Stain Technology a Bone Stain for Osteoid Seams in Fresh, Unembedded, Mineralized Bone

Fresh, ground, mineralized bone sections 75-100 μ thick are stained 90 minutes or 48 hours in the Bone Stain, a preparation containing fast green FCF, orange G, basic fuchsin, and azure II. Surface stain is then removed by grinding under running water. Sections are washed in 0.1% zephiran chloride (benzalkonium chloride) or in 0.01% mild soap and again washed in tap water, followed with distilled water. Sections are next differentiated in 0.01% acetic acid in 95% methanol, dehydrated in 95% ethanol and 100% ethanol, cleared in alcohol:xylene 1:1, 1:4, 1:9 and 2 changes of xylol, and then mounted permanently in Eukitt's mounting media.

Osteoid seams stain either green to jade green or red to dark red, incompletely mineralized bone red or orange yellow, and the zone of demarcation light green. The walls of lacunae, canaliculae, feathered bone, procedural artifacts and periosteocyte lacunar low-density versions stain red.

The method helps in the differential diagnosis of certain metabolic bone diseases in human biopsy and autopsy material.     

Modified Tetrachrome Method for Osteoid and Defectively Mineralized Bone in Paraffin Sections

A new modification of the tetrachrome method for bone osteoid in paraffin sections has been designed. The modified tetrachrome method suitable for routine use in any histology laboratory retains the simplicity of the original method and gives good results on the freshly fixed, decalcified, paraffin embedded material. Osteoid tissue is stained deep blue and normally mineralized bone is stained red. Defectively mineralized bone stains pale blue or pink and the cellular population is clearly identifiable. The ability to distinguish the osteoid tissue from mineralized bone and connective tissue and cartilage makes diagnosis of osteomalacia or osteoid producing tumors or assessment of ossification process straightforward, without the need for un-decalcified sections. By displaying simultaneously irregularities in the mineralized matrix and morphology of bone cells, the method also permits the diagnosis of conditions recently described in patients with osteoporotic fractures, such as osteocytic degeneration and bone tissue defects.     

A Tetrachrome Stain for Fresh, Mineralized Bone Sections, Useful in the Diagnosis of Bone Diseases

Fresh, unprocessed bone is ground to sections 75-100 μ thick, stained in an aqueous solution composed of fast green FCF, 0.1 gm; orange G, 2.0 gm; distilled water, 100.0 ml; and adjusted to pH 6.65, then in a mixture of 1 part alcoholic solution of 0.25% celestine blue B and 9 parts of alcoholic solution of 0.1% basic fuchsin. Surface stain is removed by grinding sections to 50 μ and washing them in 1% invert soap (Zephiran) to remove adherent debris. (Commercial detergents and alkaline soaps may interfere with chromophore groups of the dyes.) Wash in tap water; rinse in distilled water and differentiate in 1% acetic alcohol. Dehydrate in ascending alcohols, clear in xylene and mount permanently in a neutral, synthetic resin. Active osteoid seams stain dark to light green; resting osteoid seams, red to bright orange red; transitional osteoid seams, geenish-yellow, orange red to red; older, partly mineralized matrix, orange; new, partly mineralized matrix, red; osteocyte nuclei, red; osteoblasts and osteoclasts, greenish-blue to dark purple nuclei and green or light green cytoplasm. Hyper-trophic and differentiating cartilage cells are stained light pink and dark red respectively. The staining reactions are consistent; the solutions are stable.     

Staining Osteoid Seams in Thin Slabs of Undecalcified Trabecular Bone

For qualitative and quantitative study of osteoid seams in trabecular bone, 5-7 mm thick slabs were cut from the bodies of fresh, frozen, undecalcified human vertebrae. After washing out the bone marrow and soft tissue in a jet stream of water, the slabs were stained in 0.5% aqueous basic fuchsin for 30-40 hr at 18-20 C. The specimens were then trimmed by sawing off both overstained surfaces, to make a 2 mm slab which was submerged in 50% ethanol (2 or 3 changes of 10-30 min each), until the nonosteoid trabeculae became pale pink. The slab was allowed to dry in air. Osteoid seams are stained dark red and are well differentiated under a dissecting microscope with reflected illumination, either dry or immersed in water. This method permits the various types of trabculae to be separately studied in the same specimen     

A Versatile Stain for Pollen Fungi, Yeast and Bacteria

A versatile stain has been developed for demonstrating pollen, fungal hyphae and spores, bacteria and yeasts. The mixture is made by compounding in the following order: ethanol, 20 ml; 1% malachite green in 95% ethanol, 2 ml; distilled water, 50 ml; glycerol, 40 ml; acid fuchsin 1% in distilled water, 10 ml; phenol, 5 g and lactic acid, 1-6 ml. A solution has also been formulated to destain overstained pollen mounts. Ideally, aborted pollen grains are stained green and nonaborted ones crimson red. Fungal hyphae and spores take a bluish purple color and host tissues green. Fungi, bacteria and yeasts are stained purple to red. The concentration of lactic acid in the stain mixture plays an important role in the differential staining of pollen. For staining fungi, bacteria and yeasts, the stain has to be acidic, but its concentration is not critical except for bacteria. In the case of pollen, staining can be done in a drop of stain on a slide or in a few drops of stain in a vial. Pollen stained in the vial can be used immediately or stored for later use. Staining is hastened by lightly flaming the slides or by storing at 55±2 C for 24 hr. Bacteria and yeasts are fixed on the slide in the usual manner and then stained. The stock solution is durable, the staining mixture is very stable and the color of the mounted specimens does not fade on prolonged storage. Slides are semipermanent and it is not necessary to ring the coverslip provided 1-2 drops of stain are added if air bubbles appear below the coverslip. The use of differentially stained pollen mounts in image analyzers for automatic counting and recording of aborted and nonaborted pollen is also discussed.     

A Polymethyl Methacrylate Method for Large Specimens of Mineralized Bone with Implants

A simple modified polymethyl methacrylate method is described for large mineralized bone specimens with implants and bioactive materials which produces consistently good histological preservation of the interface between bone and implant. Human femoral heads, whole rabbit condyles and canine tibias and femurs containing implants consisting of hydroxyapatite, smooth polyethylene, porous polyethylene and carbon were dehydrated in ascending grades of ethanol and cleared with xylene on an automated tissue processor which alternated vacuum and pressure for 22 hr. Infiltration was done with washed polymethyl methacrylate at 4 C under vacuum for 13 days. Polymerization was carried out in wide-mouth glass jars at 38 C for 36 hr so that the total processing time was less than 20 days. The only important modification was in the polymethyl methacrylate, which had less plasticizer than usual in order to give a harder block. This enabled production of 4 μm sections with good preservation of mineralized and cellular areas for the study of metabolic bone diseases, morphometry, fluorochrome labelling and interface analysis with the implant in situ.     

A Simple Histological Method for Identification of Osteoid Matrix in Decalcified Bone

The present experiment indicated that cyanuric chloride fixation was very useful in identifying osteoid matrix, which is difficult to distinguish from mineralized matrix in sections decalcified in the routine fashion. Small slices of bone from 3 mm to 5 mm thick were fixed with 0.5% cyanuric chloride in methanol containing 1% N-methyl morpholine for from 1 to 2 days at room temperature. EDTA decalcified sections were prepared and stained with hematoxylin and eosin. The regions presumed to be osteoid matrix were intensely eosinophilic. It was shown that the eosinophilic regions correspond precisely to the unmineralized osteoid matrix which was radiolucent by microradiography and devoid of silver by the von Kossa method in undecalcified serial sections.     

Kallichrome: A New Stain for Erythroblasts

Using the obscure dye kallichrome, erythroblasts at all stages of maturation demonstrated intense yellow to yellow-brown staining of both nucleus and cytoplasm. Staining of this type was not observed in other types of normal or pathological marrow cells. As such, kallichrome may be a valuable stain for identification of erythroblasts and their distinction from other types of blood cells.     

A New Stain for Pollen Fertility Studies

The juice from the berries of Cocculus hirsutum was extracted and used for pollen fertility studies in various crops. Two stains were prepared: P. H. Ramanjini (PHR) stain and modified PHR stain. The modified PHR stain contains lactic acid and produces the best staining differentiation. The intensity of the staining was dependent on the thickness of the pollen cell walls, hence PHR stain is recommended for thick walled pollen grains and the modified PHR stain for pollen with relatively thin walls. The preparation of both the stains are very simple, quick and inexpensive.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

A Thionin Stain for Visualizing Bone Cells, Mineralizing Fronts and Cement Lines in Undecalcified Bone Sections

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored hi the dark at 7 C.     

Improved Procedure for Histological Identification of Osteoid Matrix in Decalcified Bone

Several improvements on the original method of Yoshiki and coworkers for histological identification of osteoid matrix in decalcified bone are described in this report. The first, fixation of bone with neutral buffered formalin, a popular and stable fixative, should produce better tissue morphology and ensure easy handling in any laboratory. The second is a simple test for aged cyanuric chloride. Aged reagents show poor or no solubility in methanol and have almost no effect on differential staining of osteoid matrix. The third is an application of an organic acid solution in place of neutral EDTA for bone decalcification. Reduced decalcification time with the acid results in rapid preparation of bone sections. Neutral formalin fixation, immersion in the cyanuric chloride solution, decalcification with an organic acid, and hematoxylin and eosin staining, all quite routine laboratory procedures, yield high quality results for identification of osteoid matrix in bone sections.     

新型乳杆菌素产生菌的筛选及菌株特性的研究

从酸菜汁中分离出 2 4株乳酸杆菌 ,采用牛津杯定量扩散法筛选出 1株抑菌活性较高的乳酸杆菌 ,经鉴定为戊糖乳杆菌。排除乳酸对指示菌作用的干扰 ,该菌株的离心发酵液不仅对革兰氏阳性细菌有抑制作用 ,而且对大肠杆菌 (E .coli.) ,沙门氏菌 (Salmonellatyphi)等革兰氏阴性细菌也有一定的抑制作用     

New Fuchsin-Hematoxylin-Eosin; A Stain for Acid-Fast Bacilli and Surrounding Tissue

This is a staining technique for histopathologic evaluation of tissue reaction in the environs of acid-fast tubercle bacilli (avian and bovine) in sections. Fresh tissue is fixed in 10% neutral formalin and processed in the usual manner for embedding in paraffin. Sections are cut approximately 6 μ. thick, dewaxed, hydrated, and stained with Harris' hematoxylin. They are rinsed in tap water, differentiated in add alcohol, washed in tap water, given a distilled water rinse and stained at 20-30° C in a 1% solution of new fuchsin in 5% phenol. Each slide is then handled individually by placing it directly into a saturated aqueous solution of Li₂CO₃ and agitated gently for a few seconds. This is followed by differentiation with 5% glacial acetic acid in absolute or 95% ethyl alcohol until the color stops running. Two rinses in absolute or 95% ethyl alcohol follow. The sections are then counterstained in the color add of eosin Y prepared according to the method of Schleicher (Stain Techn., 28, 119-23, 1953) and used as an 0.025% solution in absolute alcohol. Following passage through 2 changes of absolute alcohol, the sections are cleared in xylene, then mounted in Permount or similar synthetic resin. The add-fast barilli are emphasized by their bright retractile red color within a contrasting background of hematoxylin and eosin.     

New Fuchsin-Hematoxylin-Eosin; A Stain for Acid-Fast Bacilli and Surrounding Tissue

This is a staining technique for histopathologic evaluation of tissue reaction in the environs of acid-fast tubercle bacilli (avian and bovine) in sections. Fresh tissue is fixed in 10% neutral formalin and processed in the usual manner for embedding in paraffin. Sections are cut approximately 6 μ. thick, dewaxed, hydrated, and stained with Harris' hematoxylin. They are rinsed in tap water, differentiated in add alcohol, washed in tap water, given a distilled water rinse and stained at 20-30° C in a 1% solution of new fuchsin in 5% phenol. Each slide is then handled individually by placing it directly into a saturated aqueous solution of Li₂CO₃ and agitated gently for a few seconds. This is followed by differentiation with 5% glacial acetic acid in absolute or 95% ethyl alcohol until the color stops running. Two rinses in absolute or 95% ethyl alcohol follow. The sections are then counterstained in the color add of eosin Y prepared according to the method of Schleicher (Stain Techn., 28, 119-23, 1953) and used as an 0.025% solution in absolute alcohol. Following passage through 2 changes of absolute alcohol, the sections are cleared in xylene, then mounted in Permount or similar synthetic resin. The add-fast barilli are emphasized by their bright retractile red color within a contrasting background of hematoxylin and eosin.     

A Method to Stain Decalcified Bone without Loss of Structural Detail

A modification of Bodian's protargol-S technique is done on 7 μm sections of decalcified bone. Light microscopic results are greatly improved when compared to either ground bone sections or decalcified bone stained routinely with hematoxylin and eosin.     

Quad-Type Stain for the Simultaneous Demonstration of Intracellular and Extracellular Tissue Components

This technic for the simultaneous demonstration of several different tissue components works equally well on invertebrate and vertebrate tissue if they have been treated with nonchromate fixatives Sections 4-7 μ thick are stained 30 min in 1% Alcian blue, then treated with alkaline alcohol for 2 hr. They are stained in Verhoeff's hematoxylin for 4-6 hr, and rinsed in alcohol; stained in woodstain scarlet-acid fuchsin for 3 min, decolorized in 5% phosphotungstic acid for 20 min and finally stained 5-8 min in alcoholic saffron. Collagen and bone are stained yellow; elastin, myelin and nucleic acids, purple to black; muscle, chitin, cytoplasm, fibrinoid and acid secretion, bright red to lavender; ground substances and mucus, blue-green. Fibrous connective tissue, cartilage, bone and glandular epithelia are exceptionally well demonstrated by this method. Slides stained in this manner are well suited for color photomicrography and as demonstrations in the classroom.     

Effectiveness of SYTOX Green Stain for Bacterial Viability Assessment

The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations of Escherichia coli and Salmonella typhimurium. This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes. Its application to natural samples should be considered with caution.     

Lycramine Brilliant Blue Jl: A New Stain for Human Megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte ctyoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.