The Piccolyte Resins as Microscopic Mounting Media

A new series of synthetic resins, the “Piccolytes” (β-pinene polymers), is recommended for permanent mounting media in histological work. These resins, which are available with various melting points, are of correct refractive indices, very low acid numbers, are pale, non-yellowing, have good adhesion to glass, and are freely soluble in xylene. Of a considerable variety of newer synthetic resins which were tried (stimulated by the recent scarcity of Canada balsam), only these terpene resins were found entirely satisfactory. Being of controlled manufacture, they are uniform in characteristics as well as readily available and very cheap.

A partial review of the literature of synthetic resin mountants is included.     

Vinylpyrrolidone-Vinyl Acetate Copolymers as Mounting Media for Azo and Other Dyes

Vinylpyrrolidone-vinyl acetate copolymers (Antara Chemicals, General Aniline and Film Corp., New York N. Y., PVP/VA, E-735 and E-635) were employed as mounting media by adition of ethanol-water to the concentrated ethanolic solution of these plastics (25% plastic in 50% ethanol). The E-735 copolymer was frequently employed and is specifically recommended because it exhibits the highest degree of water tolerance. This type of mounting medium was found to be especially satisfactory in the preservation of azo, oxidation, and other histochemically derived dyes. The medium is useful also in the preservation of stains for fat and certain metachromatic dyes. The inexpensive nature of this mountant and its ease of application recommend it as a useful substitute for glycerol-gelatin.     

Mounting Media for phase Microscope Specimens

-Results with phase microscopy can often be improved by the proper selection of mounting media. Temporary mounts can first be made in liquid mixtures of water and glycerol, butyl carbitol and alpha-chloronaphthalene, alpha-chloronaphthalene and alpha-bromonaphthalene or alpha-bromonaphthalene with methylene iodide to determine what index liquid serves best to emphasize structures of primary interest. Once this has been determined, permanent mounts can be prepared by the substitution of a solid mounting medium that most closely approaches the refractive index of the liquid.     

Combined Fixing, Staining and Mounting Media

A number of non-volatile, water-soluble substances can be added to the usual aceto-cannine fixing fluids. These inert substances do not alter the fixation image and serve as mounting media when the volatile ingredients of the mixture evaporate. Formulae are given for solutions containing dextrin, dextrose, gelatin, pectin, sorbitol, and sucrose. Gum arabic can be incorporated in a formic-acid-carmine fixative. The limiting factor in the use of such mounting media in fixing fluids is the osmotic value they give the solution; with certain precautions, however, they can be used in place of the usual aceto-carmine treatment. The indices of refraction of these media are not as high as those of the natural balsams and the fixation images which the mixtures produce have the characteristic limitations of those secured by the aceto-carmine technic. Some of the natural balsams (Canada balsam, sandarac and Venetion turpentine) can also be incorporated in fixing fluids. These fixatives are able to hold balsam and water in solution together, and, as the volatile components of the mixtures evaporate, the fixed specimens remain in permanent balsam mounts. The addition of carmine to these fluids enables us to fix, stain, dehydrate, clear and mount a specimen in a single operation. These fixatives preserve more details of chromosome structure than the aceto-carmiae fluids, but their use is more limited; and they can be substituted for the latter only with certain favorable material, e.g., pollen mother cells of Rhoeo and Tradescantia and salivary gland chromosomes of Cbirono-mus. Some of the newer synthetic resins can be substituted for the natural balsams. Formulae are given for fixatives which contain Venetian turpentine, sandarac, Canada balsam and two synthetic resins.     

Interim Report of the Committee on Histologic Mounting Media: Resinous Media

The Fisher “Permount” naphthalene polymer, the Hartman Leddon “H.S.R.” terpene polymer resin, a Monsanto polystyrene P-1, the Will Corporation “Diaphane” and “Green Diaphane”, and the du Pont “Lucite” methyl methacrylate polymer were examined, and the possibility of use of some other plastics was also explored. The first 5 mentioned were tested for color preservation of a variety of stains in comparison with Canada balsam and Clarite X. From this point of view polystyrene, the Hartman Leddon “H.S.R.” and the Fisher “Permount” resins were the most satisfactory, then the “Diaphanes”. Both “Permount” and “H.S.R.” show some yellowing. The H.S.R. with a melting point of 115°C, the Permount with 150°C. melting point, and the Polystyrene with a thermal denaturation point above 220°C. all excell Canada balsam in heat resistance. Trimethylbenzene, cymene and monoamylbenzene appear to be the best solvents for polystyrene. Mounts made in a solution of 20 g. polystyrene in 100 ml. trimethylbenzene can be packed flat slide to slide in 24 hours after mounting without sticking together.

This report is not intended to deprecate the use of other resinous mounting media which have not as yet been tested or compared with those mentioned herein.     

False Cellular Localization of Aminopeptidase Caused by Resinous Mounting Media

Fresh frozen sections of rat submandibular gland were processed for leucine aminopeptidase localization with L-lencyl-β-naphthylamide hydrochloride and L-leucyl-β-naphthylamide. When the first substrate was utilized stained sections were dehydrated in an ethyl alcohol series, cleared in xylene, and mounted in water-insoluble (resinous) media. Sections were also removed at each stage of dehydration and cleared and mounted with glychrogel. When the second substrate was used, tissues were partially decolorized in 40% ethyl alcohol and mounted directly in glychrogel. Comparison of all sections mounted in glychrogel indicated that there was no variation in cellular localization, regardless of substrate used or degree of dehydration. Nuclei were unstained. After mounting in balsam or synthetic resin the nuclei exhibited an intense stain and the parenchymal reaction was stronger, but diffuse. Progressive staining of the nuclei was observed, microscopically, immediately after applying the resinous mounting medium. The use of an aqueous mounting medium appears to be mandatory in this procedure—glychrogel is recommended.     

Preparation of Copolymers of Isobutyl Methacrylate and Styrene for Mounting Media

Details are given for the preparation of low molecular weight copolymers of isobutyl methacrylate and styrene for use in mounting microscopical sections between the slide and cover glass. Any refractive index in the range from 1.477 to 1.590 is available, depending upon the proportions of monomers used. A refractive index of 1.550 is recommended for general purposes, though other indices might be desirable in special cases including phase microscopy. The resin gives solutions in toluene of suitable fluidity at usable concentrations, adheres satisfactorily to glass, and has excellent light-transmission. The features which make it especially valuable are its optimum refractive index and its permanent water-whiteness.

Copolymers of isobutyl methacrylate and styrene may also be used to prepare plastic mounts of thick sections or gross specimens, which are optically cleared by the plastic mountant.     

Some Synthetic Resins IN Combined Fixing, Staining and Mounting Media

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 included in the aceto-carmine and propionic-carmine fluids with the result that more nuclear detail is preserved. Lacto-gelatin has some valuable properties as a mounting medium and can be used when the specimen is stained with orcein. Carmine, which gives a permanent stain in Rezyl 7020, Amberol 750 and Bakelite BR-7160 fades in lacto-gelatin.     

Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.     

Influence of Mounting Media on the Fading of Basic Aniline Dyes in Epoxy Embedded Tissues

A simple cytophotometric technique is used to quantitate stain fading of basic aniline dye-stained epoxy-embedded tissues mounted in six different commonly used mountants. Significant fading was detected with all six mountants, although rates varied. the lowest rate of fading was observed with immersion oil and the highest rate of fading with Canada balsam. No significant differences in fading rates of four synthetic mounting preparations were observed.     

New Uses for Calcium Chloride Solution as a Mounting Medium

Fresh cross sections of stems [Psilotum nudum, Coleus blumei, and Pelargonium peltatum] and roots (Setcreasea purpurea) 120 μm thick were fixed in FPA₅₀ (formalin: propionic acid: 50% ethanol, 5:5:90, v/v) for 24 hr and stored in 70% ethanol. The sections were transferred to water and then to 1% phloroglucin in 20% calcium chloride solution plus either hydrochloric, nitric, or lactic acid in the following ratios of phloroglucin-CaCl₂ solution:acid: 25:4, 20:2, or 15:5. The sections were mounted on slides either in one of the three mixtures or in fresh 20% calcium chloride solution. A rapid reaction of the acid-phloroglucin with lignin produced a deep red color in tracheary elements and an orange-red color in sclerenchyma. Fixed and stored leaf pieces from Nymphaea odorata were autoclaved in lactic acid, washed in two changes of 95% ethanol, transferred to water, and treated with the three acid-phloroglucin-calcium chloride mixtures. The abundant astrosclereids stained an orange-red color similar to that of sclerenchyma in the sections. In addition, a new method is reported for specifically staining lignified tissues. When sections or leaf pieces are stained in aqueous 0.05% toluidine blue O, then placed in 20% calcium chloride solution, all tissues destain except those with lignified or partially lignified cell walls. Thus, toluidine blue O applied as described becomes a reliable specific test for lignin comparable to the acid-phloroglucin test.     

Effects of Mounting Media on Fading of Toluidine Blue and Pyronin G Staining in Epoxy Sections

Available mounting media cause fading of histological preparations over time. A study was designed to find the most suitable medium for durable mounting of Araldite embedded semithin sections of rabbit cerebral cortex stained with toluidine blue and pyronin G. Among four synthetic mounting media tested, only DePeX prevented fading of the sections during the first month. All mounting media tested helped preserve staining intensity after one month, since the fading rate after one year is only about half that in sections prepared without mounting medium. The average optical density of sections after one year was higher in preparations mounted with DePeX than in sections treated with the other mounting techniques tested in this study. After one year, the average optical density of sections mounted with DePeX had decreased approximately 20%.     

Xylene-Cellulose Caprate as a Histologic Mounting Medium of Low Refractive Index

A 50% solution of cellulose caprate in xylene forms a clear pale yellowish syrupy solution of proper consistency for mounting stained and unstained sections. Cover slips become immovable in 30 minutes and slides do not stick together after one-day drying. The refractive index of the xylene solution is 1.4860, of the dry resin 1.4734, and unstained sections remain easily visible. Most stains are well preserved. Prussian blue is well preserved, cobalt sulfide decolorizes in a month.     

Slide Transparencies

Abstract

Ernest Pascarella and Patrick Terenzini. How College Affects Students. San Francisco: Jossey-Bass, 1991. 792 pages. $75 hardback; $39.95 paperback.