The “Dispersion Staining” Method for the Selective Coloration of Tissue

A method has been developed for the selective coloration of fixed tissue without the use of dyes. Microtome sections of formalin-fixed material are mounted under a cover glass in a mixture of two liquids such as diethylene glycol monobutyl ether with cin-namaldehyde and examined with the dark-field microscope. The refractive index of the liquid used for mounting must be of high dispersion and equal or close to the index of the specimen.

Tissue elements, dependent on their refractive index, whether slightly lower, the same as, or slightly above the mounting medium appear colored in shades of blue, red or yellow.

The optical principles involved in this optical dispersion method are similar to those involved in the production of colored light by the Christiansen filter.     

Plastic Embedding, Orientation in the Mold and Tape-Supported Sectioning of Sclerotized Insects and Other Hard Objects

Sections of large specimens such as whole honeybees or beetle adults embedded in plastic usually are difficult to cut with a constant thickness. The sections also compress and roll. Sections of even thickness have been obtained by using a mixture of methacrylates (ethyl, 1:butyl, 3) and by firmly supporting the block in the microtome with a special holder. Scotch tape #810 applied to the block before each section is cut eliminates section compression and rolling. The sections are attached to slides with 2% celloidin in an absolute alcohol-methyl benzoate mixture (5:5-7:3); and the tape is removed with heptane. Large sections can also be cut from blocks of styrene mixed with butyl methacrylate. The specimens are oriented in the monomer in gelatin capsules by directing them into the desired plane among the fibers of a wad of absorbent cotton previously placed in the bottom of the capsule. The cotton is sectioned with the specimen but its fibers do not interfere, and remain outside the tissue.     

A Hematoxylin and Eosin-Like Stain for Glycol Methacrylate Embedded Tissue Sections

A staining procedure is described for use with glycol methacrylate embedded tissue sections which does not stain the plastic embedment or remove the sections from the glass slides. The basic dye is celestine blue B. It is prepared by treating 1 g of the dye with 0.5 ml concentrated sulfuric acid. It is then dissolved with the following solution. Add 14 ml glycerine to 100 ml 2.5 percent ferric ammonium sulfate and warm the solution to 50 C. Finally adjust the pH to 0.8 to 0.9 The acid staining solution consists of 0.075 percent ponceau de xylidine and 0.025 percent acid fuchsin in 10 percent acetic acid. Slides containing the dried plastic sections are immersed in the celestine blue solution for five minutes and in the ponceau-fuchsin solution for ten minutes with an intervening water rinse. After a final wash, the sections are air dried and coverslipped. This staining procedure colors the tissues nearly the same as hematoxylin and eosin procedures.     

The “Dispersion Staining” Method for the Selective Coloration of Tissue

A method has been developed for the selective coloration of fixed tissue without the use of dyes. Microtome sections of formalin-fixed material are mounted under a cover glass in a mixture of two liquids such as diethylene glycol monobutyl ether with cin-namaldehyde and examined with the dark-field microscope. The refractive index of the liquid used for mounting must be of high dispersion and equal or close to the index of the specimen.

Tissue elements, dependent on their refractive index, whether slightly lower, the same as, or slightly above the mounting medium appear colored in shades of blue, red or yellow.

The optical principles involved in this optical dispersion method are similar to those involved in the production of colored light by the Christiansen filter.     

Oriented Embedding of Biological Materials and Accurate Localization for Ultrathin Sectioning

Gelatin capsules with rounded ends clipped off and open ends moistened, affixed to a glass slide and sealed with a 15% gelatin solution are used to embed blocks of tissue in plastic. The surface of the slide serves as an orientation plane for structures of the tissue. The plane end of capsules of polymerized plastic containing no tissue is used in embedding frozen tissue sections. The plastic-infiltrated section is flattened against the capsule end under the weight of a 3/4 inch square of plate glass so that larger sections may be cut and surveyed. Embedding cultured cell monolayers grown on coverslips is accomplished in a comparable manner, but the square of plate glass is not needed as a weight. Block-face localization methods depend on the type of material embedded. With blocks of tissue it is achieved by moistening the face with xylene to develop relief. Thin tissue sections are examined by transmitted light, while cell monolayers are stained on the capsule end with methylene blue.     

Combined Lectin Binding and PAS/Alcian Blue Staining in Glycol Methacrylate Sections

Evaluation of cryofixation and paraffin and glycol methacrylate embedding showed that lectin binding was essentially independent of the embedding medium. Fluorescence intensity increased in the following order: glycol methacrylate, paraffin and cryostat sections, The optical resolution increased in the reverse order. Semi-thin glycol methacrylate sections provided satisfactory fluorescence intensities and the best resolution of all embedding techniques applied. Furthermore the lectin treated sections can be stained further using routine histological or specific histochemical methods. The potassium hy-droxide/alcian blue/periodic acid-phenylhydra-zine-Schiff method was used successfully to demonstrate sulfated and nonsulfated sialomucins. Lectins combined with mucin histochemistry allowed visualization of specific sugar residues in the same glycol methacrylate plastic section.     

Ultra-Thin Sectioning for Electron Microscopy

A technic is described for obtaining thin sections of animal tissue suitable for electron microscopy. Fixation is accomplished by perfusion of the whole animal with neutral formalin or alcohol formalin followed by immersion of pieces to be examined in neutralized osmium tetroxide. The embedding medium is a mixture of equal parts of n-butyl and ethyl methacrylate polymerized by ultra-violet light. Sectioning is done by means of a glass knife on an International ultra-thin sectioning microtome set at 0.1 μ. The sections are floated on warm water to spread, then placed on Formvar-coated grids, dried, and put into toluene to dissolve the plastic. The technic produces routinely usable, thin sections that show a minimum of damage owing to fixation, embedding, and sectioning.     

Cement Line Staining in Undecalcified Thin Sections of Cortical Bone

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 μm, 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.     

Wrinkle-Free Plastic Sections for Light Microscopy

Plastic sections 0.5 to 2 μm thick are routinely used for light microscopy. Although plastic sections have several advantages over paraffin or celloidin sections, a problem that is often encountered with plastic sections is wrinkling (Fig. 1). Wrinkling occurs during staining when sections dried on glass slides are covered with stain and heated to hasten the penetration of the stain. Mounted sections heated on glass slides, but not stained, ordinarily lack wrinkles, even when examined with phase contrast optics. Similarly, mounted sections covered with stain, but not heated, lack wrinkles; unfortunately, such sections fail to stain adequately. Unmounted sections floated on heated drops of stain also lack wrinkles (Millonig 1980). Thus, it is clear that wrinkling occurs only when mounted sections are covered with stain and heated.     

A polycarbonate based surface plasmon resonance sensing cartridge for high sensitivity HBV loop-mediated isothermal amplification

In this study, we report a simple, low-cost surface plasmon resonance (SPR)-sensing cartridge based on a loop-mediated isothermal amplification (LAMP) method for the on-site detection of the hepatitis B virus (HBV). For LAMP detection, a SPR based LAMP sensing system (SPRLAMP) was constructed, including a novel SPRLAMP sensing cartridge integrating a polymethyl methacrylate (PMMA) micro-reactor with a polycarbonate (PC)-based prism coated with a 50 nm Au film. First, we found that the change of refractive index of the bulk solution was approximately 0.0011 refractive index (RI) units after LAMP reaction. The PC-based prism's linearity and thermal responses were compared to those of a traditional glass prism to show that a PC-based prism can be used for SPR measurement. Finally, the HBV template mixed in the 10 μl LAMP solution could be detected by SPRLAMP system in 17 min even at the detection-limited concentration of 2 fg/ml. We also analyzed the correlation coefficients between the initial concentrations of HBV DNA templates and the system response (ΔRU) at varying amplification times to establish an optimal amplification time endpoint of 25 min (R(2)=0.98). In conclusion, the LAMP reaction could be detected with the SPRLAMP sensing cartridge based on direct sensing of the bulk refractive index.     

Health risk assessment of occupational exposure to styrene depending on the type of industry: Data from the Workplace Environmental Monitoring Program in Korea

Data from the Workplace Environmental Monitoring Program was used to evaluate the concentrations and risk of occupational exposure to styrene in different industries to identify which industries should be prioritized for styrene exposure management. Risk assessments were conducted for the five industries with several workplaces that mostly use styrene: motor vehicle and motorcycle maintenance and repair services, other chemical product manufacturing, ship and boat building, basic chemical manufacturing, and plastic products manufacturing. The highest central tendency exposure was found in the plastic products manufacturing industry (10.14 mg/m³). In addition, the hazard quotient (HQ) for central tendency exposure exceeded 1 only in the plastic products manufacturing industry. Almost two-thirds (62.2%) of workplaces in the plastic products manufacturing industry have an HQ exceeding 1. We conclude that workers in the plastic products manufacturing industry are at the highest risk for styrene exposure, and those in motor vehicle and motorcycle maintenance and repair service and basic chemical manufacturing are at the lowest risk. These results show that styrene exposure could be most effectively managed by prioritizing control measures in the plastic products manufacturing industry.     

Serial Sections of Smears for Electron Microscopy

Ultrathin sections for electron microscopy may be prepared from smears or squashes embedded in methacrylate. The cover slip or glass slide with the attached fixed cellular material is passed through alcohols to methacrylate monomer and finally to monomer containing a catalyst. The portion of the smear to be sectioned is covered with a gelatin capsule containing partially polymerized methacrylate. When polymerization is completed at 47°C, the hardened block is separated from the cover slip and trimmed under the compound microscope so as to encompass the desired area. Photographs are made of the intact smear to afford a basis for identification of cellular materials in electron micrographs of the individual ultrathin sections.     

Cement line staining in undecalcified thin sections of cortical bone.

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.     

Cement line staining in undecalcified thin sections of cortical bone

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.     

Cytochemical Staining of Sections from Plastic-Embedded Flagellates

Dinoflagellate chromosomes in sections of plastic-embedded cells were stained without removing the plastic. Azur B and Feulgen procedures were used to localise DNA. Azur B was used with Araldite or methacrylate sections by staining in 0.2% stain in 0.05 M citrate buffer at pH 4 for 1 hr at 50 C followed by rinsing in tertiary butyl alcohol to differentiate the chromosomes. Feulgen stain was used with Araldite sections by hydrolyzing in 1 N HCl at 60 C for 10 min, rinsing in water, staining for 24 hr, washing well, drying and covering. Fast green was used with methacrylate sections to stain proteins by flooding the slide with a 0.1% solution of stain in 0.06 M phosphate buffer at pH 8, allowing the stain to dry out at 40-50 C, washing well, drying and covering. Controls were carried out on material fixed in formalin and treated with nucleases or proteolytic enzymes prior to embedding, and staining.     

Understanding microwave-stimulated Romanowsky-Giemsa staining of plastic embedded bone marrow

Summary Bone marrow smears were made and fixed in methanol or formaldehyde. Marrow sections of various thicknesses were also prepared from formaldehyde fixed marrows embedded in paraffin or plastic (glycol methacrylate). The different smears and sections were then stained by a Romanowsky-Giemsa procedure. Some specimens were stained using a standard microwave-stimulated method previously used diagnostically. The effects of technical variations were studied, including degree of microwave irradiation and the staining time. Comparisons of the resulting staining outcomes showed that microwave stimulated Romanowsky-Giemsa staining of plastic sections is a rate controlled process. Unusual aspects of the staining pattern of plastic sections (namely the purple basophilic cytoplasms and nucleoli, and blue chromatin) are due to microwave stimulation and formaldehyde fixation respectively.     

Computational determination of refractive index distribution in the crystalline cones of the compound eye of Antarctic krill (Euphausia superba)

In order to understand how a compound eye channels light to the retina and forms an image, one needs to know the refractive index distribution in the crystalline cones. Direct measurements of the refractive indices require sections of fresh, unfixed tissue and the use of an interference microscope, but frequently neither is available. Using the eye of the Antarctic krill Euphausia superba (the main food of baleen whales) we developed a computational method to predict a likely refractive index distribution non-invasively from sections of fixed material without the need of an interference microscope. We used a computer model of the eye and calculated the most realistic spatial distribution of the refractive index gradient in the crystalline cone that would enable the eye to produce a sharp image on the retina. The animals are known to see well and on the basis of our computations we predict that for the eyes of the adult a maximum refractive index of 1.45-1.50 in the centre of the cone yields a better angular sensitivity and light absorption in a target receptor of the retina than if N(max) were 1.55. In juveniles with a narrower spatial separation between dioptric structures and retina, however, an N(max) of 1.50-1.55 gives a superior result. Our method to determine the most likely refractive index distribution in the cone without the need of fresh material and an interference microscope could be useful in the study of other invertebrate eyes that are known to possess good resolving power, but for a variety of reasons are not suitable for or will not permit direct refractive index measurements of their dioptric tissues to be taken.     

A Rapid Plastic Embedding Technique for Preparation of Three-Micron Thick Sections of Decalcified Hard Tissue

A 24 hour start-to-finish method is described for the preparation of three-micronthick sections of decalcified hard tissues. Following acetone dehydration, the tissue to be embedded is infiltrated under vacuum with a series of graded clearing solutions which approach the content of the final methyl methacrylate mixture. After overnight in a 35 C oven, the plastic is polymerizd by four hours heating at 42 C. Three-micron-thick sections are then easily prepared using a Jung microtome for high resolution histologic or detailed autoradiographic procedures.     

A rapid plastic embedding technique for preparation of three-micron thick sections of decalcified hard tissue.

A 24 hour start-to-finish method is described for the preparation of three-micron-thick sections of decalcified hard tissues. Following acetone dehydration, the tissue to be embedded is infiltrated under vacuum with a series of graded clearing solutions which approach the content of the final methyl methacrylate mixture. After overnight in a 35 C oven, the plastic is polymerized by four hours heating at 42 C. Three-micron-thick sections are then easily prepared by using a Jung microtome for high resolution histologic or detailed autoradiographic procedures.     

A Plastic Embedding Medium for Thin Sectioning in Light Microscopy

Tissue blocks with surface areas up to 2 cm² can be sectioned at 1 or 2 μ after embedding in a medium consisting of: methyl methacrylate, 27 ml; polyethylene glycol distearate MW 1540, 6 gm; dibutyl phthalate, 4 ml; and Plexiglas molding powder A-100, 9 gm (added last). The methacrylate mixture is polymerized at 50° C by benzoyl peroxide, 0.8 gm/ 100 ml of methacrylate. The polymerized matrix is transparent and the blocks can be cut on a rotary microtome with a steel knife. The plastic can be removed from sections with acetone prior to staining. Artifacts caused by embedding and sectioning are negligible