Fluorochrome Staining of Multilamellar Liposomes

Multilamellar liposomes can be stained with such fluorochromes as acridine orange, eosin Y, neutral red, and thiazine red. The liposomes are brought into a 1% solution of the fluorochrome; 5-10 minutes later they are centrifuged and washed by resuspending in water or phosphate buffered saline three times. The last pellet is resuspended and a drop studied with the fluorescence microscope (1000 × magnification). The fluorochrome is seen to be accumulated in the liposomal membranes.

Acridine orange could also be trapped in the aqueous compartments of the liposomes but the trapped fluorochrome was gradually lost from the liposomes. Part of the fluorochrome, however, remained associated with the liposomal membranes for a long time.

Additional experiments justify the conclusion that an equilibrium is maintained between fluorochromes in the aqueous and lipid phases.     

Nucleoli Visualized by Silver Staining Combined with A New Rna-Specific Fluorochrome

A technique for distinguishing nucleolar components by light microscopy is described. Silver staining is used for localizing nucleolar-organizing regions. A new RNA-specific fluorochrome, 4'-6-bis(2'-imidazoliny1-4'-5'H)-2-phenyl-4'-phenoxindole, visualizes the RNA-containing “partes-fibrillares et granulares” of the nucleolus. Both staining procedures can be combined to analyze nucleolar structure and function by light microscopy.     

Structures Suggesting Cell-Wall-Deficient Forms Detected in Circulating Erythrocytes by Fluorochrome Staining

Cell-wall-deficient (CWD) forms of bacteria are associated with certain cases of idiopathic septicemia. In this preliminary study of blood examined immediately after venipuncture, structures with a morphology characteristic of CWD forms were seen parasitizing the erythrocytes. These inclusions were usually circumferential, but in some cases they protruded from the red cells. The CWD forms were detected by staining with Gould's rhodamine-labeled muramidase, which reacted similarly to acridine orange but with greater specificity. A blocking test, employing unlabeled muramidase, indicated the specificity of the reaction between muramidase and the microbial substrate. Reaction of the forms with muramidase indicates their bacterial, rather than mycoplasmal, nature. Thus in vivo CWD forms have a detectable component of muramic acid, at least in certain cases. Sixty-eight individuals with a diagnosis of fever of unknown origin were tested, with 51 nondebilitated individuals serving as controls. More intraerythrocytic forms reacting with muramidase were found in the patients than in the controls. Nearly 40% of the cases had a relatively high incidence of erythrocyte parasitism. In some instances when freshly drawn blood was examined, the structures, which appear to be microbial, extended in rhizoid filaments from the erythrocytes.     

Karyotyping Human Chromosomes by Optical and X-Ray Ptychography Methods

Sorting and identifying chromosomes, a process known as karyotyping, is widely used to detect changes in chromosome shapes and gene positions. In a karyotype the chromosomes are identified by their size and therefore this process can be performed by measuring macroscopic structural variables. Chromosomes contain a specific number of basepairs that linearly correlate with their size; therefore, it is possible to perform a karyotype on chromosomes using their mass as an identifying factor. Here, we obtain the first images, to our knowledge, of chromosomes using the novel imaging method of ptychography. We can use the images to measure the mass of chromosomes and perform a partial karyotype from the results. We also obtain high spatial resolution using this technique with synchrotron source x-rays.     

黄颡鱼染色体的C─带、Ag—NORs、荧光带和复制带显带的研究

采用Giemas染色、C─带、Ag—NORs、荧光染色和复制带显带的技术对黄颡鱼染色体进行了研究。结果表明,黄颡鱼只有部分的染色体呈现阳性C─带,可分为三类,其中NORs区是染色最深、染色面积最大的区域,为深染居间C─带。其Ag－NORs位于m5q末端。CMA3染色显示NORs区呈现出明亮的荧光。中复制染色体上着丝粒区、端粒区和居间区浅染。发现核仁缢痕、深染居间C─带、Ag—NORs、CMA3明亮区和中复制带浅染NORs区位置基本一致,C─带阳性区和中复制带浅染区具有对应性。     

黄颡鱼染色体的C-带、Ag-NORs、荧光带和复制带显带的研究

采用Giemas染色、C-带、Ag-NORs、荧光染色和复制带显带的技术对黄颡鱼染色体进行了研究。结果表明, 黄颡鱼只有部分的染色体呈现阳性C-带, 可分为三类, 其中NORs区是染色最深、染色面积最大的区域, 为深染居间C-带。其Ag-NORs位于m     

黄颡鱼染色体的C-带、Ag-NORs、荧光带和复制带显带的研究

采用Giemas染色、C-带、Ag-NORs、荧光染色和复制带显带的技术对黄颡鱼染色体进行了研究。结果表明, 黄颡鱼只有部分的染色体呈现阳性C-带, 可分为三类, 其中NORs区是染色最深、染色面积最大的区域, 为深染居间C-带。其Ag-NORs位于m5q末端。CMA3染色显示 NORs区呈现出明亮的荧光。中复制染色体上着丝粒区、端粒区和居间区浅染。发现核仁缢痕、深染居间C-带、Ag-NORs、CMA3明亮区和中复制带浅染NORs区位置基本一致, C-带阳性区和中复制带浅染区具有对应性。 Abstract　Metaphase chromosomes of Pelteobagrus fulvidraco were analyzed by means of Giemsa staining, C-banding, Ag-NORs, fluorochrome staining and replication banding. Only parts of chromosomes showed C-band positive, the C-band heterochromatin was located in three regions. The NORs-bearing chromosomes had the largest and strongest C-bands. The long arms of chromosome pair 5 were the regions showing positive labeling with Ag-staining. Fluorochrome CMA3positively stained the NORs. In mid-period, negative replication bands were located in the centromere, the terminal and interstitial regions of the chromosomes. The distributions of secondary ?constri-ctions, positive C-bands, Ag-NORs, positive fluorescence bands and negative replication bands of mid-period were coincident.     

双参数人类染色体流式分析及分选

用ＦＡＣＳＶａｎｔａｇｅ型流式细胞分选仪对人二倍体成纤维细胞的单分散染色体悬液进行双参数、双激光染色体核型分析及分选。人类染色体可分出２１个集团,除９至１２号染色体外,其余均能被单独分离。经染色体特异性探针池ＦＩＳＨ鉴定,染色体分选纯度可达９０．５％。     

Selective Staining by the Fluorochrome 5,5'-Diphenyl-9-ethyl-oxacarbocyanine. II. Application to Paraffin Embedded Nervous Tissue

We present a simple and rapid method to label specific structures of neural tissue in paraffin sections. After incubation of deparaffinized sections in the cyanine dye 5,5'-diphenyl-9-ethyl-oxacarbocyanine (DEOC), three different washing and mounting procedures were performed. Incubation in DEOC followed by washes in ethanol and water and mounting in glycerol-gelatin resulted in selective labeling of myelin in the central and peripheral nervous systems. Weak labeling of myelin and axons and staining of nuclei of neurons was seen after incubation in DEOC followed by washes in ethanol and xylene, and mounting in Eukitt. Nuclear proteins, the endoplasmic reticulum and the Golgi apparatus were stained strongly and exclusively after incubation in DEOC, washes in water, ethanol and xylene, and mounting in Eukitt. Thus the absorption pattern of DEOC is changed significantly by solvents applied after the fluorochrome. In any case, fluorescence did not fade even after repeated and intense fluorescence microscopy over a period of 6 months.     

Molecular Karyotyping of Human Single Sperm by Array- Comparative Genomic Hybridization

No valid method is currently available to analyze the entire genome of sperm, including aneuploidies and structural chromosomal alterations. Here we describe the optimization and application of array-Comparative Genomic Hybridization (aCGH) on single human sperm. The aCGH procedure involves screening of the entire chromosome complement by DNA microarray allowing having a molecular karyotype, and it is currently used in research and in diagnostic clinical practice (prenatal diagnosis, pre-implantation genetic diagnosis), but it has never been applied on sperm. DNA from single human sperm isolated by micromanipulator was extracted, decondensed and amplified by whole-genome amplification (WGA) and then labeled, hybridized to BAC array, and scanned by microarray scanner. Application of this protocol to 129 single sperm from normozoospermic donors identified 7.8% of sperm with different genetic anomalies, including aneuploidies and gains and losses in different chromosomes (unbalanced sperm). On the contrary, of 130 single sperm from men affected by Hodgkin lymphoma at the end of three months of chemotherapy cycles 23.8% were unbalanced. Validation of the method also included analysis of 43 sperm from a man with a balanced translocation [46,XY,t(2;12)(p11.2;q24.31)], which showed gains and losses corresponding to the regions involved in the translocation in 18.6% of sperm and alterations in other chromosomes in 16.3% of sperm. Future application of this method might give important information on the biology and pathophysiology of spermatogenesis and sperm chromosome aberrations in normal subjects and in patients at higher risk of producing unbalanced sperm, such as infertile men, carriers of karyotype anomalies, men with advanced age, subjects treated with chemotherapy, and partners of couples with repeated miscarriage and repeated failure during assisted reproduction techniques.     

Selective Staining by the Fluorochrome, 5,5'-Diphenyl-9-ethyl-DiOC2(3). I. Physicochemical Studies of Dye-Dye and Dye-Tissue Interactions

The 5,5'-diphenyl-9-ethyl-oxacarbocyanine (5,5'-diphenyl-9-ethyl-DiOC₂(3); CD) has properties suitable for histological investigations including the spectral range for absorption and fluorescence emission, the values of the corresponding molar coefficients and fluorescence quantum yield. Furthermore, CD remains relatively unchanged over the entire pH range and interacts with protein β-sheets. The latter fact is detectable spectroscopically as a bathochromic shift. In water-containing media such as the histological stain and washes, CD exists as a monomer, a dimer and in two aggregated states. These differ in their binding affinity to tissue sections, in their solubility in water, alcohol and water/alcohol mixtures, and in their UV/VIS absorption and fluorescence emission. The ratio of the various CD states and the contrast of selectively stained tissue areas can be controlled via the staining conditions and the sequence of the washes. Furthermore, mounting in a xylene-based medium produces a solvatochromic spectral shift of the CD monomer, which leads to a marked elevation in phase contrast.     

Evaluation of Preparation, Staining and Microscopic Techniques for Counting Incremental Lines in Cementum of Human Teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.     

A Comparison of Standard Lipid Staining Techniques Used in Electron Microscopic Studies of Mammalian Tissues

Comparisons of several standard techniques for staining lipids in ultrastructural studies have been undertaken using the rat uterine epithelium as the experimental tissue. The best technique for clarity, retention of stain, and acceptability of cellular ultrastructure utilized p-phenylenediamine after primary fixation in glutaraldehyde and postfixation in osmium tetroxide. While osmium by itself stained only unsaturated lipids and p-phenylenediamine stained no lipids in spot tests, when acting together, the staining of unsaturated lipids was enhanced and some staining of saturated lipids was seen. Further, the marked extraction of stained lipids normally found during dehydration did not then occur.     

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.     

A Disrupting Factor in Silver Staining Techniques

Single-stage surface replicas of treated or fresh pollen grains can be made ready for the electron microscope in 1.5 hr. The microspores are discharged into a drop of 50% acetone on a 1 cm square of cleaved mica and air dried. Carbon is evaporated to a film thickness of 35 mμ during rotation of the mica support. The carbon film and microspores are parted from the mica with water and heated in 2-aminoethanol at 145-155 C for 10 min to 3 hr. The replicas are then washed 5 min or longer on water at 90 C and picked up on electron microscope grids. The resulting self-shadowed surface replica can be immediately observed by electron microscopy.     

Staining Secretory Capillaries of Exocrine Glands with Techniques for Specific Phosphatases

Intercellular secretory capillaries in parotid glands, eccrine sweat glands and intracellular secretory capillaries in parietal cells of gastric glands were demonstrated histo-chemically by the use of the Wachstein-Meisel adenosinetriphosphatase (ATPase) technique in the rabbit, rat and guinea pig. However, with the Wachstein-Meisel 5-nucleotidase technique, secretory capillaries were not stained. For parotid glands, optimal incubation in ATPase substrate mixture was: in rabbit, 15 min; in rat, 2.5 hr; and in guinea pig, 2 hr. For eccrine sweat glands, optimal incubation was 15 min in rabbit, 30 min in rat and 15 min in guinea pig. For parietal cells of gastric glands, optimal incubation was 3 hr for all three species. Secretory capillaries were best demonstrated in the parotid by using rabbit tissue; in eccrine sweat glands, with rat tissue, and in parietal cells, guinea pig tissue. Since ATPase activity in cell membranes of secretory cells may play a part in the mechanism of transport of secretory products from their place of formation in the acini to the excretory ducts, the Wachstein-Meisel ATPase technique can therefore be used successfully for staining secretory capillaries in many of the exocrine glands of laboratory mammals.     

Techniques for Preparing and Staining Phytophthora Oospores in Soybean Rootlets

Nylon mesh tissue carriers were constructed to hold soybean rootlets through fixing, dehydrating and embedding. Mesh pieces three centimeters square were doubled and sealed at each end by heat. Tissue samples were placed inside with an identifying piece of aluminum foil and the carrier sealed. Rootlets were fixed in Karpechenko's solution, dehydrated in an alcohol series and infiltrated with paraffin. They were embedded in paraffin after removal from the carrier, and sectioned on a microtome. Sections were mounted on glass slides and deparaffinized. A new stain was developed to differentiate oospores of Phytophthora megasperma var. sojae formed in these rootlets. The stain was prepared by dissolving 100 mg bromphenol blue in 50 ml of 95% ethanol and adding 3 g silver nitrate. Procedure: 5 sec in 95% ethanol, 30 min in silver stain, tap water rinse, 5 sec in 95% ethanol, 1 sec in saturated methylene blue in ethanol, immediate rinse in tap water, dehydration in absolute ethanol, rinse in tertiary butanol and xylene and mount. Previous clearing of the tissue was not required, and no air bubbles accumulated within the mesh carrier. This low cost, permeable carrier preserved the minute tissue specimens throughout processing, and the simple, progressive stain clearly differentiated oospores from surrounding tissue.