and Vittaria gemmae with 100 mM EGTA for 30 min nearly abolishes staining of their extruded protoplasts and also of intact cells of gemmae. The use of alizarin red S with and without chloral hydrate demonstrates different pools of protoplasmic Ca²⁺. When Onoclea spores are ruptured to extrude the protoplasm, both dye mixtures stain a peripheral, granular protoplasmic component. However, the chloral hydrate-containing dye also reveals Ca²⁺ associated with small particulate protoplasmic components. Extruded protoplasm of gemma cells stains intensely with alizarin-chloral hydrate, but does not stain with alizarin lacking chloral hydrate.     

Application of the Periodic Acid-Schiff Procedure to Tissue Blocks

The periodic acid-Schiff procedure can be used for staining en bloc by incorporating the periodic acid with the fixing fluid. After simultaneous fixation and oxidation for 48 hr at room temperature and subsequent staining in Schiff reagent the tissues are dehydrated, embedded in paraffin and sectioned. Of two fixatives used, 95% alcohol proved superior to 10% formalin. Various concentrations of periodic acid (0.1-2.0%) yielded equally good results, thus the use of the lower concentrations is feasible and preferable. Fixation and oxidation simultaneously or separately yielded equally satisfactory results and in view of the time saved in the simultaneous method the authors recommend it. Using similar time of fixation and oxidation, satisfactory results were obtained with the intestine of rat after 3 hr of exposure to Schiff reagent. A longer period of exposure (up to 48 hr) was needed for comparable results with the kidney and liver.     

Staining of Xylem Parenchyma Mitochondria with Photo-Oxidized 3,3'-Diaminobenzidine

A photo-oxidized solution of 3,3'-diaminobenzidine (DAB) is used to stain xylem parenchyma mitochondria in specimens prepared from lupin hypocotyls fixed with glutaraldebyde and osmium tetroxide and embedded in Epon. No other subcellular components, including plastids, nuclei, vacuoles or cell walls were stained when xylem parenchyma cells were exposed to this reagent for 1 hr. This reaction was stable for 20 min at 80 C, inhibited by KCN, and insensible to 3-amino-1,2,4-triazole. The outstanding sensitivity of this reaction to inhibition probes suggests that this stain is analogous to the previously described DAB/cytochrome c/cytochrome oxidase reaction in plant mitochondria, although the incubation of lupin sections with freshly prepared DAB solution (free of auto-oxidized DAB) did not result in staining. These results draw attention to the unreliability of DAB oxidation for demonstrating electron transport in plant mitochondria. However, we do recommend photo-oxidized DAB as a direct ultrastructural stain for plant mitochondria without reference to its oxidative capacity.     

Nuclear Staining of Colletotrichum Gloeosporioides F. SP. Malvae Conidia with Fluorescent and Nonfluorescent Stains

Six different staining techniques were evaluated for their suitability to stain nuclei of Colletotrichum gloeosporioides f. sp. malvae (C.g.m.) spores. Of the three fluorescent stains, DAPI (4',6-diamidino-2-phenylindole) and bisbenzimide (Hoechst 33258) stained spore nuclei well; mithramycin did not. To achieve consistent results with the bisbenzimide staining protocol, the spores had to be fixed prior to staining and the stain had to be supplemented with Triton X-100. Both safranin O and Giemsa were suitable nonfluorescent staining techniques; lomofungin was not. Safranin O staining was simple and rapid. However, reproducibility was better if the spore suspension and KOH droplets were rapidly mixed prior to adding the stain. There was no significant difference in the percentages of uninucleate and binucleate spores observed in spore preparations stained with DAPI, bisbenzimide, safranin O or Giemsa. Bisbenzimide and safranin O were found to be simple, rapid and reliable fluorescent and nonfluorescent techniques, respectively, for staining nuclei of C.g.m. spores.     

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

Fluorescein isothiocyanate-labeled β-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed.