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.     

Producing Frozen Sections of Calcified Bone

We describe a procedure for the rapid production and maintenance of fresh frozen bone biopsies which can be used for a variety of immunohistochemical techniques. Within 5 min of excision. tissue is placed in cold 5% polyvinyl alcohol, surrounded with 3% carboxymethylcel-lulose in a hand made aluminum foil embedding mold and frozen by immersion in an absolute ethanol/dry ice slurry at -70 C. The tissue block is attached to the specimen stub with cryocom-pound and installed in a -32 C cryostat whose tungsten carbide D profile knife is maintained at -70 C. Automatic controls are set at a slow cutting speed and the “sectioning window” is adjusted to fit the biopsy size. Knife angle, thickness gauge and antiroll bar are changed to produce a complete section. The block face is smoothly “papered” with a polyvinylpyrrolidone (PVP) impregnated Ross lens paper strip. A single section is cut and positioned on a sequentially numbered, acid cleaned, double dipped chrome-alum gelatin coated slide: adhesion is aided by “press-blotting” with bibulous paper. Sections are stored at -20 C or in a desiccator at room temperature. A brief fixation followed by removal of the water soluble PVP and lens paper generates fresh frozen bone sections suitable for further analysis.     

A Three-Dimensional Histological Method for Direct Determination of the Number of Trabecular Termini in Cancellous Bone

Osteoporotic fractures occur frequently in aging populations. Established methods for analyzing microarchitecture indicate that cancellous bone loss in the elderly is associated with progressive reduction in the connectivity of the trabecular network. This disconnection may explain the increased skeletal fragility that is sometimes out of proportion to the amount of bone lost. Connectivity, however, is difficult to measure and usually requires indirect methods. We describe development of a simple, inexpensive and direct procedure for counting sites of trabecular disconnection. The method is based upon preparation of 300-500 fjim thick slices of methylmethacrylate embedded material rather than the more usual thin 8 μm. histological sections. The marrow tissue is retained within the thick slice; this is essential for conservation of any detached bone fragments. In such preparations differential superficial staining of the upper and lower surfaces with alizarin red and light green, respectively, allows the two-dimensional image to be viewed at the same time as its three-dimensional counterpart. In this way, “real” (i. e., unstained) trabecular termini can be distinguished from “apparent” (i. e., stained red or green) termini that are artifacts of the plane of section. Partly polarized light enhances the microscope image. The method does not destroy the material for subsequent bone histomorphom-etry and, therefore, may be a useful adjunct to iliac bone biopsy analysis in studies of metabolic bone disease.     

Solochrome Cyanine R As An Indicator Dye of Bone Morphology

Sections of undemineralized bone embedded in a polyester resin and cut at 6 μ are stained for 10 min, without removal of the embedding matrix, in an aqueous solution composed of Solochrome cyanine R, 1 gin; glacial acetic acid, 2 ml; and distilled water, 98 ml. A pH about 2 is obtained by the acetic acid. The sections are washed and differentiated in tap water at 30 C, dehydrated in ascending alcohols, cleared and mounted in synthetic resin. “Young osteoid” stains light orange and, in the rest of an osteoid seam, two types of lamellae can be distinguished: one blue layer of ground substance or collagen and one orange layer of fibrillar collagen. The “calcification front” is sharply demarcated by its dark blue color.     

Detection of DNA in Ancient Bones Using Histochemical Methods

We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian “Casti Amanti” house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4' -' 6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.     

Autoradiography of Tracks from Beta-Particle Emitters in Tissues

Mounted paraffin sections, 2-4μ thick, ˙were stained, dehydrated, allowed to air dry, and given a thin coating of 1 % Plexi-glas solution in chloroform. The chloroform was allowed to evaporate completely in a dry atmosphere. An emulsion whose dried thickness was 100-150μ, was prepared from Ilford G5 type in gel form and glued to the section by means of a 15% solution of shellac in absolute alcohol. The surface of the emulsion was then cleaned with absolute ethyl alcohol, to remove the impermeable shellac layer. The exposure for radiation reaction was made at about 2°C and required, in the conditions of our experiment, about 24 hrs. The emulsions were processed by the “temperature-development method.” With the described procedure, autoradiographs have been obtained of various organs of albino rats, labeled with P³², S³⁵ and other radioisotopes, and very precise localizations of the origin of electron tracks was attempted. This technic has allowed the fixing and staining of the tissues by means of all the reagents commonly employed in histology, without any damage to the emulsion and the obtaining of good adhesion and minimum separation between specimen and emulsion, thus permitting reliable extrapolations of electron tracks. Due to the fact that the emulsion is fully sensitized when placed in contact with the preparation the limits of the exposure times were well defined. The uniform development at all depths of the emulsion achieved by the temperature-development method facilitated the work with fast electron tracks.     

Demonstration of the Ground Substance of Cartilage, Bone, and Teeth

A slab of bone about 5 mm. thick is decalcified in 5% HCl, washed, and placed for several days to 2-3 weeks in 3% KOH in 20% glycerin (if the bone is medium sized); for small bones the KOH should be decreased to 1%, and for large bones it may be increased to 5%. The solution is changed frequently. When the bone begins to dissociate, it should be removed and washed in water till all traces of alkali are removed. The specimen is passed through 3 changes of dioxane into paraffin, and then through a second paraffin bath into the final paraffin. Sections are cut at 10-12 μ and stained with VanGieson's picro-fuchsin or with orcein.     

A One-Solution Tannic-Aced-Iron Stain for Plant Tissue Sections

After completing the bulletin on “Actinomycetes in various parts of the potato and other plants” (Lutman, 1945) the author found the beautiful plates in the atlas to Olivier's monograph (1881) on root structure in which the same intercellular inclusions were shown. Olivier stated that he had stained his sections in “tannate of iron”. Attempts were made by the author to prepare and use such a combination but they were unsuccessful owing to the precipitate that was formed.

The formula used by the U. S. government for ink for official use was tried. This combination is composed of tannic and gallic acids with ferrous sulphate and is acidified with hydrochloric acid. When used double strength, as suggested for special blackness and permanence, the stain was very successful on sections of potato roots and tubers. It stained the Actinomyces hyphae very differentially and was decolorized from all other cell organs. Any other stains used dyed also the pectins and the Actinomyces secretions (melanins) but with this iron tannate combination in one solution, the finest hyphae could be seen and photographed. Since hydrochloric acid was used in this stain, such Actinomyces inclusions must be very tannophylic; much more so than any animal intercellular inclusions so far described.     

Staining Aspirated Human Bone Marrow with Domestic Wright Stain

The method employs the domestic Wright stain for the staining of aspirated human bone marrow. Freshly distilled water, pH 6.0 to 6.4, is used. Wright stain, 0.5 cc, is placed upon the air-dried preparation and permitted to act for two minutes. The stain is then diluted with 2 cc. distilled water and permitted to act for from 5 to 10 minutes. After washing off the stain with distilled water, the preparation is placed into a decolorizer (acetone 0.5 cc, pure methyl alcohol 5.0 cc, and 100 cc. distilled water, pH 6.0 to 6.4) for differentiation from 1 to 5 seconds, rinsed, washed under running water and permitted to air-dry. A well stained and differentiated preparation shows the “Romanovsky effect”, and the sharpness of minute structures obtained compares favorably with control preparations stained with German dyes.

The bone marrow should be prepared as described. The Wright stain marketed by the National Aniline and Chemical Co., N. Y. was found to be reliable as regards staining quality of registered batches. One photomicrograph, showing bone marrow cells from pernicious anemia, is included.     

The Staining of Radulae

A review of the various methods of staining and mounting radulae is given. Normally the radula should be extracted with 0.5 to 1% sodium hydroxide solution, and the associated tissues removed before staining. Two staining methods are recommended for facilitating the interpretation of radulae. Newly formed teeth and the bases of older ones are well stained by saturated aqueous chlorazol black E (up to 10 minutes). A more uniformly stained specimen) in which the cusps of all but the young teeth are alone stained, may be obtained by using the “oxidation-dahlia technic”. The radula is oxidized in N/10 potassium permanganate solution until black and subsequently decolorized in saturated aqueous oxalic acid. It is then stained in 0.1% aqueous dahlia (Hofmann's violet), the staining time varying from 10 to 30 minutes, according to the material. It is then dehydrated and passed through xylene and clove oil into Canada balsam. Other mountants may be employed but glycerin jelly is only recommended for the rapid examination of unstained radulae. Several other staining methods are mentioned, and general precautions to be observed while mounting are discussed.     

A Modified Mallory-Cason Staining Procedure for Large Cryosections

The classic Mallory-Cason staining procedure has been modified for application to sections “on tape” obtained from large deep frozen tissue specimens. These 20 μm cryosections are collected on tape from a large heavy duty cryomicrotome. The stained sections provide anatomical details that are not revealed by other techniques. The merit of this procedure is found in the support of modern medical modalities, both for research and educational purposes.     

Microwave Irradiation as a Generator of Heat for Histological Fixation

The kitchen appliance known as the “radar oven” generates heat quickly in materials containing water. Protoplasm exposed to the irradiation can thus be denatured. The amount of heat generated is a function of the time of exposure and the intensity of the irradiation, and the size and specific heat of the tissue or organism being irradiated. But docs such heating have applicabiity to histological technique? One of four carcas temperaturea (approximately 60°, 70°, 77°, and 85 C) was generated in anaesthetized, adult hairless mice of both sexes. “Control” animals were not irradiated. Specimens of liver, kidney, lung, and (from males) testis were taken from the five groups; the tissue spedmens were dehydrated in tetrahydrofuran, embedded in paraffin, sectioned at 9 μm, and stained with hematoxylin and eosin. The preparations were suitable for histological examination. Each organ had an optimum temperature for histological fixation under the conditions of this experiment: liver, ∼70°; kidney, ∼77°; lung, ∼77°; and testis, ∼85 C. Heat fixation by microwave irradiation also shows some applicability to electron microscopical studies and to investigations of the blood vascular arrangements of organs.     

A comparison of eating habits between retired or semi-retired aged subjects and younger subjects in full-time work

An analysis of eating habits in older retired subjects (“No Work group”) and younger subjects employed in full-time work (“Work group”) has been carried out. It used a questionnaire that assessed why individuals chose to eat or not to eat meals during the course of the day, and subjective responses to the meals. The questionnaire was answered every three hours over a “typical week” which, for the Work group, entailed working during the weekdays and resting at the weekend. For the “No Work” group, breakfast was the most frequently taken meal of the day whereas, for the “Work” group, this meal was often missed. Patterns of meal intake were not significantly different between the weekdays and weekend for the “No Work” group, but the “Work” group ate more hot food at the weekend. The reasons cited for eating/not eating a meal and for choosing the type of meal eaten were dominated by hunger/lack of hunger in both groups. In addition, whereas habits were also important for the “No Work” group, it was time availability or the lack of it that was of major importance to the Work group, though this was significantly less important at the weekend. Meals which required more time for preparation and cooking were appreciated significantly more (appetite before the meal, enjoyment during it, and satiety afterwards) than meals such as snacks and cold food, which could be prepared more quickly. Some implications of these results, with regard to regular meals acting as a social zeitgeber in older subjects and the additional constraints imposed upon night workers, are considered.     

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.     

Principles in the selection of inorganic elements by organisms — Application to molybdenum enzymes

Some basic principles were delineated with regard to the selection of inorganic elements by organisms; they are “basic fitness rule”, “abundance rule”, “efficiency rule” and “evolutionary pressure”. The basic fitness rule was then applied to the case of molybdenum, to show how inherently fit molybdenum is to the biological functions carried out by those enzymes dependent on it, including xanthine oxidase, sulfite oxidase, nitrate reductase and nitrogenase.     

Histomorphometric analysis of adult articular calcified cartilage zone

The aim of this study was to carry out a histomorphometric analysis of calcified cartilage zone (CCZ) and its interfaces between hyaline cartilage and subchondral bone. The study used 40 donated normal human femoral condyles, from which paraffin-embedded sections were prepared after fixation and decalcification. The histomorphology of the CCZ were qualitatively and quantitatively observed by staining, scanning electron microscopy (SEM) and three-dimensional (3D) reconstruction. The hyaline cartilage and CCZ were stained red with Safranin-O, and the subchondral bone was stained blue with Fast green. CCZ was stained black after von Kossa staining. The hyaline cartilage was interlocked tightly in the manner of “ravine-engomphosis” by the CCZ. The surface roughnesses of tidemark and cement line were 1.14 ± 0.04 and 1.99 ± 0.38. The maximum, minimum and mean thicknesses of CCZ were 277.12 ± 8.6, 9.83 ± 6.72 and 104.162 ± 0.87 μm, respectively. The cell density of CCZ (51.25 ± 21.26 cells/mm²) was significantly lower than that of the hyaline cartilage (152.54 ± 35.77 cells/mm²) (P < 0.05). The subchondral bone was anchored tightly in the manner of a “comb-anchor” by the CCZ in our 3D reconstruction model. Thus, we discovered two junctional interfaces of CCZ using different histomorphometric methods. The upper interface of CCZ is a “ravine-engomphosis” shape, while its lower interface is a “comb-anchor” shape.     

Temperature profiles, and the effect of sleep on them, in relation to morningness-eveningness in healthy female subjects

There were 15 healthy female subjects, differing in their position on the “morningness-eveningness” scale, studied for 7 consecutive days, first while living a sedentary lifestyle and sleeping between midnight and 08:00 and then while undergoing a “constant routine.” Rectal temperature was measured at regular intervals throughout this time, and the results were subjected to cosinor analysis both before and after “purification” for the effects of physical activity. Results showed that there was a phase difference in the circadian rhythm of core temperature that was associated with the morningness score, with calculations that “morning types” would be phased earlier than “evening types” by up to about 3h. This difference in phase (which was also statistically significant when the group was divided by a median split into a “morning group” and an “evening group”) could not be attributed to effects of waking activity and existed in spite of the subjects keeping the same sleep-wake schedule. Moreover, it persisted when the subjects' data had been purified and when the data were obtained from the constant routine. That is, there was an endogenous component to this difference in phase of the core temperature. The morning group also showed a greater fall of core temperature during sleep; this was assessed in two ways, the main one being a comparison of constant routine and nychthemeral data sets after correction for any effects of activity. Even though the morning group was sleeping at a later phase of their circadian temperature rhythm than was the evening group, neither group showed a fall of temperature due to sleep that varied with time elapsed since the temperature acrophase. It is concluded that another factor that differs between morning and evening types is responsible for this difference. (Chronobiology International, 18(2), 227-247, 2001)     

The history of essentialism vs. Ernst Mayr's “Essentialism Story”: A case study of German idealistic morphology

Idealistic morphology as perhaps the most important historical manifestation of typology is very suitable for a historical analysis of Ernst Mayr's “Essentialism Story”, which postulates an antagonism between “typological thinking” and “population thinking”. We show that German-language idealistic-morphological theories consisted of two clearly distinguishable parts. The cornerstone of these theories was the concept of the type as an abstract pattern representing a certain class of phenomena and embodying the norm of this class. The primary objective of pure typology was to create a non-phylogenetic classification system for living organisms based on structurally explicable characters. Thus, typology, as a non-phylogenetic foundation of idealistic morphology, was conceptually neutral with respect to hypotheses of evolutionary mechanisms. Typology was often accompanied by concepts such as Lamarckism, orthogenesis, creationism, essentialism, etc. These peripheral (with respect to pure typology) concepts were autonomous constructions and did not represent a direct logical consequence of typology. In our view “population thinking”, as part of the Darwinian theory of evolutionary mechanism, could not be directly opposed to “typological thinking”. Rather, it was peripheral concepts such as essentialism or creationism that led to conflicts between the Modern Synthesis and idealistic morphology.     

Preparation of Thin Undecalcified Bone Sections by Rapid Manual Method

Sections from 3 μ to over 100 μ thick of fresh, unfixed, unembedded, unde-calcified and undehydrated bone are made by grinding 1 to 2 mm slabs of the desired orientation on waterproof carborundum abrasive paper, grit No. 320, 360 or 400. The manner of controlling the section is the crux of the technique. The section is held by wrapping a fresh strip of sandpaper around a 3' × 1' slide and accomplishing the grinding on a used piece of paper. The abrasive points on the fresh paper effectively prevent the section from sliding off the slide. The specimen is kept wet with water during the entire procedure. Sections are then stained, and excess surface stain can be ground off in similar fashion. After washing in dilute detergent solution to remove adherent derbis, the section is air dried and mounted in any nonacidifying resinous media. The method is suitable for wood and for fruit pits also.     

Influence of sex, slaughter weight and carcass weight on “non-carcass” and carcass quality in segureña lambs

The effects of sex, slaughter weight and carcass weight on carcass characteristics and meat quality traits were evaluated using 100 Segureña lambs. The management of all lambs was similar prior to slaughter at 19–25 kg. Slaughtered animals with a hot carcass weight below 20 kg were assigned to class B, and those greater than 22 kg to class C. Carcass weight had a significant influence on “non-carcass” components, dressing percentage, subjective carcass conformation, fat deposits, carcass fatness, bone and most carcass measurements. Sex had a significant effect on age at slaughter, “non-carcass” components, rib measurements, dressing percentage, fat deposits, and neck and shoulder percentage. As the weight increased, the carcass measurements also increased. Concurrently, while improving the conformation indices of the carcass, leg and dressing percentages, neither the commercial cuts of the animal nor tissue composition was significantly affected. Sex primarily affected the quantity of all types of fat deposits.