Handling Small Numbers of Marine Eggs Through Dehydration and Embedding

A method is described whereby small numbers of eggs can be embedded compactly. Handling through reagents is achieved in 6 mm. glass tubing having a linen mesh affixed to one end, up to and including paraffin. The egg mass is then obtained and reembed-ded in a pre-cut paraffin block in which a well has been drilled. Perfect seal of the two masses is achieved with a hot needle. Some advantages of the method are described.     

Punched-Out Agar Petri Plates for Systematic Fixation and Embedding of Multiple Small Tissue Samples

A 5% agar solution is poured into Petri dishes, jelled, and a pattern of cylindrical holes punched out according to the number and size of tissue samples to be processed. These punched-out Petri plates withstand neutral formalin, water, benzene, and paraffin immersion at 60 C; thus providing an adaptable container for processing small tissue samples. Reduced handling of the tissues, minimized risk of loss, and easy identification of samples by means of the map-like arrangement of the holes are among the virtues of such plates. An optimal fixative-volume: tissue-weight ratio can be attained by using wells of varying diameter and depth according to the sample size.     

Effect on Tissue Volume of Various Methods of Fixation, Dehydration, and Embedding

A new indirect method is described for following volume changes of homogeneous pieces of tissue during fixation, dehydration and embedding, and area changes during sectioning, staining and mounting. Pieces of rabbit kidney cortex were compared after fixation in Destin's, Orth's, Petrunkevitch's cupric-paranitrophenol, Bouin's, SUSA, Zenker-formol, 10% formalin in distilled water, formalin in saline, Burke's pyridine formalin, CaCOy neutralized formalin, MgCO₃-neutralized formalin, Bensley's vacuum distilled neutral formalin in distilled water, and Bensley's neutral formalin in saline; during dehydration in ethyl alcohol, dioxan, and tertiary butyl alcohol and clearing in xylol and chloroform; and after infiltration and embedding with parowax, with paraffin-nitrocellulose double embedding technic, with hot nitrocellulose, and with cold nitrocellulose. The H-ion concentration of these fixatives was followed during tissue fixation.

Altho all fixatives showed specific merences, SUSA and Bouin's gave the best general results and neutral formalin mixtures, especially pyridine-formalin, the poorest. Isotonic saline was found superior to distilled water as a formalin diluent, reducing tissue swelling during formalin fixation. Reagents producing marked decreases in tissue volume render such tissues less susceptible to shrinkage during subsequent procedures. Shrinkage of tissue during dehydration and infiltration with hot parffin may exceed that produced by fixation alone. Excessive heat causes tissue distortion and shrinkage. Inijltration with hot paran causes considerable shrinkage, with hot nitrocellulose Iess, and with cold nitrocellulose the least sbrinkage.     

Effects of Fixation and Dehydration Procedures on Marine Nematodes

Different combinations of fixation and dehydration procedures for the preparation of permanent mounts of marine nematodes of the subfamily Oncholaiminae were tested and compared. Qualitatively, the best specimens resulted from Seinhorst''s killing method and fixation in FAA; the dehydration procedure was of less significance. Quantitatively, no significant modification of measurements resulted from any of the methods used. Sources of error in measurements are discussed.     

Albumen Embedding and Individual Mounting of One or Many Mammalian Ova on Slides for Fluid Processing

For studies of ova with the light or electron microscope, as well as for autoradiographic and histochemical studies, these cells need to be sectioned. The handling of individual, often hard-to-obtain, cells through fluid processing by micropipettes is time-consuming and can easily cause damage or loss of valuable specimens. A number of interesting methods have been described for handling ova or free-floating cells. In these methods cells are commonly handled in containers with fine-mesh, wire cloth bottoms, when a number of cells are involved. Unfortunately they all require special equipment not readily or easily available (Buchanan 1965; Rinaldi et al. 1966; Izquierdo 1967; Shands 1968). In our method, egg white provides a supporting matrix for mouse ova and allows one or several specimens to be mounted on a slide.     

An Improved Method for the Fixation, Embedding and Immunofluorescence Labeling of Resin-Embedded Plant Tissue

The use of antibodies as direct probes for specific macromolecules in plant cells and tissue is a well-established and extremely powerful technique and is of particular use in the post-genomics era. In this paper, we present an improved fixation, embedding, and immunofluorescence technique suitable for fixing “difficult” plant tissues such as pistils and inflorescence stems, which possess many trichomes and a thick hydrophobic cuticle. The key modification of the fixative used in the current study was the addition of a small amount of sucrose, CaCl₂, and detergent into a 4% (v/v) formaldehyde and 1% (v/v) glutaraldehyde mixture without the requirement to vacuum infiltrate. The modified immunofluorescence labeling method featured an amended blocking buffer, increased number of washing steps, and the use of an aqueous mounting medium which produced intense immunolabeling signals with extremely low background. Moreover, the immunocytochemistry methodology described in this study has proven to be suitable for use on two widely studied plant species, namely, Vicia faba and Arabidopsis thaliana, and may, therefore, be applicable for use in studies of a wide range of angiosperms.     

An Improved Method for Rapid Fixation and Embedding of Pteridophyte Plant Materials

A rapid method for fixation and embedding of plant materials, especially pterido-phytes, is suggested. Addition of tannic acid following osmication improved the visualization of membranes. Staining en bloc with uranyl acetate between osmication and tannic acid is suggested for tissues infected with fungi and bacteria.     

Nongraded Dehydration and Low-Pressure Infiltration for Rapid Celloidin Embedding of Brain Tissue

Various ways of shortening single steps in the celloidin process have been combined to form a routine method which may be completed, for tissues of average size, within a week following fixation. Fixed, washed tissue slices 5 mm thick are dehydrated in 1 or 2 changes of absolute ethanol and acetone, 1:1. This requires 24 hr in an incubator at 37 C, or 12-16 hr if a magnetic stirrer is used. After ether-alcohol for 4 hr. the tissues are transferred to 5% celloidin and infiltrated in a vacuum desiccator attached to a filter pump. When the volume of celloidin is reduced to half the original amount (about 2 hr), the tissues are removed from the infiltrating fluid and embedded in 10% celloidin. The blocks are hardened in chloroform and cleared by suspending them in 2 or 3 changes of terpineol agitated by a magnetic stirrer. Sections are cut in terpineol, using any type of microtome. After washing in 95% alcohol, they are mounted on albumenized slides for staining.     

A Mordanting Fixation for Intense Staining of Small Chromosomes

A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)₃ per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)₃ in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.     

A Mordanting Fixation for Intense Staining of Small Chromosomes

A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)₃ per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)₃ in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.     

Simultaneous Formalin Fixation and Edta Decalcification, With Carbowax Embedding for Preservation of Acid Phosphatase

Carbowax serial sections from pubic symphyses of female mice, fixed and decalcified in a 10% formalin-5% Versenate solution for 18 hr at 4 C, pH 5.2, were incubated for 30 min with Burstone's simultaneous coupling reagent (pH 5.2); substrate: naphthol AS-TR and the diazonium salt, fast red violet L.B. All sections were counterstained with 1% methyl green at pH 4.0 in a phospho-citrate buffer. Inhibition by 0.01 M NaF, 0.0002 M CuCl₂, 10% tartaric acid and 0.01 M NaCN, as well as substrate-deficient and heat-inactivated controls, demonstrated conclusively that acid phosphatase was functionally preserved. Strong enzymatic activity was exhibited by osteoclasts, chondroclasts and free multinucleated giant cells. In addition, megakaryocytes, histiocytes, plasma cells, and monocytes exhibited moderate activity. The results demonstrated the technique to be consistently reproducible.     

A Simple Method for Paraffin and Plastic Embedding of Protozoa

SUMMARY. To avoid multiple centrifugation and considerable losses of material in preparing protozoa for paraffin and plastic embedding a simple method has been worked out, requiring only two centrifugations, one before and one after fixation. During the second centrifugation, which is done at 700 g for 10 minutes, the organisms form a cohesive pellet which may be removed from the centrifuge tube by means of a fine spatula and handled as a piece of tissue through the whole process of dehydration and embedding.     

Polystyrene Embedding: A New Method for Light and Electron Microscopy

Polystyrene embedments of histological specimens can be Obtained with a solution 1 : 4 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue. which are then glued to a Plexiglas support Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections, with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy dons on a slide heated on a hot plate to 80 C; those can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fired for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron-microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and give acceptable results.