Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

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 Technic for Salivary-Gland Chromosomes

This technic was worked out for making permanent mounts of salivary-gland squashes of strains and species of Drosophilidae, which yield material difficult to work with. It includes a prior fixation in a modified Carnoy's fluid, staining in solutions of orcein in 60% or 70% acetic acid, part dehydration in the vapor and liquid of a 95% solution of alcohol, and direct mounting in Euparal by means of the drainage-method.     

The Smear Technic in Plant Cytology

The following method of making permanent smears of pollen mother cells is in general use and gives excellent results. Determine the stage of meiosis from aceto-carmin mounts. Smear the pollen mother cells on a dry slide. Fix in Navaschin's or a modified Flemming's solution from 1 to 2 hours. Wash in 10 to 20% alcohol from 15 to 30 minutes. Stain in 1% aqueous crystal violet from 1 to 5 minutes. Rinse in water and pass thru 30 to 50% alcohol, about 15 to 20 seconds in each. Transfer to 80% alcohol containing 1% iodine and 1% potassium iodide for 30 seconds. Destain with absolute alcohol, followed by clove oil. xylol, balsam and cover.

Permanent smears for chromosome counts can be quickly made by smearing pollen mother cells on a dry slide, fix and stain with aceto-carmin, dehydrate with mixtures of absolute alcohol and acetic acid, follow with xylol, balsam, and cover.     

A Method for Injecting Insect Tracheae Permanently

By the use of an alcohol insoluble dye (trypan blue), acetic acid, and a detergent (“Santomerse No. 3”), a resulting dye solution is obtained which will completely penetrate the tracheal system of an insect. The dye is injected by the use of a vacuum and by the pressure produced when the air is allowed to re-enter the dye vessel. The dye itself is permanently fixed in the tracheae by means of a fixing solution containing alcohol, acetic acid and barium chloride as its components. The material must be properly preserved after staining. It may be stored indefinitely in 70% alcohol, xylol, cedar oil or clove oil, depending on whether the material is to be used for sectioning or for whole mounts. Injected material may be sectioned in either celloidin or paraffin, or may be cleared and mounted in toto.     

THE SWELLING PRESSURE OF GELATIN AND THE MECHANISM OF SWELLING IN WATER AND NEUTRAL SALT SOLUTIONS

1. A method is described for measuring the swelling pressure of solid gelatin. 2. It was found that this pressure increases rapidly between 15° and 37°C., and that the percentage change is nearly independent of the concentration of gelatin. 3. It is suggested that this pressure is due to the osmotic pressure of a soluble constituent of the gelatin held in the network of insoluble fibers, and that gelatin probably consists of a mixture of at least two substances or groups of substances, one of which is soluble in cold water, does not form a gel, and has a low viscosity and a high osmotic pressure. The second is insoluble in cold water, forms a gel in very low concentration, and swells much less than ordinary gelatin. 4. Two fractions, having approximately the above properties, were isolated from gelatin by alcohol precipitation at different temperatures. 5. Increasing the temperature and adding neutral salts greatly increase the pressure of the insoluble fraction and have little effect on that of the soluble fraction. 6. Adding increasing amounts of the soluble fraction to the insoluble one results in greater and greater swelling. 7. These results are considered as evidence for the idea that the swelling of gelatin in water or salt solutions is an osmotic phenomenon, and that gelatin consists of a network of an insoluble substance enclosing a solution of a soluble constituent.     

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 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.     

Modifications to clearing methods used in combination with vital staining of roots colonized with vesicular-arbuscular mycorrhizal fungi

Leek, maize, and pigmented soybean roots colonized by vesicular-arbuscular mycorrhizal (VAM) fungi were assessed for succinate dehydrogenase (SDH) activity using the nitro blue tetrazolium chloride (NBT)-succinate method. NBT-succinate-reacted roots, cleared in a 55° C drying oven in 5% (w/v) KOH for 24 h or longer and observed as whole mounts, revealed signs of intraradical VAM fungus colonization more clearly than roots cleared by the standard 20% (w/v) boiling chloral hydrate method. Combined clearing of NBT-succinate-reacted roots using boiling chloral hydrate followed by clearing in 5% KOH at 55° C for prolonged periods also improved the visualization of intraradical fungal structures. Bleaching of NBT-succinate-reacted roots using the standard NH₃-H₂O₂ method removed pigmentation from roots and did not alter the viability indicator, formazan. Pigmented, field-collected soybean roots were successfully cleared and bleached to reveal signs of viable and nonviable intraradical fungal structures. Counterstaining of NBT-succinate-reacted roots with acid fuchsin clearly revealed both viable and nonviable intraradical fungal structures. The NBT-succinate solution infiltrated all intraradical fungal structures after 24 h; formazan products were observed at similar concentrations in viable structures after 24, 36, and 48 h.     

Dissection and Preparation of Whole Mounts of Endosperm from the Seeds of Grevillea (Proteaceae)

It is sometimes very desirable to supplement the examination of serially cut microtome sections by dissection and study of whole mounts of objects. In the case of the seeds of Grerillea the endosperm forms a curious worm-like structure, the vermiform appendage, in elucidating the nature of which microtome sections alone are inadequate. The dissection of the endosperm for demonstrating this structure is very necessary; otherwise the structure is altogether overlooked. In dissecting out the endosperm, which is easily done, either fresh seeds or previously preserved seeds may be used; the former are easier for handling and yield more satisfactory results. In the case of fresh material, an immediate killing is necessary and any one of the ordinary killing fluids may be used; Bouin's fluid was tried by the writer and was entirely satisfactory. After killing, the material is stained and dehydrated, the several stages of which are best done on the slide alone on which the material is placed. Mounting is done in any of the mounting media, and Canada balsam answers the purpose quite well.     

A Schedule for Chromosome Counts M Some Plants with Small Chromosomes

A schedule is given which produced excellent results for somatic chromosome counts on some British species of Galium, These species present great difficulties owing to the small size of the chromosomes (ca. 1.5μ long), the large somatic numbers (up to 96) and the slenderness of the root tips. Special features of the schedule, which is the result of much experiment with various technics, are: fixation with Belling's Navashin type fixative, which was quite the best tried; the modification of Randolph's card mount method to overcome the difficulty of the small diameter of the root tips by mounting together a number from the same plant, so that they can be embedded and sectioned almost as easily as much larger root tips; staining with dilute (0.1%) crystal violet, the most critical stain used, after mordanting with 1% aqueous chromic acid to intensify the stain in the small chromosomes; and the addition of an extra stage of differentiation in absolute alcohol diluted with xylol to remove strands of stain, which are often left in the cytoplasm between the chromosomes, since clove oil, usually the last differentiating fluid used, differs from alcohol in removing crystal violet more rapidly from the chromosomes than from the cytoplasm. It is suggested that this schedule will be valuable in chromosome counts of other plants where similar difficulties arise.     

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and ‘roll’ mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card, tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.     

A Quick-Freeze Method for Making Smear Slides Permanent

Many types of smear slides can be made permanent rapidly and effectively by substituting for the usual dehydration series a single-step process of freezing the slide on a block of dry ice, placing it immediately in 95% or absolute alcohol, and then mounting it. Advantages of the technic are its speed, the ease of separation of cover slip from slide with a minimum loss of cells, and the superiority of the resulting permanent slides.     

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.     

Orseillin Bb for Staining Fungal Elements in Sartory's Fluid

A method is described for the squashing and permanent mounting of preparations, similar to the nucleal squash technic, to be used after hematoxylin, coal tar dyes and other stains. The colored pieces are immersed in glycerin, squashed or smeared, the glycerin is slowly removed with water by capillarity and the water is afterwards similarly substituted by alcohol. The cover glass is then removed and after a rapid rinsing in alcohol the materials are permanently mounted in alcohol-soluble resin.     

A Simple Method For Squashing And Mounting Preparations After Any Stain

A method is described for the squashing and permanent mounting of preparations, similar to the nucleal squash technic, to be used after hematoxylin, coal tar dyes and other stains. The colored pieces are immersed in glycerin, squashed or smeared, the glycerin is slowly removed with water by capillarity and the water is afterwards similarly substituted by alcohol. The cover glass is then removed and after a rapid rinsing in alcohol the materials are permanently mounted in alcohol-soluble resin.     

Advantages of Epoxy Resin as a Mounting Medium for Light Microscopy

The need for very durable mounting is especially felt in the teaching of parasitology and mycology; otherwise, the availability of microscope slides may depend on the use of fresh specimens. Resinous mounts and those in aqueous media sealed with fingernail lacquer, paraffin or asphalt do not preserve specimens satisfactorily. Polyvinylpyrrolidone (pvp), a water-soluble mounting medium described by Burstone (1962), cannot be applied directly for mounting of insects and certain other parasites which have water-repelling integuments; moreover, pvp bleaches eosin. Grimley et al. (1965) prepared large epoxy sections of tissues from which areas for electron microscopy could be selected. This procedure however is designed for electron microscopic techniques whereas the present paper describes a direct epoxy mounting method to produce permanent mounts for light microscopy.     

A Rapid Method for Making Permanent Mounts of Nematodes

A rapid method which can be used to mount and clear nematodes and their eggs is presented. Permanent mounts of certain nematodes and parasite eggs have been prepared using a medium consisting of 56 parts of a stock solution of polyvinyl alcohol (“PVA”), 22 parts phenol and 22 parts of lactic acid. The stock solution of PVA is prepared by dissolving 15 grams of PVA in 100 ml. of distilled water. This medium can be used on material killed and fixed in 10% formalin, any concentration of alcohol, alcohol-glycerin or glycerin. Results have been very satisfactory in most instances. An accompanying plate of photographs shows some of the preparations obtained by using this method.     

Basic Dye Counterstaining of Sections Impregnated by the Golgi-Cox Method

Celloidin blocks of Golgi-Cox impregnated material are cut at 50 μ, the sections collected in 70% alcohol, transferred to a 3:1 mixture of absolute alcohol and chloroform for 2 min, and then stored in xylene or toluene for at least 3 min, or up to 2 wk until processed further. Mounting is done on glass slides which have been coated with fresh egg albumen diluted in 0.2% ammonia water (or a 0.5% solution of dry powdered egg albumen) and then dried at 60°C overnight. For attachment to these coated slides, sections are first soaked for 2-3 min in a freshly prepared mixture of methyl benzoate, 50 ml; benzyl alcohol, 200 ml; chloroform, 150 ml; and then transferred quickly to the slides by means of a brush. After 2-3 min the chloroform evaporates and the celloidin softens. The slides are then immersed in toluene which hardens the celloidin and anchors the sections to the slides. Alcohols of descending concentrations to 40% are followed by alkalinizations, first in: absolute alcohol, 40 ml; strong ammonia water 60 ml, for 2 min, then in: absolute alcohol, 70 ml; strong ammonia water, 30 ml, for 1 hr. Excess alkali is then removed by 70% and 40% alcohol, 2 min each, and a 10 min wash in running tap water. Bleaching in 1% Na₂S₂O₃, for 10 min and washing again in tap water for 10 min completes the process preliminary to staining. The preparations are then stained for 90 min in an aqueous solution of either 0.5% cresylecht violet, neutral red, or Darrow red, buffered at pH 3.6. Dehydration and differentiation in ascending grades of alcohol, clearing with toluene or xylene, and applying a cover glass with a mounting medium having a refractive index of about 1.61 completes the process.