A coverslip culture technique for preparing permanent fungus mounts

A coverslip culture technique for the growth of fungi, with subsequent preparation of permanent mounts for microscopic examination, is described. This simplified technique allows examination at different stages of development of the fungus, or can be used in the preparation of large numbers of similar mounts for teaching purposes.     

A simple device to rapidly prepare whole mounts of murine intestine

Many mouse models of neoplasia and pre-neoplasia require the examination of whole mounts of the gastrointestinal tract. A simple device has been produced to facilitate the rapid preparation of mouse intestines for subsequent quantification of tumours and pre-neoplastic lesions such as aberrant crypt foci. The device greatly speeds up the production of whole mounts and also provides far more consistent and better-quality preparations.     

The History of Staining Cochineal Dyes

Of the dyes used in early histological work, none was so highly prized as carmin. Even today biologists would be loath to part with it. It is still valuable to the histologist and embryologist as a bulk stain of great permanency, and for the preparation of in toto mounts. To the cytologist, also, it is invaluable as a medium for the rapid staining and examination of chromosomes in fresh material. Its permanence has always been a great advantage. To the early histologists, however, it was the dye, par excellence.     

The History of Staining Cochineal Dyes

Of the dyes used in early histological work, none was so highly prized as carmin. Even today biologists would be loath to part with it. It is still valuable to the histologist and embryologist as a bulk stain of great permanency, and for the preparation of in toto mounts. To the cytologist, also, it is invaluable as a medium for the rapid staining and examination of chromosomes in fresh material. Its permanence has always been a great advantage. To the early histologists, however, it was the dye, par excellence.     

Vascular labelling with monastral blue B

Vascular labelling is an established technique of experimental pathology whereby leaky vessels can be identified in vivo. A suspension of a suitable colloidal pigment is injected intravenously; the pigment is then trapped in the wall of the leaky vessels. The colloidal preparation of carbon black, which has been used for many years for this purpose, is no longer commercially available. This communication introduces a substitute: Monastral blue B which gives beautiful preparations in whole mounts, is readily visible in paraffin and plastic embedded histologic sections, has a distinctive appearance in electron micrographs, and is nontoxic in the required dosage.     

Vascular Labelling with Monastral Blue B

Vascular labelling is an established technique of experimental pathology whereby leaky vessels can be identified in vivo. A suspension of a suitable colloidal pigment is injected intravenously; the pigment is then trapped in the wall of the leaky vessels. The colloidal preparation of carbon black, which has been used for many years for this purpose, is no longer commercially available. This communication introduces a substitute: Monastral blue B which gives beautiful preparations in whole mounts, is readily visible in paraffin and plastic embedded histologic sections, has a distinctive appearance in electron micrographs, and is nontoxic in the required dosage.     

A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells

The fine structure of resting and activated platelets was compared using two approaches novel to this dense cytoplasm. First, rapid lysis of platelets on carbon-coated grids was following by negative staining of the "cytoskeleton." Second, a brief, minimal fixation of platelets in plasma was coupled with partial lysis and examination of the unstained whole mounts at 200 kV. The results showed that the dense ground cytoplasm of discoid, fully resting platelets appeared granular or amorphous, and microfilaments were not observed. A coiled microtubule terminated in one, free, straight end. When any slight degree of activation occurred, microfilaments could be detected in the platelets. In fully spread specimens, the amorphous character of the resting cytoplasm was strikingly altered into an interconnected network of microfilaments. Stereo views of the whole mounts showed that dense granules, 100-250 nm in diameter, appeared as if suspended in the filament nets. The results support the view that platelet activation involves a major assembly of microfilaments from amorphous precursors. The change can only be seen convincingly when stringent precautions are taken during preparation because the platelets are very easily activated by thermal or mechanical stimuli.     

Techniques for the rapid preparation of permanent slides of microscopic algae

Because most techniques for the preparation of permanent mounts of algae have hitherto involved long alcoholic dehydration series, which frequently result in cell collapse if cut short, a method has been developed which is anhydrous from the start and avoids these series. It can be used to produce rapidly, permanent, generally-stained mounts of most microscopic algae in which the degree of shrinkage of the cell or its contents is reduced to a minimum.

A method for the cleaning and mounting of diatoms is also given.     

A modified gold chloride technique for optimal impregnation of nerves within corneal whole mounts and dura of the albino rat

Ranvier's method of staining tissue whole mounts with gold chloride to visualize nerve fibers was modified by lengthening the incubation time in gold chloride and reducing the time in acidulated water. These simple modifications of an old technique give consistent impregnation of nerve fibers with light background staining in whole mounts of cornea and dura.     

A Simple and Rapid Wholemount Staining Method with Methyl Green-Pyronin G Applied to A Molluscan Brain Preparation

Subesophageal ganglia of molluses have been stained as whole mounts with methyl green-pyronin G to display the relative location of individual neurons. Nuclei appear blue, perikarya red. Expose the ganglion cells by dissection of the connective tissue in dl Ringer. Transfer the ganglion to a fixative of 2.5% glutaraldehyde in 0.1 M Na cacodylate pH 7.1 at 4 C for 12-24 hours, and wash in distilled water for 1 1/2 hours. Stain with methyl green-pyronin G for 1/2-1 hour ad differentiate in 96% ethanol wing many rapid changes. Transfer the ganglion to absolute ethanol for 2 1/2 hours and clear in xylene for 3 hours before embedding in Depex in a suitable dish. When the Depex has hardened, the preparation can be stored, and is readily available for subsequent examination. The method may be applicable to other invertebrate tissues, and may be useful in preparing objects for teaching purposes.     

A Technique for C-banding Chromosomes with Pinacyanol Chloride

A technique is presented for rapid C-banding of eukaryotic chromosomes with pinacyanol chloride. This technique has given excellent definition and clarity to chromosome bands in plant and animal chromosomes. In permanent mounts the chromosomes did not fade after storage for up to two years.     

A simple and rapid whole-mount staining method with methyl green-pyronin G applied to a molluscan brain preparation.

Subesophageal ganglia of molluscs have been stained as whole mounts with methyl green-pyronin G to display the relative location of individual neurons. Nuclei appear blue, perikarya red. Expose the ganglion cells by dissection of the connective tissue in snail Ringer. Transfer the ganglion to a fixative of 2.5% glutaraldehyde in 0.1 M Na cacodylate pH 7.1 at 4 C for 12--24 hours, and wash in distilled water for 1 1/2 hours. Stain with methyl green-pyronin G for 1/2--1 hour and differentiate in 96% ethanol using many rapid changes. Transfer the ganglion to absolute ethanol for 2 1/2 hours and clear in xylene for 3 hours before embedding in Depex in a suitable dish. When the Depex has hardened, the preparation can be stored, and is readily available for subsequent examination. The method may be applicable to other invertebrate tissues, and may be useful in preparing objects for teaching purposes.     

Antiserum to lucifer yellow: preparation, characterization, and use for immunocytochemical localization of dye-filled retinal neurons

We present a new method for the preparation of antisera to Lucifer Yellow, and these antisera are here shown to be particularly suitable for immunocytochemical localization of multiple dye-injected cells in large pieces of vertebrate retina. The method involves the preparation of covalent conjugates of the VS isomer of Lucifer Yellow with keyhole limpet hemocyanin (KLH) or rabbit serum albumin (RSA), and their use as immunogens in rabbits. Both carrier protein conjugates yielded robust antibody responses. Antiserum to the KLH-LY conjugate contained precipitating antibodies against LY and KLH, although activity to the latter did not interfere with immunocytochemical staining. Rabbit antiserum to the RSA-LY conjugate contained precipitating antibody only against LY. When used for immunocytochemical staining of large retinal pieces containing many LY-filled cells, both antisera yielded well-stained, darkly filled cells similar to those seen with the Golgi technique; even very fine dendritic processes of retinal ganglion cells could be followed for long distances. LY immunocytochemistry provides a useful alternative to photooxidation for the analysis of multiple dye injected cells, especially in whole mounts. This approach may also be useful for immunocytochemical identification of cells filled with LY after tissue fixation.     

Lamination of Thermoplastic Sheets as a Means of Mounting Histological Material

Permanent preparations of squashes, whole mounts and stained sections can be made by lamination of thermoplastic sheets. Classical procedures of staining and dehydration for sectioned material were used although dehydration after staining was not required for root tip squashes. Arranging the specimen with the identification label between two pieces of clear Vinylite plastic, 15 mils thick, tightening the preparation in a Photo-Seal Kit electric press and laminating for 3 min gave a finished preparation without the use of glass slides and cover slips. For root tip squashes, the stained tip was placed with a drop of stain between two pieces of plastic, squashed and then laminated. This insured retention of all the tissue which is sometimes lost during mounting processes. Preparations of unstained whole mounts were similarly laminated, with an identification label added between the plastic sheets. Stained sections were placed between two sheets of plastic but the identification label was placed on top of the preparation and a third piece of plastic added. This prevented the label from absorbing excess stain and the increased thickness allowed the slide to be used in a mechanical stage. Well preserved slides 18 mo old indicate that the laminated plastic slide is quite durable. It saves time, reveals good cytological detail and avoids some of the laborious features of other methods. It is a technic that can be used in introductory microtechnic and in the preparation of slides for class use in histology.     

Simultaneous demonstration of adrenergic and non-adrenergic nerve fibres

Summary A technique for simultaneous demonstration of adrenergic and non-adrenergic nerve fibres is described, using methylene blue staining and fluorescence microscopy after formaldehyde treatment. The procedure is applicable to whole mounts as well as to microtome sections.     

Effects of Fixation and Dehydration Procedures on Marine Nematodes

Different combinations of fixation and dehydration procedures for the preparation of permanent mounts of marine nematodes of the subfamily Oncholaiminae were tested and compared. Qualitatively, the best specimens resulted from Seinhorst''s killing method and fixation in FAA; the dehydration procedure was of less significance. Quantitatively, no significant modification of measurements resulted from any of the methods used. Sources of error in measurements are discussed.     

A Method of Permanently Mounting Biological Tissue Cleared in Herr's Four-And-A-Half Clearing Fluid

A technique which should be generally applicable for preparing permanent mounts of tissue cleared in Herr's four-and-a-half clearing fluid is described. This technique involves transferring plant or animal tissues through a series of solutions consisting of Pienarr's fixative, Herr's clearing fluid, chloral hydrate, acetone and finally polyester resin for mounting. Material prepared using this method is exceptionally transparent and well preserved, and is suitable for either phase contrast or Nomarski interference microscopy.     

The Slide-Sealing Compound "Glyceel"

"Glyceel" has been considered for many years to be the best sealant for whole mounts of soil, plant-parasitic, freshwater, and marine nematodes. However, "Glyceel" has not been available since the mid 1980s when its production was halted. Currently available substitutes are inadequate. The original formula for "Glyceel" has been found in the literature and is given here with a method of preparation. "Glyceel" prepared in this way by the author has been used and appears to function well.     

Card Mounts for Handling Root Tips in the Paraffin Method

A method is described for utilizing card mounts to facilitate the handling of root tip samples and similar material in the paraffin method. The freshly collected roots are attached to temporary card mounts having dimensions of approximately 2 cm. × 2.5 cm. with DuPont Household Cement or a similar adhesive that hardens rapidly in the ordinary aqueous fixing fluids and is insoluble in the lower grades of alcohol. After fixation and dehydration to 75% alcohol, the roots are transferred to permanent card mounts on which they are carefully oriented for sectioning. Mucilage or glue which hardens rapidly in 85% alcohol and is insoluble in the ordinary dehydrating and infiltrating media is used in making the permanent card mounts. Detailed instructions are given for preparing and handling the card mounts, and a system of labeling the mounts is also suggested.     

Laboratory Hints from the Literature: A Department Devoted to Abstracts of Books and Papers from Other Journals Dealing with Stains and Microscopic Technic in General

A technique which should be generally applicable for preparing permanent mounts of tissue cleared in Herr's four-and-a-half clearing fluid is described. This technique involves transferring plant or animal tissues through a series of solutions consisting of Pienarr's fixative, Herr's clearing fluid, chloral hydrate, acetone and finally polyester resin for mounting. Material prepared using this method is exceptionally transparent and well preserved, and is suitable for either phase contrast or Nomarski interference microscopy.