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.     

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.     

Simplified Permanent Aceto-Carmine Smears with A Water-Miscible Mountant

By employing a water-miscible mountant with a good refractive index, permanent aceto-carmine smears can be made from any of a variety of killing, fixing, and staining schedules. Clear-col, a commercial preparation, orginally developed for mounting fungi from various water-containing media, is employed for anther smears by placing the medium directly on the final stage of aceto-carmine staining schedule. Thus, no dehydration and clearing are necessary. By controlling amounts of acetic acid either mixed with Clearcol or on smears, clear, well-stained preparations can be produced.     

The Venetian Turpentine Mounting Medium

As a mounting medium to follow aceto-carmine the following modification of Zirkle's is suggested: Venetian turpentine, 25 ml.; phenol, 50 ml.; propionic acid, 35 ml.; acetic acid, 10 ml.; water, 20 ml. The technic can be employed with either root-tip or pollen-mother smears, and has been used with quite a variety of plants. It is especially valuable where it is desired to make temporary mounts permanent. The method is simple, and with reasonable care no displacement of marked cells occurs.     

On the Composition of “Viscol” and the Employment of Gum Arabic for Mounting Media

“Viscol”, a water soluble permanent mounting medium hardening through evaporation of water under the cover glass, has been analyzed. It proves to consist of a mixture of gum arabic, glycerol, phenol and water and is especially suitable for simple botanical preparations. The use of gum arabic for hardening permanent mounting media is reviewed. Instead of a glycerol-phenol-water mixture a lactophenol, potassium acetate or zinc chloride solution mixed with gum arabic may be used for a permanent mounting medium. However, gum arabic contains calcium, magnesium and potassium ions giving crystals with the solutions mentioned. In the case of lactophenol and potassium acetate, the calcium and magnesium ions must be removed beforehand, which is done by precipitation with sodium or potassium carbonate. In the case of zinc chloride the potassium ions must be removed, which is done by dialysis with zinc chloride. It is pointed out that the same principles may be used for a great variety of different mounting media.     

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.     

Chlorazol Black E As An Aceto-Carmine Auxiliary Stain

In making chromosome counts on plants and plant parts treated with colchicine it was found that in cases where aceto-carmine alone is not satisfactory—as in axillary buds of apple, pear, plum, peach, apricot, and cherry—the following method was effective : Dissect out the meristematic parts of the axillary bud under a binocular (or cut free-hand sections) and transfer the dissected tissue immediately to a solution of 3 volumes alcohol to 1 volume acetic acid for killing and fixing. Let the fixative act at least 10 minutes; a longer time, 12-24 hours, improves the staining quality. Wash in at least 3 changes of 70% alcohol to remove most of the acid. Stain for 5-25 minutes in 1% chlorazol black E² in 70% alcohol. Rinse in 3 changes of 70% alcohol to remove excess stain. Transfer the material to a slide, cover with a drop of aceto-carmine, and if necessary, dissect further under a binocular. Cover with cover glass, heat, flatten and seal, or run Zirkle's fluid under the cover for permanent mounting. For smears of sporocytes, chlorazol black E may also be employed alone, or in combination with aceto-carmine, if a dark purple nuclear stain is desired.     

Alcoholic Hydrochloric Acid-Carmine as a Stain for Chromosomes in Squash Preparations

A regressive bulk carmine staining schedule was adapted from a formula proposed by P. Mayer. The stain is made by boiling gently 4 gm of certified carmine in 15 ml of distilled water to which 1 ml of concentrated HC1 has been added. After cooling, 95 ml of 85% alcohol is added, and the solution filtered. Fixed tissue is soaked in the stain until thoroughly penetrated; squashes are then prepared as usual, but plain 45% acetic acid is used as the temporary mounting medium instead of aceto-carmine. The advantages of this method are: intense, precise staining of chromosomes coupled with a lightly stained cytoplasm; consistency and uniformity of results; simplicity of use.     

Preparation of Permanent Whole Mounts of Marine Centric Diatoms to Permit Observations of Cytoplasmic Inclusions

Marine centric diatoms are highly vacuolated plant cells which often exhibit a marked tendency to plasmolyze during fixation or other manipulatory stages required to make permanent preparations which pemit the observation of cytoplasmic inclusions. Altmann's, Schaudinn's, vom Rath's, 5% acrolein, and Allen's PFA₃ are fixatives which have been found to induce relatively little plasmolysis in the species studied. If, following fixation, the fixative is removed and desalting and dehydration are carried out in a filter assembly which allows gentle suction to make gradual changes in reagent concentration and if the cells are then placed in dilute mounting medium, only a relatively small percentage of them will be plasmolyzed. In such preparations, depending upon the fixatives employed, various cytoplasmic organelles or other inclusions will be visible in the unstained material when examined with phase contrast. Fixation with Altmann's fixative and mounting the material in Zeiss phase-contrast mounting medium (L-15) after gradual desalting and dehydration gave the best results. Standard cytochemical techniques for the detection of saccharides, protein, and lipids may be incorporated in this procedure; or, all of the steps needed for embedding the material in resins, excepting polymerization, are feasible also.     

Aceto-Carmine-Lactophenol Preparations Of Paramecium Aurelia

Paramecia were killed and stained by adding a sat. soln. of carmine in acetic acid to a small drop of culture, cleared with 45% acetic acid as soon as the nuclei became darkly colored, and mounted in lactophenol (phenol crystals, 20 g.; lactic acid, 20 ml.; glycerol, 40 ml.; distilled water, 20 ml.). The mounting mixture was prepared by warming to dissolve the phenol and 20 drops of aceto-carmine added after cooling. Cover glasses were ringed with colorless nail polish or with asphaltum after slides had stood several days.     

Root-Tip Smears Following Fixation with Boiling Water

A convenient, quick method of preparing fresh root-tips for the detailed study of the critical stages of mitosis is presented. The cells of fresh-cut root-tips are killed and fixed instantaneously by immersion in boiling water. To effect maceration, the root-tips are transferred to a mixture of 95% ethyl alcohol and concentrated HCl. The root-tips are then smeared in a drop of aceto-carmine with glass instruments. Application of mineral oil makes the preparations permanent or semi-permanent. Characteristically, anaphasic chromosomes in the fresh materials thus prepared appear as structures composed of a swollen transparent matrix in which are embedded spiral interlocking chromonemata. Suggestions are offered as to the advantages and possibilities of the technic.     

Preparation of Chromosomes of Tetrahymena Pyriformis for Photomicrography

Conjugating animals of the protozoan, Tetrahymena pryiformis, were affixed to cover slips by means of Nissenbaum's fluid, followed immediately by 1:3 acetic-alcohol for 18-24 hr. After fixation, the material was transferred through a descending alcohol series to water, then hydrolyzed in 1 N HCl, washed in water, followed by immersion in 45% acetic acid and subsequent mounting in aceto-carmine. Photomicrographs were made using a phase-contrast microscope and Microfile film. The schedule resulted in preparations with abundant material, adequate spacing of chromosomes in a single plane, and excellent differentiation of the chromosomes from the cytoplasm.     

Preparation of Chromosomes of Tetrahymena Pyriformis for Photomicrography

Conjugating animals of the protozoan, Tetrahymena pryiformis, were affixed to cover slips by means of Nissenbaum's fluid, followed immediately by 1:3 acetic-alcohol for 18–24 hr. After fixation, the material was transferred through a descending alcohol series to water, then hydrolyzed in 1 N HCl, washed in water, followed by immersion in 45% acetic acid and subsequent mounting in aceto-carmine. Photomicrographs were made using a phase-contrast microscope and Microfile film. The schedule resulted in preparations with abundant material, adequate spacing of chromosomes in a single plane, and excellent differentiation of the chromosomes from the cytoplasm.     

Production of Pine Resjn Balsams for Microscopical and Optical Uses

The authors describe a method of obtaining balsam from the crude resin of Yugoslav pines. The quality and the refraction of those balsams are very favorable and they may be used instead of Canada balsam and cedar oil for microscopic work and in some cases for the optical industry. When placed in the above mentioned balsams, histological preparations dyed with very sensitive colors remained unaltered. The authors are of the opinion that the best of all is Balsamum Pini peuce. The balsams of Pinus halpensis, Pinus leucodermis, Pinus nigrae and Pinus silvestris are very similar in their properties to the balsam of Pinus peuce. Tables are given to show: some physical properties of these balsams; the yield obtained in preparing them; their composition in respect to resin and turpentine.     

Non-aqueous permanent mounting for immunofluorescence microscopy

It is generally assumed that an aqueous mounting medium is necessary for the preservation of immunofluorescent-labelled microscopical preparations and polyvinyl alcohol-based solutions (e.g. Mowiol) being the most frequently used mounting media; however, both the quality and intensity of the fluorescence signal in most immunolabelled preparations after aqueous mounting slowly diminish with time, and finally, samples become unsuitable for examination. In the present work, we describe a very simple and rapid non-aqueous mounting procedure for cultured cells and tissue sections, which preserves the fluorescent signal in an excellent way after immunodetection or use of other specific labelling methods. It is based on the current histological protocol in which, after fluorescence labelling, preparations are dehydrated in ethanol, cleared in xylene and mounted in DePeX. Using this non-aqueous mounting medium, the fluorescent signal remains high and stable, allowing a suitable and permanent preservation of labelled and counterstained microscopical preparations.     

Iron-Alum Aceto-Carmine Staining for Chromosomes and Other Anatomical Features of Rhodophyceae

The chromosomes, certain intracellular structures and gross anatomical details of many red algae, which, as a class, have proved technically difficult material, can be demonstrated by staining with aceto-carmine after a mordant bath of iron alum. Acetic-alcohol mixtures are used as nuclear fixatives and formalin-acetic-alcohol and other similar fluids for preservation of anatomical features. The tougher more cartilaginous thalli of some species can be softened, if squashes are desired, by prolonging fixation (24-48 hr.) in acetic alcohol and subsequent washing. The fixatives are washed out of the material before the latter is transferred to 0.5-5.0% ferric ammonium sulphate, the concentration of which may be altered according to the material. Excess mordant is removed by washing and the material stained in Belling's aceto-carmine containing a trace of ferric acetate as a “ripener”. The degree of heating before covering is critical as it controls the quality of the staining. Squashing must be very thorough to spread the chromosomes which are usually very small but only slight controlled pressure is necessary when diffuse structures such as carposporophytes, nemathecia or medullary filaments are being demonstrated. Paraffin sections mounted on slides can also be stained by this method.     

Impact of plant cover on the cavity‐nesting ant Temnothorax crassispinus

1. Plant communities influence the availability of important resources for ants, such as nest sites and food, as well as environmental conditions. Thus, plants affect the abundance and distribution of ants. 2. In a field experiment, the influence of plant cover on the settlement of nest sites and per‐capita productivity of sexual individuals by the ant Temnothorax crassispinus was analysed. In July 2014, in five areas with patches of alien balsam Impatiens parviflora, and another five of native balsam I. noli‐tangere, transects composed of artificial nests were established; the nest sites were situated inside patches of balsams, and outside of them. Four hundred and forty artificial nests were used. One year later, the nests were collected. 3. Colonies of the ants three times more often inhabited nest sites outside the patches of both balsams. Besides, colonies with queens were more frequently found in nest sites located away from balsams. The per‐capita productivity of sexual individuals was higher in nests collected in patches of balsam, and the colonies from patches of alien balsam produced a more female‐biased sex ratio. 4. In terms of the impact on the ant, no clear differences were found between the alien balsam and the native one. The most important factor affecting the fitness of ants in areas dominated by balsams is the presence of herbaceous plant cover rather than whether the plant is alien or native.     

Ecology of a plant growth-promoting strain of Pseudomonas fluorescens colonizing the maize endorhizosphere in tropical soil

Pseudomonas fluorescens strain BR-5 stimulated the growth of maize in a natural soil and inhibited fungal root pathogens in vitro. Strain BR-5 was detected inside plant cells, indicating that it is able to colonize the endorhizosphere. No significant effect was detected on soil or ectorhizosphere microbial population after inoculation of strain BR-5 onto seeds.     

Root-Tip Smear Method for Difficult Material

A technic is outlined for the preparation of difficult material for the study of chromosome number and morphology in root-tip smears. The chief objective is to obtain polar views of the metaphase plates rather than equatorial views. To achieve this end it is recommended that fresh root-tips be cut free-hand into thin cross sections. The important features are: thin freehand cross sectioning of the fresh root-tips; fixing in Belling's iron aceto-carmine solution; maceration for 2-5 minutes in 50% HCl in 95% alcohol; and mounting in “Diaphane.”     

Handling Human Chromosomes by A Coumarin Technic

Small pieces of nonneoplastic and neoplastic human tissues, mainly from cervix uteri and soon after removal by biopsy, are transferred to a saturated aqueous solution of coumarin for 5-10 min, after which they are put into the regular aceto-carmine mixture for about 1 hr. A squash preparation is made with moderate pressure and the cover glass is sealed by a mixture made of equal volumes of Canada balsam and hard paraffin. These temporary preparations are suitable for the study of chromosomes for about 15 days, after which they can be made permanent by following one of the usual procedures that employ dehydration and clearing.