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A satisfactory di-ammine-silver hydroxide solution may be repeatedly and consistently prepared by adding 9 or 10 volumes of 10% silver nitrate solution to 1 volume of 28% ammonia water, running in the first 6 or 7 volumes rapidly and proceeding cautiously from then on, shaking until clear after each addition, until a faint permanent turbidity is reached.The essential nature of Gomori's iron alum treatment and of Wilder's uranyl nitrate step following the Weigert permanganate-oxalic-acid sequence appears to be an oxidation, since the same results may be achieved with chromic acid, hydrogen peroxide, sodium iodate and elemental iodine, and since this step is better omitted on previously chromated material.

(1946). A Gold Chloride Method for Motor End Plates. Stain Technology: Vol. 21, No. 1, pp. 23-25.

A method nearly identical with that used by Rafalko on small amoebae, oocyte prophases of Habrobracon and several yeasts, has been found confirmatory to his results when applied to mammalian testicular tissue. The method is described, additional preparational notes are given, and several questions raised on possible improvement of the technic.

Tannin-iron Iii, as a technic, differs very little from Salazar's tannin-iron I, and must be considered as a new modification of the latter1. But the results obtained are very different and its use in plant cytology will be found very advantageous due to its specificity for certain cellular organs.

Many of the recently devised plasticizers and resins can be utilized to advantage in cytological technics. Some of them have solubilities which enable us to incorporate them in such fixing and staining solutions as aceto-carmine and propiorric-carmine. They are non-volatile, do not alter the fixation images of the fluids with which they are mixed, and serve as mounting media as the volatile components evaporate. Thus it is possible to make a permanent slide in a single operation. These newer compounds are better adapted for this technic than are the natural balsams which have been used previously, as their greater tolerance for water provides a much greater margin of safety. Procedures are described for the utilization of (1) Rezyl 7020, a water-soluble resin (now, unfortunately, not available), which dries to form a water insoluble film, (2) Amberol 750 and (3) Bakelite BR-7160, two alcohol soluble resins, more miscible in solutions containing water than are the natural balsams. Formaldehyde can be inclu...

Three different bacterial stains were prepared using ethyl, isopropyl and methyl alcohols as solvents for the dry stain. The stains thus prepared were tried against various organisms and their staining qualities noted. Stains prepared with methyl alcohol were comparable, in ease of preparation and staining quality, with those prepared using ethyl alcohol. It was concluded that stains prepared with methyl alcohol, instead of ethyl, would be entirely satisfactory for routine procedures. This confirms the findings of Conn and Darrow.

Three fixing solutions causing least distortion and bright staining of plant tissues are named. Glycerin dehydration causes less distortion than a series of alcohol concentrations; 95% alcohol removes some of the glycerin, sets the protoplasm and improves the staining. Absolute alcohol causes distortion and should be avoided. Pure chloroform, as a paraffin solvent, is followed by brighter staining but more distortion than are the butyl alcohols. A schedule resulting in minimum distortion is given. The results are shown in photomicrographs. Brightest staining follows the use of C. P. iron alum and hematoxylin. The use of a paper cup for very gradual change from one liquid to another and as a labor saver is described.

The influence of the commonly used tissue fixing reagents, individually and in various combinations, on subsequent staining by protargol was studied. The reagents used were formalin, formamide, picric acid, acetic acid, paranitrophenol, pyridine and chloral hydrate. Parraffin sections from intestine and peripheral nerve of cat, dog, monkey and rat were stained with protargol after fixation in various experimental mixtures of the fixing reagents. Satisfactory nerve stains of intestine were not obtained with regularity after any one fixing and staining procedure. (Good fixation and staining appeared to be influenced by properties inherent in the tissue itself and showed marked variations from animal to animal even in the same species.)Stains of nerve fibers in peripheral nerve trunks were much more easily obtained than in the intestine where good stains were sporadic and unpredictable. The use of a mixture of 0.5% protargol and 0.1% fast green FCF, is proposed as a silver-dye staining medium.

The methods commonly used for staining developing bone (Lundvall, 1905; Spalteholz, 1914; Batson, 1921; Dawson, 1926; Richmond and Bennett, 1938; Cumley, Crow, and Griffin, 1939; Williams, 1941), have involved the use of 1% or 2% Aqueous KOH without employing any bleaching agent. The introduction of hydrogen peroxide into the technic proves to be a distinct advantage, and in the concentration here proposed, does not result in the production of fine bubbles. The maceration process can be hastened through the use of an increased concentration of potash in the absence of a fixative, although this in itself is one of the hazards of the technic. However, if the preparations are carefully watched, this difficulty may be largely avoided. The method works admirably and gives a very clear muscle tissue so that the skeletal elements are very sharply outlined. The proposed method (arranged in the form adopted in Staining Procedures) is herewith described.

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 r...

To apply the bromodeoxyuridine (BrdU) labeling method using a monoclonal antibody to the study of cell proliferation in the mouse uterus, methods of fixation and embedding of tissues and of immunofluorescent staining were compared in terms of the rate of detection of labeled cells and specificity and stability of fluorescence obtained. BrdU was administered intravenously 2 hr before death and uterine blocks were embedded in polyester wax and Technovit resin after fixation in formalin and periodate-lysine-paraformaldehyde, respectively. The indirect method with anti-BrdU and fluorescein isothiocyanate (FITC) conjugated antimouse IgG antisera and the direct method with FITC conjugated anti-BrdU antibody were applied to both wax- and resin-embedded sections. Labeled and total cells were counted in luminal and glandular epithelia and stomata adjoining them. Counterstaining with hematoxylin for counting total cells produced intense fluorescence over the whole of resin sections and made counting of labeled cells impossible. On wax sections, on the other hand, the results were satisfactory, although the number of labeled cells detected was decreased slightly. In wax sections fluorescence due to nuclear incorporation of BrdU in the indirect method could be easily distinguished from the cytoplasmic or extracellular emission seen in some cells by its location and characteristic color. In resin sections, however, more careful observation was needed since the second antibody used in the indirect method cross-reacted with IgG in eosinophils and produced cytoplasmic fluorescence of the same color. By the indirect method greater numbers of labeled cells were detected in wax sections than in resin sections. The difference was distinct in tissues with extensive cell proliferation. By the direct method the fluorescence obtained was weaker and apt to fade more quickly than that obtained by the indirect method; use of the direct method reduced the number of labeled cells detected in both wax- and resin-embedded sections.

A combined method for clearing soft tissues, staining cartilage and bone, and injecting the vascular system of small mammals was developed using Mus musculus (house mouse). Mammalian muscle tissue remains milky or even opaque after "clearing" by previous techniques due to the relatively high content of intramuscular fat. A method employing chloroform-ethanol successfully renders soft tissues of mammalian specimens translucent without damaging or bleeding color from the latex injected in the circulatory system. Resulting specimens yield an excellent view of the skeletal system and the injected vascular system without obstruction by opaque tissues or disruption by physical removal of connective tissue.

The classic Mallory-Cason staining procedure has been modified for application to sections "on tape" obtained from large deep frozen tissue specimens. These 20 microns 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.

Traditionally, blood and bone marrow cells have been identified based on their characteristic shapes and colors when stained with one of several panoptic stains including Wright's or Giemsa's. As questions arose regarding the origin of normal and leukemic cells, cytochemical stains were developed. These stains help identify cells on the basis of a distinctive metabolite or enzyme. As part of an ongoing tradition in which textile dyes are used for biological staining, several new stains have been applied to hematologic staining. These include C.I. basic blue 41, basic blue 141, basic blue 93, and an asymmetrical polymethine dye. As additional cell-selective stains are developed, we can anticipate further improvements in our ability to identify normal and malignant hematopoietic cells.

By treating ultrathin sections with H2O2 prior to normal uranyl acetate and lead citrate staining, a strong increase in contrast of cutinized and suberized plant cell walls can be achieved.

The fluorescent DNA probes DAPI and Hoechst 33258 produce superior images to the traditional acetocarmine stain of the small chromosomes of the woody shrub Thryptomene calycina at all stages of microsporocyte meiosis and microspore mitosis. Hoechst 33258 was slightly superior to DAPI because of reduced background fluorescence. Binding with the DNA-specific probes required a fixative containing chloroform to remove autofluorescent materials, a pretreatment with acetic acid and a pH of least 6 during treatment. The nucleoli did not fluoresce after treatment with DAPI or Hoechst 33258. Superior resolution of chromosomes after treatment with the fluorochromes enabled easy determination of the haploid number at metaphase I, metaphase II and at metaphase of the microspore mitosis.

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.

A technique for demonstrating cement lines in thin, undecalcified, transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.

The ontogeny of early microspore-derived embryo development was followed using three stains. The stain 3,3'-diethyloxadicarbocyanine iodide, which previously had been reported to be specific for mitochondria, was observed also to demonstrate the exine of developing microspores of Brassica napus. It provided high contrast when used in combination with Tinapol 5 BM, a stain for cellulosic cell walls, and aided identification of microspores with embryogenic potential. Hoechst 33342, a nuclear stain, alone or in combination with either or both of the other stains, could be used to highlight the nuclear developmental stage of the microspores. This paper describes procedures using these materials for the specific staining of exine, cell wall/intine and nucleus, thereby permitting their fate to be followed during the early phases of microspore-derived embryo development.

This report investigated the quantitative distribution of radio labelled compounds in the central nervous system of the rate using autoradiographic techniques. To complement our autoradiographic images, we searched for a suitable fiber stain delineating myelinated structures using fresh frozen tissue. Surprisingly, the majority of staining procedures were exclusive to fixed tissues. These techniques applied to fresh frozen brain tissue produced poor differentiation of myelinated and nonmyelinated areas and were generally unsatisfactory. Therefore, we preformed a series of studies using lipid soluble dyes such as oil red O, Luxol fast blue, hematoxylin and Nile blue in an effort to enhance differentiation of myelin. We found Sudan black B to be superior to enhance differentiation of myelin. We found Sudan black B to be superior for our purpose.

Gastrointestinal mucus occurs partly as a stable, translucent water-insoluble gel adherent to the mucosal surface, and partly in a water soluble form in the lumen (Allen and Carroll 1985). The adherent pre-epithelial mucus gel (PMG) has been shown in unfixed sections of rat stomach as a continuous layer of median thickness 80 μm (Kerss et al. 1982), and 145 μm (Sandzen et al. 1988), respectively.

p-Phenylenediamine (pPD) can be used en bloc to preserve and differentiate cell lipids in aldehyde-fixed peanut plant tissues treated with osmium tetroxide during dehydration in 70% ethanol. Semithin plastic sections for light microscopy need no further staining and can be mounted in Histoclad after drying on a slide. Brown staining above background differentiates lipid-containing structures. Nonspecific staining can be distinguished in control preparations extracted en bloc with lipid solvents.

The histochemical demonstration of quantitative differences in myofibrillar ATPase activity at the selective pH optima of the various types of human skeletal muscle fibers is the most widely used technique for their differentiation. The basis of the reaction is the deposition of insoluble salts of inorganic phosphate cleaved from ATP by myofibrillar ATPase(s) followed by substitution of the phosphates with less soluble chromogenic salts. Doriguzzi and associates reported using metachromatic dyes to demonstrate quantitative differences in phosphate deposition among different fiber types. Following routine ATPase histochemistry and staining with either azure A or toluidine blue, fibers with low ATPase activity (and low phosphate content) were stained metachromatically while fibers with high ATPase activity (and high phosphate content) were orthochromatic with the intensity of color proportional to the content of insoluble phosphate. The metachromasia was readily lost after immoderate washing in aqueous solutions or routine dehydration in ethanol, with consequent diminished fiber type distinction. A critical modification of this technique is reported in which incubation of frozen sections of human skeletal muscle in ATP-containing medium is carried out at room temperature (22-24 C), rather than the usual 37 C, followed by a revised washing and dehydration protocol. With these modifications, the four human skeletal muscle fiber types (types I, IIA, IIB, and IIC) can be identified rapidly and reliably in single sections, obviating the need for examination of serial sections. The tinctorial differentiation allows fiber type identification even in black and white photographs.

Development of a resin-dissolving solution for use at low alkali concentrations is described. Crown ether dissolved in dimethyl-sulfoxide produces a superbasic alkoxide anion. A five minute treatment resulted in complete resin removal from kidney biopsy specimens embedded in Epon 812. Specimens were well stained by Loeffler's methylene blue. Periodic acid-methenamine silver and Giemsa stains yielded good results. Application of PAS reaction and subsequent hematoxylin counterstaining was practicable for diagnosis.

A trichrome staining technique using safranin-indigo-picrocarmine (SIPC) can be used to distinguish the various stages of the cell cycle in onion root tip. When the tissue was fixed first in formalin followed by picric acid and stained in SIPC, a clear differentiation of interphase nuclei into four color classes, viz., green, orange, red and brown can be recorded. Replacing crystal violet for safranin produces a similar pattern of differentiation of interphase nuclei into green, light blue, blue and deep blue. Autoradiographic study using 3H-thymidine as a DNA precursor demonstrates the reliability of the SIPC staining technique. All the orange and red nuclei are found to be labelled and therefore are in S phase of the cell cycle. Almost all the green nuclei are unlabelled and may be assigned to G1. The larger brown nuclei which are mostly unlabelled can be considered in G2 phase.

Standardized specimens with reproducible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied +/- 5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as +/- 5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.

The effect was examined of the chemical decomposition of the potassium stain sodium hexanitrocobaltate (III) (SHC), on its ability to produce stain granules of consistent size that could be used to estimate the K+ contents of stomatal guard cells. Stomata in detached epidermis from leaves of Vicia faba (fava bean) were stimulated to accumulate K+ by treating them with fusicoccin. Stomatal apertures and the fraction of guard cell area covered by K+ precipitate granules (K+ score) were measured by digitizing photographic enlargements, and K+ scores were correlated with the age of stain that had been stored either in open or closed containers. The ability of stain aged in open containers to produce consistent fractional cell coverage was compared to 1) the ability of identically treated stain to precipitate K+ from solutions of KCI, and to 2) the kinetics of decomposition of SHC. It was found that the fractional coverage of guard cells of stomata opened to the same apertures decreased with a first order rate constant of 2.3 x 10(-5)/sec. The mass of precipitate formed by treatment of KCl solutions was unchanged for 2 hr after initial preparation of the SHC, and decreased thereafter with a first order rate constant of 1.0 x 10(-5)/sec. When stored in tightly sealed containers, nearly 100 hr were required for an occasionally opened bottle of SHC to decay to the same efficacy as a solution left open to the air for 8 hr.

The application of Miller's dilute elastic stain followed sequentially by Gill's III hematoxylin and a fast green counterstain produced a reliable and consistent method for differentially staining elastic fibers, nuclei, muscle and collagen in glycol methacrylate tissue sections. Evaluation of different methods of fixation and conditions of staining on animal tissue sections showed that elastic fibers in both perfusion and immersion fixed tissues can be intensely stained. The stability of Miller's elastic stain offers the potential of a commercially available histological stain reagent for coarse and fine elastic fibers in glycol methacrylate tissue sections.

An improved method for preparing and staining ground tissue-implant sections for light microscopy is presented. Undecalcified tissue blocks with titanium implants were dehydrated in an ascending series of ethanol and stained in toto with basic fuchsin. Specimens were infiltrated and embedded in methyl methacrylate and sections were prepared using a cutting-grinding-system. The polished surface was counterstained with light green or anilin blue. Light polymerizing resin was used as slide mounting medium and for mounting the coverglass. The sections obtained were 10-15 microns thick with tissue architecture which clearly differentiated structures at the tissue-implant interface. The method was very useful for computer assisted morphometric analysis.

An improved routine method for embedding tissue, especially hard tissue, in polymethyl methacrylate (pMMA) is described. The improvements were: the final dehydration step before MMA infiltration was performed with methanol in a Soxhlet apparatus; the stabilizer hydroquinone was not extracted from the monomer (MMA), and more important, the commonly used polymerization initiator, benzoyl peroxide (bpo), was replaced by the initiator, bis (4-tert-butylcyclohexyl)peroxydicarbonate (bbpd). Bbpd is preferred to bpo because it is not explosive, far less is needed and it has a suitable half life. Moreover, bbpd, as obtained from the manufacturer, needs no further purification, in contrast to bpo. Temperatures during bbpd initiated polymerization did not exceed 48 C. In bbpd initiated pMMA, bubbles were almost never generated.

Normal formalin-fixed gelatin-embedded cerebral hemispheres were serially sliced and the 25 to 30 slices form each hemisphere were batch stained by a modification of Mulligan's method. following washing in water the slices were immersed in Mulligan's acid/copper sulfate/phenol solution for 20 minutes at room temperature, treated with a xylene/Polyclens mixture for 20 seconds and immediately transferred to a 2% sodium hydroxide solution for 10 seconds. Final staining was by immersion in 2% potassium ferrocyanide which was followed by washing in tap water. The grey matter was stained a brick red color while the whiteness of the white matter was accentuated. Following staining the slices were stored between sheets of black paper in 2% aqueous formalin prior to measurement of the respective areas of grey and white matter using a Quantimet 720 image analyzing computer. The method is rapid and color stable, and reduces the risk of exposure to toxic fumes by eliminating the need for hot phenol solutions. This technique is also suitable for the macroscopic demonstration and quantitation of demyelinating conditions in the brain.

A technique without negative effects on tissue preservation that allows precise identification and subsequent removal of central nervous system nuclei for ultrastructural analysis is described. The procedure uses 200 microns thick Vibratome-cut sections of glutaraldehyde fixed brains. These sections are stained for 25 seconds with a methylene blue solution and stored for 4 hours in 0.2 M pH 7.4 phosphate buffer in 4% sucrose for optimal visualization at the light microscopic level. The stock solution of 1 g methylene blue and 1 g sodium borate in 100 ml of distilled water, is filtered through a Millipore filter and diluted 5:95 with distilled water immediately prior to use. Regions of specific interest are then processed for electron microscopy.

Methyl green-pyronin is a notoriously difficult stain to reproduce. Although very useful in detecting cells containing substantial amounts of RNA, it is of limited use in broader problems of cell identification. By careful standardization of the proportions of methyl green to pyronin and combination of these stains with hematoxylin to enhance nuclear contrast and with orange G to improve connective tissue staining, it was possible to produce a consistently reliable staining preparation in which it is possible to identify all the component cells of a mixed inflammatory infiltrate in routine paraffin sections.

One of the monoazo dyes reported by us in an earlier study (Lillie et at. 1972), amidol brown NAP, yielded an oxidation product, amidol black I, which was an excellent elastin stain. The present study revises the synthesis of amidol black I to a more convenient form giving much larger yields. Briefly, diazotized 4-nitro-2-aminophenol is azo-coupled into 2, 4-diaminophenol (amidol) and the product is oxidized to a quinoneimine with nitric. The product stains elastin black from an acid alcohol bath according to a Taenzer-Unna (1890) orcein type technic. The stain may be combined with a Van Gibson collagen stain or with an oil red O fat stain. The synthesis is presented in sufficient detail to permit its repetition in a hospital pathology laboratory.

For progressive staining 1 g mordant blue 3, 0.5 g iron a alum and 10 ml hydrochloric acid are combined to make 1 liter with distlled water. Paraffin sections are stained 5 minutes blued in 0.5% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely results very resemble a good hematoxylin and eosin.

Plastic embedding preserves tissue structure much more faithfully than does paraffin. Acrylic polymerization is innocuous to dye-binding groups in sections. The water solubility of glycol methacrylate monomer and the hydrophilic properties of the polymer allow for convenience in dehydration and for versatility in staining sections. Five years of experience with glycol methacrylate (GMA) embedding for light microscopy is summarized. Methods for purifying GMA monomer are cited. Procedures for fixing, dehydrating, embedding, polymerizing, sectioning and staining, using GMA, are explained. A method is provided for making glass knives long enough to cut large blocks. Simple, reliable, quick staining methods are outlined. When compared with paraffin, GMA offers opportunities for simpler, quicker procedures and yields sections of superior quality, greater information content, and less distortion.

The present work introduces a method for the localization in situ of the abdominal paraganglia. After treating retroperitoneal tissue blocks with a near-neutral glutaraldehyde/ potassium dichromate solution following routine glutaraldehyde perfusion, intra- and extra-adrenal chromaffin tissues develop a pronounced brown color from the interaction of glutaraldehyde/potassium dichromate with amines. In this manner, visualization of the abdominal extra-adrenal chromaffin organs is enhanced at the same time that cellular ultrastructurr is preserved. Subsequent examination of the dichromate-reacted tissues with the electron microscope confirms that they represent the amine-rich paraganglia. This method offers an effective alternative to extensive sampling of plastic-embedded blocks for localizing peripheral chromaffin tissue and has been used to define the exact distribution of abdominal paraganglia in the rabbit.

The effect of pH on silver staining of the nucleolus organizer regions (NORs) of human chromosomes has been investigated between pH 6.5 and 12.0. Nonvolatile mixtures of ethanolamine and ethanolammonium nitrate replaced the ammonia of standard procedures. The optimal NOR staining obtained at pH 3.5 by the silver staining procedure of Howell and Black served as a standard; this procedure stained all ten NORs in 90% of mitoses. Similar NOR staining was found in 75% of mitoses stained at pH 11.7 or 11.8, but only in 10-15% of mitoses stained between pH 11.6 and 10.0. Between pH 10.0 and 9.0 NOR staining was incomplete, and between pH 8.5 and 6.5 there was no NOR staining.

Double staining techniques were devised to study Escherichia coli attachment to mucopolysaccharide-coated urinary tract epithelial cells. In addition, vital stains were used to distinguish between viable and nonviable epithelial cells. Alcian blue was used to confirm the presence of glycosaminoglycans and periodic acid Schiff was used to distinguish a further group of polysaccharides, proteoglycans, neutral mucosubstances and glycolipids. The staining methods enabled investigations to be carried out concerning the possible importance of mucopolysaccharides in the attachment of bacteria to mucosal surfaces. Staining techniques were also used to investigate whether the presence of a mucopolysaccharide coat is related to epithelial cell viability. The combinations of vital and mucopolysaccharide stains were found to give reproducible results when cell preparations were evaluated by three individuals. Both in vivo and in vitro certain populations of epithelial cells have been found with a large number of bacteria preferentially attached. The double staining techniques described here may help to reveal the nature of these target cells.

Staining of oxytalan fibers in marsupial, eutherian and human periodontal ligaments was surveyed with 65 different dyes. Using the criteria of response to preoxidation, distribution, and morphologic appearance, 27 dye preparations in addition to the Gomori aldehyde-fuchsin, Taenzer-Unna orcein, and Weigert resorcin-fuchsin techniques displayed oxytalan fibers. With two exceptions all dyes were cationic and reacted with varying degrees of excellence with different animals. Most dyes produced their best staining results as concentrated solutions in 3% acetic acid, suggesting involvement of oxidatively engendered polyanions predominantly associated with an acid mucopolysaccharide component of the oxytalan fiber. The significance of carboxyl and sulfur-containing groups should not be overlooked in further studies aiming to elucidate oxytalan fiber chemistry and microstructure. This study supported the view that oxytalan fibers belong to the family of elastic tissues and represent a biologically important system within the periodontal ligament.

After reconsidering the physicochemical mechanisms involved in the so-called degeneration methods for the demonstration of axons and nerve terminals, the method of Eager was fundamentally modified in order to stabilize the staining process. This resulted in a simple and reliable method which stains degenerating terminals and lysosomes with a high degree of selectivity and sensitivity. Frozen sections 30 to 50 micrometers thick are prepared from material fixed with formaldehyde by cardiac perfusion. The staining procedure consists of 5 steps: 1) alkaline pretreatment (pH 13), 2) silver impregnation, 3) washing, 4) development at pH 5.0-5.5 monitored by an indicator, and 5) washing in acetic acid. Possible faults can be easily detected by their specific effects on the staining results. Primary submicroscopic silver precipitates are localized selectively in the osmiophilic parts of lysosomes and those degenerating presynaptic elements that are surrounded by glial processes. In degenerating axons, precipitates originating from mitochondria can usually be distinguished from terminal degeneration by their different size, shape, or characteristic arrangement. Nonspecific staining is restricted to glial fibrils, erythrocytes, and single cell nuclei. Dark field illumination can be applied routinely and television image analysis can be used for quantitative evaluation because of low background staining.

Bodian and Nissl procedures were combined to stain dissociated mouse spinal cord cells cultured on coverslips. The Bodian technique stains fine neuronal processes in great detail as well as an intracellular fibrillar network concentrated around the nucleus and in proximal neurites. The Nissl stain clearly delimits neuronal cytoplasm in somata and in large dendrites. A combination of these techniques allows the simultaneous depiction of neuronal perikarya and all afferent and efferent processes. Costaining with little background staining by either procedure suggests high specificity for neurons. This procedure could be exploited for routine network analysis of cultured neurons.

A number of techniques are presented which permit precise selection and efficient preparation of individual cultured cells for electron microscopy. Techniques described include marking of the living and embedded cells, drilling and mounting cores of embedded material, and improved technique for marking of the selected area on the block face before trimming.

Nondissected ovaries of tuber-bearing Solanum sp. were stained with Mayer's hemalum, a positive stain for chromatin and nucleoli, and then optically cleared with methyl salicylate, a clearing agent. Clarity, resolution and contrast within the ovules dissected from ovaries were comparable to those of sectioned, paraffin embedded ovaries. Contrast within ovules greatly exceeded that of unstained and nonspecifically stained clearings, and eliminated the need of special optics, i.e., Nomarski interference-contrast optics, for optimal viewing and photography. Much less time and labor were required than for embedded specimens. Usefulness of the technique for cytogenetic and cytological research was verified by analyzing meiosis and other features of megasporogenesis and megagametogenesis in normal, and in two meiotic mutants, of Solanum. The results illustrate the usefulness of combined Mayer's hemalum staining and methyl salicylate clearing, and suggest additional stain-clearing agent combinations have potential for cytological and cytogenetic research. Preliminary results with other species suggest the technique may also be useful for classroom instruction.

A photo-oxidized solution of 3,3'-diaminobenzidine (DAB) is used to stain xylem parenchyma mitochondria in specimens prepared from lupin hypocotyls fixed with glutaraldehyde and osmium tetroxide and embedded in Epon. No other subcellular components, including plastids, nuclei, vacuoles or cell walls were stained when xylem parenchyma cells were exposed to this reagent for 1 hr. This reaction was stable for 20 min at 80 C, inhibited by KCN, and insensible to 3-amino-1,2,4-triazole. The outstanding sensitivity of this reaction to inhibition probes suggests that this stain is analogous to the previously described DAB/cytochrome c/cytochrome oxidase reaction in plant mitochondria, although the incubation of lupin sections with freshly prepared DAB solution (free of auto-oxidized DAB) did not result in staining. These results draw attention to the unreliability of DAB oxidation for demonstrating electron transport in plant mitochondria. However, we do recommend photo-oxidized DAB as a direct ultrastructural stain for plant mitochondria without reference to its oxidative capacity.