A Modification of Mayer's Hemalum

A survey of histological literature shows that Delafield's hematoxylin is one of the most widely used histological stains. Personal interviews with workers in this field elicit comments which seldom appear in print. Most workers confess that it usually requires several unsuccessful attempts to make a usable stain. The experienced technician is no more successful than the self-taught beginner. I have examined, at various schools, preparations stained with so-called Delafield's stain and the range of colors and degrees of muddiness found were surprising. A somewhat greater degree of certainty and uniformity is desirable in a stain which is to be used by classes in elementary histology. Mayer's hemalum has not enjoyed the popularity which it deserves perhaps because the formula given in a widely used text is incorrect. This was found to be a source of discouragement to self-taught students in some smaller colleges.     

Newer Applications of the Histological Stain Prepared from Pterocarpus Santalinus

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn, has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.     

Newer applications of the histological stain prepared from Pterocarpus santalinus

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn. has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.     

Immunofluorescence Identification of Thermopolyspora polyspora,the Causative Agent of Farmer's Lung

Farmer''s lung is a serious disabling pulmonary disease found in agricultural workers. The disease is believed to be a hypersensitivity to the thermophilic actinomycetes, principally Thermopolyspora polyspora. This organism is difficult to stain with the usual bacteriological stains and thus far has not been demonstrated in the lung tissue by microscopic methods. In this paper, it is demonstrated that the fluorescent-antibody technique is a simple method for the positive identification of T. polyspora. The technique can also be used as a rapid screening test for the detection of antibodies to T. polyspora in the patient''s serum. In addition, it opens up the possibility of the identification of T. polyspora in the lung tissue of patients with farmer''s lung and makes available a means for the study of the immunological reaction in the lung parenchyma. No false positive or cross-reactions with Thermoactinomyces vulgaris or Streptomyces griseus could be demonstrated.     

Safranin and Anilin Blue with Delafield's Hematoxylin for Staining Cell Walls in Shoot Apexes

The following technic is suggested for staining cell walls in shoot apexes: After the usual preliminary steps through 50% ethyl alcohol, stain in 1 % safranin 0 for 24 hours. Rinse in tap water and place in 2% aqueous tannic acid for 2 minutes. After rinsing in tap water, stain for 2 minutes in 1 part Delafield's hematoxylin to 2 parts distilled water and rinse in tap water. Remove excess hematoxylin with acidified water (1 drop cone. HC1 in 200 ml. water), then place slides in 0.5% lithium carbonate for 5 minutes. Dehydrate through an ethyl alcohol series, then transfer from absolute alcohol to a saturated solution of anilin blue in “methyl cellosolve” for 5-10 minutes. Wash in absolute alcohol, rinse in a solution of 25% methyl salicylate, 33% xylene, 42% absolute ethyl alcohol and clear for 10 minutes in a solution of 2 parts methyl salicylate, 1 part xylene, 1 part absolute ethyl alcohol. Transfer through two changes of xylene and mount in “clarite” or suitable alternate. The resulting preparations will have clearly defined, dark-staining cell walls and will photograph well when “Super Panchro-Press, Type B” film (Eastman Kodak Co.) is used in conjunction with suitable Wratten filters.     

Selective Light Microscopic Demonstration of the Specific Granulation of the Rat Atrial Myocardium by Lead-Hematoxylin-Tartrazine

MacConaill's lead-hematoxylin as modified by Solcia et al. was found to be a highly selective stain for the specific granulation of atrial cardiocytes in the rat. The specific atrial granules were stained blue-black. Contrast was enhanced by counterstaining in a saturated solution of tartrazine in Cellosolve. The stain is compatible with several fixatives and may be used with paraffin or Epon-embedded material.     

Histological and histochemical examination of American Indian scalps, mummies, and a shrunken head

The histological and histochemical detail remaining in Jivaro shrunken heads, North American Indian scalps, and mummified remains from Peru, Chile, and the American southwest, is remarkable when these specimens are rehydrated and fixed by Sandison's methods. Special techniques and stains used frequently in the histochemistry of the skin brought out details which were less evident or not apparent in the routine hematoxylin and eosin preparations. The polarizing microscope was particularly useful in demonstrating keratin and collagen fibers. Use of the silver stain for melanin not only aided in demonstrating hairs, but also outlined the pattern of epidermal detail by supranuclear accumulation and melanin dust in the stratum corneum. The hematoxylin-phloxine-alcian blue orange G stain for prekeratin, keratin, and mucin showed long persistence of the prekeratin and keratin staining properties. The stain identified strands as epidermis where they would otherwise have been overlooked. The Luxol Fast Blue stain, birefringence, and appearance with phase contrast microscopy are promising means for evaluating the degradation of skin collagen under conditions of dry preservation. The first loss of histological information occurs in extravascular cells of the dermis; mast cells, eosinophils, and other perivascular cells could not be identified in any of the specimens. Fine elastic fibers near the epidermis disappear before the larger, mid-dermal elastic fibers. Some of the disease processes which could be diagnosed in very old mummified skin are discussed in terms of the remaining structures.     

Metallic Lakes of the oxazines (Gallamin Blue,Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2–3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3–5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

The Status of Wright's Stain and Some Suggestions as to its use

A questionnaire was sent to a number of the larger hospitals and laboratories of the United States in order to determine the degree of satisfaction experienced in employing Wright's stain. From a survey of the answers received, there seems little justification for poor results with this stain. It is probable that the great majority of unsatisfied users work in laboratories where the stain is used so seldom that proper manipulation is not always attained. The writer offers a few simple, but very important precautions to be observed.     

Effects of field size when using Kerley's histological method for determination of age at death

The field size at which a bone is read affects the results obtained when using Kerley's histological method for age estimation, even after applying the recommended correction factor. Whereas there is no tendency for any one of three field sizes tested to consistently underestimate or overestimate age, a field size closest to that used by Kerley in his original study had significantly lower variances for its age estimates, and thus provides greater reliability. This particular field size yields more precise estimates because it is sampling a pattern and number of structures more similar to that of Kerley. Correction factors cannot equalize the counts of osteons and osteon fragments because of spatial variations in the distributions of these histological structures. A field size similar to that used by Kerley in gathering the data from which he developed his regression equations must be used to assure that the same pattern and number of structures is being sampled. For this reason, we suggest a field size as close to 2.06 mm² as possible be used when employing Kerley's method.     

Demonstration of Urinary Eosinophils in Schistosoma haematobium: a Comparative Study among Three Different Stains

Three stains, Hansel's stain, alkaline erythrocin B (AEB) and naphthalene black (NB), were used to demonstrate eosinophils in the urine of patients infected with Schistosoma haematobium. Hansel's stain was superior to the other two stains; it stained eosinophils bright red and their nuclei faint blue, and they were easily differentiated from neutrophils, lymphocytes, macrophages and epithelial cells. The method using AEB took longer than Hansel's stain and 10% of the specimens were lost during staining with this method. Like eosinophils, the neutrophils took up NB stain and their nuclei stained poorly with the counterstain.     

Functional morphology of the mandibular gland of queens of the ant Monomorium pharaonis (L.)

In this study, the functional morphology of the mandibular glands of pharaoh ant queens is investigated through histological and behavioural observations. The mandibular glands of queens of different age stages and mating status were examined at the light microscopical as well as the ultrastructural level. The results clearly show a high activity of the gland at the time of hatching, followed by a rapid degeneration independent of the queens' mating status. We therefore hypothesize the glandular secretion serves to stimulate workers to remove the queens' pupal skin. Experiments to compare hatching ability of isolated pupae and pupae assisted by workers confirmed the necessity of worker assistance, which is even more crucial to allow proper wing formation in queens and males. Together with the histological data, this suggests that the mandibular gland may indeed play a role in the social facilitation of hatching, although bioassays are required to give decisive answers about the mandibular gland's function.     

A Metachromatic Stain for Neural Tissue

The need for rapid histological feedback on neural tissue is ever present. Although there are several stains which can be readily used for staining either cell bodies or fiber tracts, adequate contrasting stains which are both rapid and easy to apply are not generally available. In 1936 Chang presented a technique for whole brains utilizing the metachromatic properties of thionin. Unfortunately this procedure was very time consuming. For the last several years we have worked with several variations of this stain and have found that thionin can be reliably used as a polychrome stain for sections of neural tissue obtained from a freezing microtome.     

Effect of heat-staining procedure on the gram staining properties of mycobacteria]

Since the establishment of Gram stain by H.C.Y. Gram in 1884, it has been widely and routinely used as an aid for differentiation of bacteria. The bacteria are divided into three categories by the staining properties; Gram-positive, -negative, and -indefinite. All the text books in the world describe that mycobacteria such as M. tuberculosis are Gram-positive. By the merest chance, however, it was found that M. lepraemurium grown in tissues was not stained by the routinely used Gram staining method. Therefore, we tried to stain some of the mycobacteria by the Gram staining procedure which is widely used at present. The results obtained indicated that the mycobacteria tested were divided into three groups; the unstainable group such as M. leprae and M. lepraemurium, the Gram-positive and difficult-to-stain group which involves such slow growing mycobacteria as M. tuberculosis, M. avium, and M. intracellulare, and the Gram-indefinite group which contains such rapid growing mycobacteria as M. phlei, M. smegmatis, and M. chelonae. However, if Gram stain is carried out by the heating procedure at the first staining step, all the mycobacteria would become Gram-positive. Therefore, we emphasize that Gram staining of mycobacteria should be performed by the heating procedure.     

A Gelatin Fixative for Paraffin Sections

Mayer's albumen fixative, of which the active principle is white of egg, is used almost universally for affixing paraffin ribbons to the slide. About eight years ago the writer's attention was called to a gelatin fixative which has proved to be so superior to albumen that he has used it almost exclusively ever since in the making of a great variety of botanical preparations, and has recommended it to a number of other workers whose experience with it subsequently has been just as satisfactory. The gelatin method was first described by Szombathy¹ and later discussed by Artschwager,² but it does not seem to have received the attention in the literature which its importance deserves. It certainly merits a wide spread use among both botanists and zoologists.     

Demonstration of Elastic Tissue and Iron or Elastic Tissue and Calcium Simultaneously

For the concomitant demonstration of iron and elastic tissue Perls' test solution was used, followed by Verhoeff's stain or Gomori's aldehyde fuchsin. When Perls' and Verhoeff's stain were used in sequence, the iron deposits were greenish blue and the elastic lamellae were black. When Perls' test solution was combined with aldehyde fuchsin the iron deposits were blue and elastic tissue purple. Calcium salts and elastic tissue were demonstrated concomitantly by using von Kossa's method followed by Gomori's aldehyde fuchsin. With such combined staining, the calcium salts appeared brownish black and elastic tissue purple. With these procedures, it was possible to see the exact relationship of calcium and iron deposits to the elastic tissue.     

Pinacyanol Erythrosinate as A Stain for Mast Cells

An alcoholic solution of the compound dye, pina-cyanol erythrosinate when diluted to the optimum dissociation point is a differential tissue stain which, in addition, selectively stains and differentiates mast cells. It can be made up and used like any other compound dye (e.g., Bowie's stain, neutral gentian, etc. or like a blood stain). It can be used after any of the common fixatives and has the advantage of selectively staining all types of mast cells in their various functional phases, even in those species (notably rabbit and man) in which they may be difficult to demonstrate with other mast cell stains after aqueous fixatives.     

Differential Stain for the Human and Animal Anterior Hypophysis

A staining method is described for human and animal pituitary glands which is based on a combination of a modification of the phosphomolybdic acid, orange G stain of Berblinger and Burgdorf's (1935) method, with an iron hematoxylin stain for the nuclei and with the periodic-acid-Schiff routine of Hotchkiss and McManus. The cytochemical part of the procedure demonstrates both muco-protein precursors of the gonadotropins and, it is thought, these hormones themselves. By comparison with other methods, finer differences in the cytology of the cyanophils can be appreciated. Quantitative cell counts are readily performed and large numbers of cells, appearing by Mallory and other histological methods to be chromophobes, are found to belong to the cyanophil series. Counts done by older methods, therefore, are considered to be inaccurate and misleading.     

Susceptibility of male reproductive system to bisphenol A,an endocrine disruptor: Updates from epidemiological and experimental evidence

Bisphenol A (BPA) is an omnipresent environmental pollutant. Despite being restrictions in-force for its utilization, it is widely being used in the production of polycarbonate plastics and epoxy resins. Direct, low-dose, and long-term exposure to BPA is expected when they are used in the packaging of food products and are used as containers for food consumption. Occupationally, workers are typically exposed to BPA at higher levels and for longer periods during the manufacturing process. BPA is a known endocrine disruptor chemical (EDC), that causes male infertility, which has a negative impact on human life from emotional, physical, and societal standpoints. To minimize the use of BPA in numerous consumer products, efforts and regulations are being made. Despite legislative limits in numerous nations, BPA is still found in consumer products. This paper examines BPA's overall male reproductive toxicity, including its impact on the hypothalamic-pituitary-testicular (HPT) axis, hormonal homeostasis, testicular steroidogenesis, sperm parameters, reproductive organs, and antioxidant defense system. Furthermore, this paper highlighted the role of non-monotonic dose–response (NMDR) in BPA exposure, which will help to improve the overall understanding of the harmful effects of BPA on the male reproductive system.     

Glandular Epithelium as a Possible Source of a Fertility Signal in Ectatomma tuberculatum (Hymenoptera: Formicidae) Queens

The wax layer covering the insect''s cuticle plays an important protective role, as for example, uncontrolled water loss. In social insects, wax production is well-known in some bees that use it for nest building. Curiously, mated-fertile queens of the ant Ectatomma tuberculatum produce an uncommon extra-wax coat and, consequently queens (mated-fertile females) are matte due to such extra cuticular hydrocarbon (CHC) coat that covers the cuticle and masks the brightness of the queens'' cuticle while gynes (virgin-infertile queens) are shiny. In this study, histological analysis showed differences in the epidermis between fertile (i.e., queens or gynes with highly ovarian activity) and infertile females (gynes or workers with non developed ovaries). In fertile females the epidermis is a single layer of cubic cells found in all body segments whereas in infertile females it is a thin layer of flattened cells. Ultrastructural features showed active secretory tissue from fertile females similar to the glandular epithelium of wax-producing bees (type I gland). Different hypotheses related to the functions of the glandular epithelium exclusive to the E. tuberculatum fertile queens are discussed.