Effects of Fixation on the Morphology of Paneth Cell Granules

Mouse Paneth cell granules fixed with formalin have been reported to be composed of a core consisting of a polysaccharide-protein complex which is surrounded by a shell or “halo” of acid mucopolysaccharide. Electron micrographs of granules fixed with 1% OsO₄ in veronal-acetate buffer show that the halo may be merely a result of shrinkage of the granule. Similar results obtained after fixation with either 1% OsO₄ in s-collidine buffer containing 1% sucrose (OsO₄) or with 3% glutaraldehyde in cacodylate buffer containing 1% sucrose (GAS) strengthen the belief that the halo is a shrinkage space. Fixation in GAS for 1-4 hr followed by fixation for 1 hr in OsO₄, however, reduced the halo to negligible proportions. This sequence of GAS and OsO₄ improved the preservation of other cytoplasmic structures also. Staining with Alcian blue failed to demonstrate acid mucopolysaccharide in the halo; therefore, the separation of granule components into a core and a halo is probably a fixation artifact.     

Straight Oso4 versus Glutaraldehyde-Oso4 In Sequence as Fixatives for the Granular Vesicles in Sympathetic Axons of the Rat Pineal Body

Pineal bodies were removed immediately after death from 6 rats: representing both sexes, and adult and 21-day postnatal ages; cut into 2 or 3 pieces, and subjected to experimental fixations at pH 7.3, 0-4 C as follows: 1-2 hr in 1% OsO₄, with veronal-acetate buffer of phosphate buffer; 3-4 hr in 3% or 6% glutaraldehyde in 0.1 M or 0.2 M phosphate buffer, with or without 1% sucrose. Specimens from OsO₄ were dehydrated, and embedded in epoxy resin; those from glutaraldehyde were allowed to soak in buffer for 12-16 hr, then transferred to 1% OsO₄ at 0-4 C for 2 hr, and embedded in the same manner as the ones fixed directly in OsO₄. Representative electron micrographs of postganglionic sympathetic endings were studied for the morphology and frequency of granular vesicles. No consistent difference was shown between vesicles fixed in OsO₄ buffered by phosphate or by veronal-acetate, nor was there any effect caused by the different concentrations used for the glutaraldehyde solution; however, vesicles fixed by the glutaraldehyde-OsO₄ sequence showed an enhancement in the graininess of their membranes, were slightly larger, and had a much larger dense core than those fixed by OsO₄ alone. After glutaraldehyde-OsO₄, granular vesicles showed a frequency of 81%, whereas after direct fixation in OsO₄, only 40% without significant change their number per unit area. Therefore, glutaraldehyde-OsO₄ seems to be more effective than straight OsO₄ for the demonstration of granular vesicles in the autonomic nervous system.     

Differential Staining of Tannin in Sections of Epoxy-Embedded Plant Cells

A staining procedure is described for the light microscopic localization of ergastic tannins in epoxy sections of plant cells embedded for study by transmission electron microscopy. Callus and cell suspensions of Pseudotsuga menziesii and Pinus taeda fixed in glutaraldehyde:acrolein and then OsO₄, followed by epoxy embedding, were sectioned 0.5 μn thick, stained on a glass slide with ethanolic Sudan black B at 60 C as described by Bronner, and then mounted in Karo syrup. Tannin deposits stained brownish-orange and were easily distinguished from lipid bodies of similar size, which stained dark blue to black, and from starch grains, which were unstained. The significance of this differential polychromasia was confirmed by transmission electron microscopy. This staining proadure should prove valuable in the cytoplasmic evaluation of the plant cell ergastics (especially tannins) via light microscopy whether or not electron microscopic examination is intended.     

Keratin in the Egg Shells of Amphistomes; Histochemical Differentiation from Quinonetanned Protein in Other Trematoda

Various combinations of the oxidation method for demonstrating keratin in shell material of amphistomes were tried. Acidified permanganate worked more efficiently than performic and peracetic acids, and Alcian blue and aldehyde fuchsin excelled other basic dyes for subsequent staining. For the permanganate-Alcian blue reaction, sections of material fixed in Susa or Bouin were oxidized in 0.3% permanganate in 0.3% H₂SO₄ for 5 min., decolourized in 1% oxalic acid, stained in 3% Alcian blue in 2 N H₂SO₄ and counterstained with eosin. The shell globules stained a deep blue. For permanganate aldehyde fuchsin staining, the sections were stained in aldehyde fuchsin for 1 hr, after oxidation with permanganate. The shell globules then stained a deep magenta. The catechol and fast red reactions were negative in amphistomes and the specimens lack the characteristic amber colour due to quinone tanning.     

Preservation of fine Structures in Yeast by Fixation in a Dimethyl Sulfoxide-Acrolein-Glutaraldehyde Solution

The primary fixative containing 2% acrolein, 2% glutaraldehyde in 50% aqueous dimethyl sulfoxide (DMSO) buffered at pH 7.4, was applied for 7 hr in the cold. After a short wash in 0.02 M s-collidine buffer, pH 7.4, containing 0.2 M sucrose and 0.001 M CaCl₂, the yeast cells were postfixed in 3% OsO₄ at pH 4.0 (veronal acetate buffer). This method preserves many cytoplasmic features such as lipid deposits and ribosomes which are usually destroyed by permanganate fixation. DMSO apparently acts as a permeating agent allowing maximum penetration of the cell wall by the fixative without disrupting cellular fine structure     

Handling Germinated Pollen on Millipore Membrane During the Feulgen and Autoradiographic Procedures

To prevent loss of pollen during the Feulgen's procedure, the pollen was grown on an autoclaved membrane filter (Millipore AA WP 025 00) in contact with a sterilized medium containing agar 0.5-1%, sucrose according to the genus (Malus 0.3-0.5 M; Persica and Tulipa 0.4 M), and H₃BO₃, 0.01%. To fix the germinated pollen of most species, the membrane was placed for 2 hr to overnight at 2-4 C on filter paper wet with the following mixture: OsO₄, 1 gm; CrO₃, 1.66 gm; and distilled water, 233 ml. To fix Persica pollen, 10% of glacial acetic acid had to be added to the fixative. Washing with distilled water and bleaching with a mixture of 3% H₂O₂ and sat. aq. ammonium oxalate, 1:1, were performed also on filter paper. Similarly, the preparation was processed for Feulgen staining by use of pieces of filter paper wet with the required fluids. Hydrolysis preceding the Schiff's reagent was performed at room temperature with 5 N HCl for 18 min. The differentiation after the Schiff's action was with 2% K₂S₂O₅ buffered to pH 2.3 with 9 ml of phosphate buffer (KH₂PO₄, 1.4 gm; conc. HCl, 0.35 ml and distilled water to make 100 ml). The stained pollen was floated off the membrane with a drop of glacial acetic acid to a gelatinized or an albumenized slide, and squashed. When the coverslip is removed the preparation may be either dehydrated and mounted or coated with autoradiographic film.     

The Quantitative Determination of Osmium Tetroxide in Fixatives

This rapid spectrophotometric method for determining the OsO₄ concentration in fixative and stock solutions is based on the reduction of OsO₄ by acidified KI to the blue species of OsI₆ =, which is then determined at 649 mµ. The salt K₂OsI₆ has been isolated from the reaction mixture and characterized. Method: A I ml aliquot of the solution, containing up to 3% OsO₄, is diluted to 100 ml with distilled water. To 1 ml of the diluted solution is added, in order: distilled water, 2 ml; 1 M HCI, 1 ml; and 1 M KI, 1 ml. Optical density at 649 mµ is read from 10-120 min thereafter. OsO₄ concentration is calculated from the measured molecular extinction coefficient of OsI₆ =, 4400 liter/mole cm.     

Selective Staining of Neurosecretory Material in Semithin Epoxy Sections by Gomori's Aldehyde Fuchsin

The epoxy resin was removed from semithin (1 μm) sections by immersing them for 30 sec in sodium methoxide (Mayor et al., J. Biophys. Biochem. Cytol., 9: 909-10, 1961) and then processed as follows: (1) left for 1-3 hr at 60 C in a mixture of formalin, 25 ml; glacial acetic acid, 5 ml; CrO₃, 3 gm; and distilled water, 75 ml: (2) oxidized 10 min in a 1:1:6 v/v mixture of 2.5% KMnO₄, 5% H₂SO₄ and distilled water: (3) bleached in 1% oxalic acid, and (4) stained for 15 min in aldehyde fuchsin, 0.125% in 70% alcohol, or in a 1% aqueous solution of toluidine blue. The neurosecretory material is selectively stained.     

A morphological and immunolabeling study of freeze-substituted Bacteroides forsythus

We used a rapid freezing and freeze-substitution technique without glutaraldehyde and OsO₄ fixation for the electron microscopic immunocytochemical demonstration of the surface structure of Bacteroides forsythus, an anaerobic Gram-negative periodontopathogen. Cells were applied to pieces of filter paper and freeze-substituted by plunge-freezing in liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR White resin. The membrane ultrastructure of B. forsythus was preserved well, and the labeling density of the freeze-submitted cells was compared to a conventional processing method. Our results show the usefulness of the freeze-substitution method for immunohistochemical studies of B. forsythus.     

Fixation of Microscopic Fresh-Water Green Algae by 31% OsO4 in CCI4 in an Unbuffered, Two-Phase Fixative System

The fixative was prepared by dissolving 0.1 gm of OsO₄ in 0.2 ml of CCI₄. It was applied to microscopic green algae, concentrated by centrifugation, in a volume equal to that of the residual culture medium and allowed to act for 45 min at either 20 or 4 C. Such fixation has proved of value in producing an electron micrographic picture of particular use in the study of cytoplasmic microtubules. The fixative acts by diffusion of OsO₄, from the nonaqueous to the aqueous phase and thus provides a condition similar to that of vapour fixation but with a much higher concentration of OsO₄     

The Use of Dimethylsulfoxide for Fixation of Yeasts for Electron Microscopy

Conventional methods of chemical fixation are often inadequate for preserving yeast ultrastructure. The thick cell wall severely limits penetration of fixatives rendering poor detail of the cell wall, membranes, and overall anatomy. Dimethylsulfoxide (DMSO) enhances penetration of chemicals and has been added to fixatives to improve cell preservation. At high concentrations (5 to 50%), however, it affects ultrastructure unpredictably. We found that adding 0.1% DMSO to fixatives greatly improved retention of yeast ultrastructure. Candida albicans, C. glabrata and Aspergillusfumigatus were fixed for 3 hr in 3% paraformaldehyde, 1% glutaraldehyde, 1 mil MgCl₂, 1 mM CaCl₂, 0.1% DMSO in 0.1 M sodium cacodylate buffer followed by 1% OsO₄, 1% K₂Cr₂O₇, 0.85% NaCl. 0.1% DMSO in the same buffer. Thin epoxy sections were post-stained in uranyl acetate and lead citrate. The multilayered character of the cell wall was distinct and well structured. Addition of ruthenium red or alcian blue to the fixatives further enhanced the outer fibrillar layer. The plasma membrane was contiguous and tightly adjacent to the inner manno-protein layer of the cell wall. The cytoplasm was well preserved and the overall preservation of the yeast ultrastructure was significantly improved.     

A Simple Polychrome Stain for Conventionally Fixed Epon-Embedded Tissues

The described technique, based upon a one-step Mallory-Heidenhain stain, can be applied as a routine stain for glutaraldehyde or OsO₄ fixed, Epon embedded tissues of various organs. The technique consists of a short treatment of the sections with H₂O₂, a nuclear staining with celestine blue B and a final staining in a modified Cason's solution. The different tissue and cell components are displayed as follows: dark brown nuclei, yellow cytoplasm, red collagen fibers and blue elastic' fibers. Intra cytoplasmic components as glycogen and mucus are stained respectively blue and violet, whereas other inclusions such as leucocyte granules are colored orange to red.     

Phosphotungstic Acid-Chromic Acid as a Selective Electron-Dense Stain for Plasma Membranes of Plant Cells

A mixture consisting of 1% phosphotungstic acid (PTA) in 10% chromic acid (CrO₃) selectively stains the plasma membrane of plant cells. Whole tissue or pelleted cell fractions are prepared for electron microscopy using conventional methods including glutaraldehyde fixation and OsO₄ postfixation, dehydration in acetone and embedding in Epon. To stain the plasma membrane, thin sections are transferred with a plastic loop to the surface of a 1% aqueous solution of periodic acid for 30 min for destaining. Following transfer through 5 distilled water rinses, the sections are exposed to the PTA-CrO₃ mixture for 5 min, rinsed and mounted on grids for viewing with the electron microscope. The selectivity of the stain is retained in homogenates and serves to identify the plant plasma membrane in cell fractions.     

Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

Effects of Inorganic Oxides, Sulfides, Chlorides, and Acid Chlorides on Metachromasia and Other Staining Properties

The ability of 17 inorganic compounds (POCl₃, PSC1₃, PC1₃, P₂O₅, P₂S₅, P₄S₃, P₄S₇, PC1₅, Sb₂O₅, As₂O₅, BiOC1₂, SeOC1₂, SO₂C1₂, Sb₂S₅, VOC1₂, SiC1₄ and CrO₂Cl₂) dissolved in pyridine or 2,2,4-trimethyl pentane, to enhance subsequent staining of tissue components with toluidine blue, phosphotungstic acid-hematoxylin (PTAH), leukofuchsin, and dihydroxydinaphthyl-disulfide (DDD) was studied. Eight of these compounds were also tested for ability to enhance staining with Alcian blue 8GN and Luxol fast blue MBS. Nine of the 17 compounds produced increased staining of certain tissue components with leukofuchsin, 13 with toluidine blue, 16 with PTAH, and 16 with DDD. The results suggest additional approaches to identification of tissue entities by induced metachromatic basophilia and leukofuchsin positivity as well as by the other stains studied, and also suggest a number of hitherto unstudied modes of reaction between the dyes used and reactive groups of tissue components. Many reactions of the compounds tested, with reactive groups known to be present in tissue components, are basecatalyzed, so that choice of solvent can influence the results obtained.     

Staining Cells on a Membrane Filter with Giemsa Stock and Tap Water

Cells grown on type AM 6 Alpha Metricel Gelman filter membranes are fixed 1-2 min with OsO₄ vapor, washed in distilled water, dehydrated 4 min in 95% and 4 min in absolute ethanol. They are then stained for 2 min in a 1:1 dilution of Giemsa stock solution and an approximately neutral, low mineral content tap water. The stain is removed from the filter by 1 min in 50%, 1 min in 95%, 1 min in absolute ethanol and 1 min in a mixture of absolute ethanol and xylene 1:1. The filter is finally cleared in xylene and mounted with a synthetic resin. This procedure gave polychrome staining on neonatal thymic cells from C57 black pedigreed mice and on human leucocytes, with little or no stain retained by the filter membrane.     

Staining Methods For Studying Ingredient Distribution In Baked Cakes

A method is described for preparing cake crumb for sectioning and staining. Previous to embedding, the fat was stained and fixed by exposing small blocks of cake to the fumes from a 5%, freshly-prepared, aqueous solution of osmic acid (OsO₄). This was followed by dehydration in ethyl alcohol and tertiary butyl alcohol, removal of air under vacuum and infiltration with paraffin.

Sections were cut 20 and 9Op thick and mounted with water.

Wax was removed by immersion in xylene. The sections were rehydrated in a series of ethyl alcohol dilutions, from concentrated to dilute, then transferred to distilled water.

Protein was then stained pink by immersion of the slides in an acidified 0.04% water solution of eosin Y, or starch was stained blue with a dilute aqueous solution of iodine. Ten grams iodine and 10 g. KI were dissolved in 25 ml. distilled water. This stock solution was diluted for use one to two hundred times.

The relationship between protein and starch was demonstrated by staining the sections with eosin, differentiating in 50% alcohol and staining with iodine.

When slides of cake crumb were prepared in this way, the fat was stained black, the protein bright pink and the starch granules a dark blue.     

Staining Myelin Sheaths of Optic Nerve Fibers with Osmium Tetroxide Vapor

OsO₄ solution in water, long regarded as the best fixing and staining agent for myelin sheaths, has poor penetrating power. This peculiarity has limited its use to very small pieces of tissue. The vapor from an aqueous solution is known to have a much greater penetrating power for non-neural tissues than the solution itself but nothing has been recorded about its advantages for fixing and staining myelin sheaths of nerve fibers. Difficulties in securing adequate staining of the myelin sheaths in vertebrate optic nerves were overcome largely by the use of the vapor of OsO₄. The technic is carried out as follows: 1) suspend a portion of the nerve above a 2% solution of OsO₄ for 12-24 hours in an air-tight container at room temperature; 2) wash 4-6 hours in distilled water, dehydrate in ethyl alcohol (50% for 2 hours, 70% for 2 hours, and finally 95% overnight), and transfer to n butyl alcohol (2 changes of 2 hours each); 3) embed in paraffin, section, mount and cover in balsam in the customary manner.     

Staining Tissue of the Central Nervous System with Luxol Fast Blue and Neutral Red

The tissue is fixed in 10% neutral saline formalin for 1 day to 3 wk depending on the size of the block, dehydrated and embedded in paraffin. The sections are stained at 57° C for 2 hr, then at 22° C for 30 min, in a 0.0125% solution of Luxol fast blue in 95% alcohol acidified by 0.1% acetic acid. They are differentiated in a solution consisting of: Li₂CO₃, 5.0 gm; LiOH-H₂O, 0.01 gm; and distilled water, 1 liter at 0-1° C, followed by 70% alcohol, and then treated with 0.2% NaHSO₃. They are soaked 1 min in an acetic acid-sodium acetate buffer 0.1 N, pH 5.6, then stained with 0.03% buffered aqueous neutral red. Sections are washed in distilled water, 1 sec, then treated with the following solution: CuSO₄·5H₂O, 0.5 gm; CrK(SO₄)₂·12H₂O, 0.5 gm; 10% acetic acid, 3 ml; and distilled water, 250 ml. Dehydration, clearing and covering complete the process. Myelin sheaths are stained bright blue; meninges and the adventitia of blood vessels are blue; red blood cells are green. Nissl material is stained brilliant red; axon hillocks, axis cylinders, ependyma, nuclei and some cytoplasm of neuroglia, media and endothelium of blood vessels are pink.