Confocal scanning laser microscopy, a new technique used in an embryological study of Dactylorhiza maculata (Orchidaceae)

Ovules of Dactylorhiza maculata , fixed in FPA₅₀ and made transparent in Herr's clearing fluid, were investigated with confocal scanning laser microscopy. This new technique makes it possible to obtain thin optical serial sections in perfect alignment without damaging the material. It made possible an interpretation of the development of the embryo sac differing from that based on previous investigations with traditional technique. Using the new technique we found that the young embryo sac generally contains seven nuclei, two of which fuse, and the mature sac is 6-nucleate. The pollen tube does not penetrate any of the synergids when entering the embryo sac. Double fertilization takes place, and all nuclei are still alive at that moment. Four to six endosperm nuclei are formed, but later they degenerate and the growing embryo fills the entire embryo sac.     

Epoxy Embedding, Sectioning and Staining of Plant Material for Light Microscopy

By using a formula which gives a relatively soft epoxy embedding medium, it is possible to cut sections of plant material with a sliding microtome equipped with a regular steel knife. Blocks having a cutting face of 10 × 10 mm, giving sections of 4-10 μm, can be used. Tissues are fixed in Karnovsky's fluid, postfixed in 1 or 2% OsO₄, embedded in Spurr's soft epoxy resin, Araldite, or Epon mixtures. 5% KMnO₄, followed by 5% oxalic acid, then neutralized in 1% LiCO₃, are used to mordant the sections. Some of the stains used are Mallory's phosphotungstic acid-hemotoxylin, acid fuchsin and toluidine blue, or toluidine blue. Mounting is done with whichever soft epoxy resin was used in casting the blocks.     

The application of the avidin-biotin technique to previously stained histological sections

Using the sensitive avidin-biotin peroxidase technique, a wide variety of tissue antigens can be detected in standard histological sections of both normal and pathological tissues previously stained with hematoxylin and eosin. There appears to be no detectable reduction of sensitivity with this method of "restaining" compared to the standard immunoperoxidase procedure applied to unstained tissue sections. This technique makes it possible for retrospective identification of tissue antigens when insufficient unstained material is available.     

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.     

The Application of the Avidin-Biotin Technique to Previously Stained Histological Sections

Using the sensitive avidin-biotin peroxidase technique, a wide variety of tissue antigens can be detected in standard histological sections of both normal and pathological tissues previously stained with hematoxylin and eosin. There appears to be no detectable reduction of sensitivity with this method of “restaining” compared to the standard immuno-peroxidase procedure applied to unstained tissue sections. This technique makes it possible for retrospective identification of tissue antigens when insufficient unstained material is available.     

The Application of Acetolysis for Releasing Leaf Cuticular Membranes of Pandanus in Taxonomic Studies

Leaf cuticular membranes can be released cleanly and in one piece after a 10 min treatment of leaf in a 100 C acetolysis mixture: acetic anhydride, 9; and concentrated H₂SO₄,1 v/v. This method has been used till now only for studying pollen and spore morphology but was found quite appropriate for a taxonomic study of the leaf surface of the genus Pandanus.     

Memorization of nuclear atmospheric tests by rhizomes and scales of the mediteranean seagrass Posidonia oceanica (Linnaeus) Delile

Determination of the age of rhizome sections of Posidonia oceanica (Linnaeus) Delile (Potamogetonaceae) by the examination of their dead leaf scales (lepidochronology), makes it possible to cut out the rhizomes in precisely dated 1- or 5-year sections. With increasing age of the section, the annual section length and the C:N ratio increase, while the dry weight per unit length and the ratio of dry weight:ash weight decrease. At a site where the only significant ¹³⁷Cs contamination can be ascribed to global nuclear fallout bombs, the activity of ¹³⁷Cs was measured i n 5-year age groups of rhizomes and scales separately. The maximum activity of ¹³⁷Cs in scales occurred in groups produced between 1960 and 1964, a period during which a peak of activity occurred in fallout. The distribution of ¹³⁷Cs activity in rhizome age groups indicates an apparent lag, perhaps owing to transport of material in the rhizome. In situ dead rhizome scales of P. oceanica thus constitute a memory, at least 30-years long, of an environmental chemical pulse. Posidonia oceanica could prove a valuable tool for marine-pollution surveys.     

Acetolysis of Pichia pastoris IFO 0948 strain mannan containing alpha-1,2 and beta-1,2 linkages using acetolysis medium of low sulfuric acid concentration

To obtain manno-oligosaccharides containing beta-1,2-linked nonreducing terminal groups from the mannan of Pichia pastoris IFO 0948 strain by acetolysis, an attempt was made to establish the reaction conditions under which cleavage of the alpha-1,6 linkage took place preferentially leaving manno-oligosaccharides composed largely of beta-1,2 linkages. By the action of an ordinary acetolysis medium, a 10/10/1 (v/v) mixture of acetic anhydride, acetic acid, and sulfuric acid at 40 degrees C for 13 h or at 25 degrees C for 120 h, the O-acetyl derivative of this mannan gave mannose, mannobiose, mannotriose, and mannopentaose. However, treatment of the same O-acetyl mannan with a 50/50/1 (v/v) acetolysis medium at 40 degrees C for 15 h gave a mannotetraose in addition to mannose, mannobiose, mannotriose, and mannopentaose. Use of a 100/100/1 (v/v) acetolysis medium at 40 degrees C for 36 h gave a more satisfactory result, a mixture of oligosaccharides, from mannose to mannopentaose, which contained more mannotetraose than mannopentaose. Because both mannotetraose and mannopentaose contained alpha-1,2 and beta-1,2 linkages, it was concluded that an acetolysis medium containing a low concentration of sulfuric acid, up to 0.5% (v/v), facilitates the preferential cleavage of the alpha-1,6 linkage, leaving manno-oligosaccharides containing the beta-1,2 linkage which was found to be labile to the action of the 10/10/1 (v/v) acetolysis medium.     

A new look at the acetolysis method

The acetolysis method intreduced byGunnar Erdtman is still a very welcome and highly successful technique in palynology. However, acetolysis destroys all pollen material with the exception of sporopollenin that forms the outer pollen wall, the exine. Modern palynology in its application to plant systematics and phylogeny must consider all sporoderm characters, not only those of the exine. The neglect of the intine may distort some principal palynological aspects. This is illustrated by cases of total breakdown or gross modification of thin exine structures (e.g. inBeilschmiedia, Strelitzia) and by the clarification of apertures (e.g.,Polyalthia, Fissistigma, Calluna). In our view the investigation of both acetolysed and non-acetolysed pollen is obligatory for a well balanced view of pollen structure and function.     

Measurement of Osteoblastic Activity in Diaphyseal Bone

Fresh undecalcified sections are made and stained with basic fuchsin by Frost's methods. Only complete cross sections cut accurately perpendicular to the diaphyseal longitudinal axis may be measured. The percentage of the longitudinal vessels containing osteoid seams is measured by a counting technique. The average number of longitudinal channels/mm² of the cross section is next measured by a similar technique. Multiplying the seam percentage by the channels/mm² gives the seams/mm². For reasons which are discussed this is equivalent to seams/mm³. A suitable sampling method is used to obtain useful precision.     

Demonstration of the Microvasculature of the Spinal Cord by Intravenous Injection of the Fluorescent Dye, Thioflavine S

In the past, thioflavine S has been used for visualizing blood vessels and patterns of blood flow (Schlegel 1949; Schlegel and Moses 1950; Oliver et al. 1951). Methods employed have involved an intravenous injection of the dye, immersion of hand-cut sections in glycerol and examination of sections under incident Wood's light. With improved techniques it is possible to obtain microtome-cut sections and to use a more intense light source for enhancing fluorescence and resulting visualization of small vessels. Occlusion of arterioles by undissolved dye particles is prevented by ultracentrifugation of the solution to be injected.     

A Simplified Nissl Stain with Thionin

Recently two articles on the use of thionin as a cell stain for neurological materials have appeared. One utilizes a solution buffered in the acid range³; the other uses a “steaming” staining solution⁴. For some time we have been using thionin as a routine stain after either formalin or alcohol fixation and our method is so simple and has given such satisfactory results with a variety of brands of thionin that it seemed to be worthy of more general use. Briefly the method consists of placing the celloidin sections in a 0.05% solution of Li₂CO₃ (the percentage of Li₂CO₃ is non-critical) for about 5 minutes and then grossly overstaining in a 0.25% solution of thionin in a 0.05% solution of Li₂CO₃ in distilled water. The overstaining is necessary if all the stain is to be removed from the background. The sections are then passed through distilled water, 70 or 80% alcohol, two changes of butyl alcohol, two changes of xylene and mounted with Clarite. For most material, split mica cover-slips are quite satisfactory. The time of differentiation may be considerably lessened by the use of the differentiator recommended by Neumann (1942) except that we find the chloroform superfluous and transfer the sections to the aniline solution from 95% alcohol. Less fading seems to occur if the aniline differentiator is followed by a saturated solution of Li₂CO₃ in 95% alcohol.     

Holding Plastic Embedded Specimens During Dissection: Two Improvements

Sectioning of tissue specimens of aligned cells (e.g., muscle, cochlea, retina) for micrographs, often requires the capsule containing the tissue to be positioned at a precise angle during sectioning. The correct angle can be set by trial and error, but the process can be shortened if the gross anatomy of the cell system is used as a guide to orient the embedded sections as closely as possible in the optimal plane. Thick sections are then cut in this plane with a razor blade, and these sections are re-embedded in preparation for thin sectioning. This technique eliminates the large angles of the capsule in the microtome which occur when the gross tissue is poorly aligned in the first embedding.     

Electron Microscopy of Mummified Material

It has proved possible to cut ultrathin sections of mummified material obtained from an American Indian burial (approximate age unknown). Small pieces of tissue were placed for 48 hr in a softening fluid consisting of 96% ethyl alcohol, 30 vol.; 1% aqueous formalin, 50 vol.; 5% aqueous Na₂CO₃, 20 vol. During this period the fluid was changed twice. The tissue was then cut with a razor blade into cubes of 1 mm per side or less, dehydrated in graded ethanols, infiltrated and embedded in methacrylate and the plastic polymerised by placing in the oven at 58°C overnight. The blocks were trimmed to a truncated cone leaving a surface area of 0.5 mm² or less, and cut on a Porter Blum ultramicrotome using a glass or a diamond knife.     

Histoautoradiographic Localization of Calcium in Oat Plant Tissues

The leaching of water-soluble and exchangeable calcium in histoautoradiog-raphy of oat tissue can be prevented by using acetone as the dehydration fluid (freeze substitution technique) and by keeping the tissue sections, while stretching on water, embedded in the methacrylate matrix. Ca⁴⁵ was either added to the mineral solution on which the oat plants were grown (75 μc), or applied on the leaf surface (8 μc). After freezing in melting isopentane, specimens of 1-2 mm dimensions are fixed for 24 hr in an acetone-OsO₄ (1%) solution at—80 C. Dehydration is obtained by transferring the material every day for 6 successive days to a fresh acetone solution at—80 C. The material is infiltrated by a three-time renewed monomer methacrylate mixture (methylmethacrylate I, butylmethacrylate 4) at—50 C. The specimens are embedded in the polymerizing methacrylate mixture at room temperature. Sections of 4-8 μ are easily cut with a rotating microtome. If the methacrylate is not removed from the sections, they can be stretched on water without leaching of calcium. The presence of methacrylate in no way hinders microscopic observation nor effective histoautoradiography.     

Recent Advances in Microtechnic. I. Methods of Studying the Development of the Male Gametophyte in Angiosperms

Among methods used for a study of nuclear details in the development of pollen grains, the following were found to be very satisfactory: (1) warming the entire grains in aceto-carmine and then clearing with chloral hydrate; (2) making smear preparations stained with crystal-violet-iodine or iron alum hematoxylin. For paraffin sections, a counterstain with dilute alcoholic erythrosin is often very useful after the usual iron hematoxylin technic.

A method of making cultures of pollen tubes on slides coated with thin films of sugar agar is described in detail. The tubes can be fixed by immersing the slide in formol-acetic-alcohol and then stained by any desired schedule. Iron alum hematoxylin was found to be the most satisfactory, but the Feulgen reaction is very valuable in such cases where the nuclei are obscured by the density of the pollen tube cytoplasm. Living pollen tubes can be kept under observation by dissolving a small quantity of neutral red or other vital stain in the sugar agar before it is spread on the slide.

For studying stages in fertilization or gametogenesis, styles should be fixed and sectioned only after a preliminary study with iodine-chloral-hydrate or safranin-anilin-blue or aceto-carmine. Once the extent to which pollen tubes grow in a given time in the stylar tissues has been determined, it is possible to fix material with some knowledge of what it is going to show.

Some other methods, that have not been tried by the authors but appear to be valuable, are also briefly described.     

Rate of Incorporation of R3hlthymidine In Various Tissues of the Mouse A basis for the evaluation of genotoxic effects of chemicals

Abstract. [³H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible.
The present paper deals with some practical aspects on the use of [³H]thymidine in vivo. [6-³H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol Were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and dNA-associated radioactivities. A rapid increase of the [³H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities asśociated with DNA closely eorrelated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [³H]thymidine incorporation into DNA.     

Preparation of Fossil Bone Specimens for Scanning Electron Microscopy

A method is described for preparing fossil bone specimens for scanning electron microscopy. To obtain bone surfaces suitable for study, material was embedded in Epon 812 and selected faces exposed by grinding were subjected to controlled etching with a 4:1 mixture of 5% HNO₃ and 1% OsO₄, Surfaces thus prepared were further processed by the so-called clearing replicas technique. As a result of this procedure the bone surfaces revealed a network of anastomosing vascular canals the inner surface of whose walls could be examined in the scanning electron microscope. By etching extremely thin ground sections of bone stuck to plastic tape the contents of vascular canals as well as osteocytes can be isolated. This method ensures the good preservation of spatial relations between bone elements essential for studies of fossil bones, which an sometimes very brittle.     

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.     

A Simple and Rapid Staining Technique for Plastic Embedded Cartilage and Bone

In this report we describe a simple and rapid staining technique for cartilage and bone embedded in Araldite. Semitbin sections of embryonic vertebrae obtained from 15 to 17 day mouse fetuses were stained using an aqueous solution 0.25% with respect to methylene blue, 0.25% with respect to azure A, and 0.5% with respect to Na₂ CO₃, then counterstained with 1% aqueous pararosaniline chloride (MAP). Results were compared with toluidine blue stained sections. MAP permitted good discrimination of developmental stages of both cells and extracellular matrix within vertebral ossification centers during endochondral ossification. The technique is simple, rapid and applicable to plastic embedded sections, and can be used prior to ultrastructural examination.