A simple and highly reproducible staining method for fungi and other polysaccharide-rich microorganisms in animal tissues

A newly devised, simple and highly reproducible method for fungal staining is reported. Grocott's method, in which methenamine-silver nitrate solution is employed, has been widely used for the staining of fungi in tissue sections, but it frequently produces heavy background staining because of sudden and progressive reaction in the methenamine-silver nitrate solution. We therefore replace the latter solution with an ammoniacal silver nitrate solution. This new method yields more consistent results in fungal staining without background staining, since the reaction time in the ammoniacal silver nitrate solution is prolonged. The present method is considered superior to Grocott's method with regard to its simplicity and reproducibility.     

A simple and economical modification of the Masson-Fontana method for staining melanin granules and enterochromaffin cells

Enterochromaffin cells from the small intestine of man, guinea pig, dog, chicken, rabbit, cat and rat were stained using the Masson-Fontana ammoniacal silver method with varying dilutions of silver nitrate solution (0.25 to 5 g per 100 ml of distilled water) and incubation temperatures (60 C and 75 C). The 0.5% solution of silver nitrate gave an argentaffin pattern similar to that of the 5% solution and had two major advantages: economically, since much less silver nitrate is used, and methodologically, since low background resulted with tissue of those species (rat, cat and rabbit) that required unusually long incubation. The staining of melanocytes was similar for all dilutions at the usual staining time (15-30 min).     

A Simple and Economical Modification of the Masson-Fontana Method for Staining Melanin Granules and Enterochromaffin Cells

Enterochromaffin cells from the small intestine of man, guinea pig, dog, chicken, rabbit, cat and rat were stained using the Masson-Fontana ammoniacal silver method with varying dilutions of silver nitrate solution (0.25 to 5 g per 100 ml of distilled water) and incubation temperatures (60 C and 75 C). The 0.5% solution of silver nitrate gave an argentaffin pattern similar to that of the 5% solution and had two major advantages: economically, since much less silver nitrate is used, and methodologically, since low background resulted with tissue of those species (rat, cat and rabbit) that required unusually long incubation. The staining of melanocytes was similar for all dilutions at the usual staining time (15-30 min).     

银染技术在生殖细胞研究中的应用

新近对传统的银染技术作出改良,以氨银反应观察精子发生及受精过程中碱性蛋白的更替,以Ａｇ－Ａｓ反应观察精子发生过程中ＮＯＲ,嗜银细胞器,细胞骨架及其它嗜银成份的变化以及皮层皮应中嗜银成分的变化。     

Histochemical observations on Pneumocystis carinii: Selective demonstration of honeycomb forms

Summary Histochemical investigations of pulmonary lesions indicated selective coloration of membranes of honeycomb stages of Pneumocystis carinii by the periodic acid — sodium bisulfite — resorcin-fuchsin reaction for basement membranes; mucus, fibrin and other deposits in respiratory pathways did not react. These membranes were colored selectively also by the picro-Sirius Red F3BA method for collagens; fungi in tissues from patients with candidiasis remained unstained. For simultaneous demonstration of honeycomb and cyst forms of Pneumocystis carinii, sections were prestained with Grocott's modification of Gomori's methenamine-silver nitrate technic and then treated with the periodic acid-Schiff (PAS) or picro-Sirius Red F3BA reaction. In contrast to other Gram-positive microorganisms, cysts of Pneumocystis carinii were immediately decolorized by acetone-ether mixtures; this indicates differences in the mode of dye binding. Frequently, only one stage of Pneumocystis carinii was found in a given area. Hence a combination of reactions showing different stages is recommended for studies of small tissue samples.     

Differentiation of Myofibrillae, Reticular and Collagenous Fibrils in Vertebrates

A procedure for the differentiation of the mesenchymal derivatives, myofibrillae, reticular and collagenous fibers is presented. Formol-Zenker fixation (5-12 hours) is followed by the washing, iodinization, dehydration and paraffin embedding steps routine for that fixative with the following modifications. Zirkle's butyl alcohol series is used for dehydration and infiltration with paraffin as well as in the alcohol slide series. Embedding paraffin used is Parawax plus 8-10% bayberry wax. Tissue-exposed surface of paraffin block is soaked in water overnight before cutting serial sections at 3-5μ. Sections are mounted using the dilute albumen method, and the slides, thoroughly dried at 37^oC. overnight, are left at 60^o for 10 minutes to melt the paraffin of the sections. Before staining, the sections are given a preliminary treatment with potassium permanganate and oxalic acid. For reticular staining a 10% silver nitrate bath is succeeded by an ammoniacal silver carbonate solution followed by reduction in 1% neutral formalin, toning in gold chloride and fixing in sodium thiosulphate. Myofibrillae, the sacroplasmic limiting membrane and other sarcous elements are stained by Heidenhain's azocarmine solution, adult tissues at room temperature and fetal tissues at 50 ^oC. Differentiation in phosphotungstic acid is followed by the staining of collagenous fibers. For adult tissue, light green SF (C.C.) is used and for fetal tissue, fast green FCF (C.C). A discussion of the preparation of ammoniacal silver solutions is included. Both stock and used solutions of ammoniacal silver have been in use by the author for over a period of two years.     

Histochemical observations on Pneumocystis carinii: selective demonstration of honeycomb forms.

Histochemical investigations of pulmonary lesions indicated selective coloration of membranes of honeycomb stages of Pneumocystis carinii by the periodic acid--sodium bisulfite--resorcin-fuchsin reaction for basement membranes; mucus, fibrin and other deposits in respiratory pathways did not react. These membranes were colored selectively also by the picro-Sirius Red F3BA method for collagens; fungi in tissues from patients with candidiasis remained unstained. For simultaneous demonstration of honeycomb and cyst forms of Pneumocystis carinii, sections were prestained with Grocott's modification of Gomori's methenamine-silver nitrate technic and then treated with the periodic acid-Schiff (PAS) or picro-Sirius Red F3BA reaction. In contrast to other Gram-positive microorganisms, cysts of Pneumocystis carinii were immediately decolorized by acetone-ether mixtures; this indicates differences in the mode of dye binding. Frequently, only one stage of Pneumocystis carinii was found in a given area. Hence a combination of reactions showing different stages is recommended for studies of small tissue samples.     

Demonstration of polysaccharides by silver impregnation (2)

To demonstrate carbohydrate-containing tissue elements becoming argyrophil by treatment with chromic acid and periodic acid respectively, the author proposes instead of the methenamine-silver nitrate solution the successive application of a silver nitrate solution, adjustable to the optimal pH, and of a physical developer.     

Rapid Bielschowsky silver impregnation method using microwave heating.

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Observations of aspergillosis in the brains of turkey poults using different histopathological staining techniques.

Different staining methods were evaluated for studying aspergillosis of the central nervous system (CNS). The pathological changes and fungal elements in cerebrum and cerebellum of 17 turkey poults with aspergillosis were examined and described. Tissue sections were stained with hematoxylin-eosin (HE), Kluver-Barrera's and Grocott's methods, and periodic acid-Schiff (PAS). Focal granulomatous reactions with central necrosis were observed in the HE stained slides. Fungal hyphae were easily demonstrated using Grocott's method and PAS. These two methods, however, were not suitable for describing detailed histopathological changes. The Kluver-Barrera method was used to demonstrate the neural tissue reaction. Neurons were found to be sensitive to aspergillosis, in contrast to glial cells that showed fewer pathological changes. The fungal elements were clearly visible with the Kluver-Barrera method, resulting in better information about the interactions of neural tissue, the inflammatory response, and the fungus. The use of the Kluver-Barrera method for this purpose has not been documented previously.     

Selective Axonal Argentaffin Staining in Rat Central Nervous System after Protein Mercuration

The mercury-silver (Hg-Ag) argentaffin technique, known to stain specifically proteins in the lateral components of triads/diads in striated muscle cells, was applied to the central nervous system of adult rats. Following fixation in glutaraldehyde, axons in white and gray matter were selectively stained, but not perikarya or their proximal axon and dendrites. Neural tissues were postfixed 24 hr in 5% (w/v) mercuric acetate in 2% (v/v) acetic acid in distilled water, stained for 12-24 hr in darkness at 37-43 C with ammoniacal silver nitrate solution, freshly prepared by adding concentrated ammonia to 60% (w/v) silver nitrate solution until a small amount of silver oxide precipitate remained undissolved. Samples were then washed with freshly prepared 5% (w/v) sodium sulfite and distilled water. All steps were carried out using dark-colored glass flasks. Samples were dehydrated with ethanol and embedded in Paraplast or Poly Bed. Electron microscopy showed the silver-reducing protein inside the axons. Methylation abolished Hg-Ag axonal reactivity indicating that carboxyl groups were necessary for silver staining. Proteins with solubility properties characteristic of neurofilament proteins were involved in Hg-Ag staining. In the cerebellum the plexus of parallel fibers in the molecular layer were not stained, while basket cell axonal processes reacted intensely. The method appears to distinguish neuronal protein variants related to cytotypic differences in cytoskeletal neurofilaments.     

The von Kossa reaction for calcium deposits: silver lactate staining increases sensitivity and reduces background

Summary The classical von Kossa method has been modified: the high silver nitrate concentration in the original was replaced by 0.05% silver lactate with hydroquinone remaining the reducing agent of choice. The present modification stained calcification nodules with a sensitivity comparable to the original von Kossa reaction, but resulted in a reduced background staining in cultured osteoblasts. The method works well also with plastic- or paraffin-embedded tissue sections.     

Electron-microscopic study of silver staining of nucleoli in growing oocytes of rat ovaries

The nucleoli of dictyate-stage growing oocytes in rat ovaries were examined both with routine electron microscopy and electron microscopy after silver nitrate and ammoniacal silver nitrate (Ag-AS) staining. The nucleoli of the unilaminar follicular oocytes consist of twisted strands of dense fibrillar components, aggregates of granular components, and small fibrillar centers. After Ag-AS staining, silver grains are numerous on the dense fibrillar strands, fewer on the fibrillar centers, and very sporadic on the granular aggregates. The same stainability of three nucleolar components with the Ag-AS method was also confirmed in the nucleoli segregated by actinomycin D. During the transition of growing oocytes from bilaminar to plurilaminar follicle stage, the nucleolar dense fibrillar strands gradually conglomerate and are transformed into large and compact spherules. The stainability of dense fibrillar components with the Ag-AS method was lost along with this nucleolar transformation. These results may provide some new clues on the functional significance of Ag-AS-positive proteins in the nucleoli.     

Modified pyrogallol-initiated immunogold-silver enhancement technique applicable to prokaryotes

A modified immunogold-silver enhancement technique that was designed to reduce the nonspecific granular background staining, particularly for application on prokaryotic organisms, is reported. Aerial oxidation of pyrogallol contained in the commercial silver enhancer solution was effectively controlled during storage and in the reaction mixture. A combination of strategies such as storing the reagent under argon, modifying it using 0.5% (w/v) anhydrous sodium sulfite, reducing the concentration of silver ions in the reaction mixture and limiting the length of the silver enhancement reaction considerably reduced the granular background staining. The modified technique was demonstrated on the bacterium Xanthomonas campestris pv. malvacearum (Smith) Dye. A 7-min silver enhancement step produced little background staining, while optimal silver intensification of the bacterium pre-treated with the immunogold label was achieved.     

Localization of histones and their synthesis by ammoniacal silver reaction in meristematic root tip cells of Allium cepa

Summary The ammoniacal silver reaction was used for localization of histones in meristematic root tip cells of Allium cepa. Electron microscopic observations showed that yellow or brown colour of interphase and prophase nuclei and brown nucleolar colour produced in the reaction coincides with the appearance of silver grains, about 400 Å in diameter, in the interphase and prophase chromatin and nucleoli. This together with the complete absence of staining reaction and silver grains in the cytoplasm could mean quite a specific reaction with histones and might suggest also that in these cells the site of histone synthesis is in the nucleolus.     

Improved microradiographic contrast for bone stain-historadiography.

Microradiographs of 5-micron sections of methyl methacrylate embedded undemineralized bone show poor resolution, but prestaining with silver nitrate increases the radioopacity of the calcified tissues to soft x-rays without masking regional differences in microscopic mineralization. Identical results are achieved using aqueous (pH 5.8 and 7.5) or ammoniacal solutions (pH 7.9). Atomic absorption spectrometry detected no loss of calcium from the sections during staining. Osteoid in plastic-embedded bone is not rendered radiopaque by this technique even after prolonged staining times (5 min-2 hr).     

Improved Microradiographic Contrast for Bone Stain-Historadiography

Microradiographs of 5-micron sections of methyl methacrylate embedded undemineralized bone show poor resolution, but prestaining with silver nitrate increases the radioopacity of the calcified tissues to soft x-rays without masking regional differences in microscopic mineralization. Identical results are achieved using aqueous (pH 5.8 and 7.5) or ammoniacal solutions (pH 7.9). Atomic absorption spectrometry detected no loss of calcium from the sections during staining. Osteoid in plastic-embedded bone is not rendered radiopaque by this technique even after prolonged staining times (5 min-2 hr).     

Examination of Induced Sputum In the Diagnosis of Pneumocystis Carinii Pneumonia

The results of the examination of sputum induced by the inhalation of nebulized hypertonic saline in the diagnosis of Pneumocystis carinii pneumonia (PCP) are presented. In suspected cases of PCP in patients who were either HIV antibody positive or were receiving immunosuppressive therapy, 46 induced sputum specimens were stained using both Grocott's modified Gomori methenamine silver nitrate (GMS) and immunofluorescence staining. In 12 specimens P. carinii cysts were detected by both methods, in four specimens by GMS staining only and in five specimens by immunofluorescence only. The sensitivity of induced sputum examination in the detection of P. carinii cysts was increased by using both of these staining methods on each sputum specimen and the need for more invasive methods of diagnosis was reduced.