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.     

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.     

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.     

Chromosomes from Herbarium Sheets of Impatiens

In northwestern Himalayan species of Impatiens, the pollen grains at shedding possess a crescent shaped generative nucleus in prometaphase from which the chromosome number is easily determined. The fresh or fixed pollen grains are studied by the usual aceto-carmine squash method. The generative nucleus generally remains in the same state in pollen of the herbarium specimens. Anthers from such material are soaked overnight in a saturated solution of iron acetate in 45% acetic acid, rinsed and squashed in dilute aceto-carmine. Preparations are heated several times to boiling. Reliable counts of chromosomes have been made from such slides.     

Smear Methods for the Study of Chromosomes in Ascomycetes

Several modifications of the aceto-carmine smear technic used by the author in studies of the meiotic chromosomes of Neurospora are outlined. The critical role of the staining solution is stressed and suggestions are given for preparing a satisfactory carmine solution. The ease and rapidity of this method suggests its use in cytologic work on other Ascomycetes.     

Procedure to Facilitate Chromosome Counts in Difficult Plant Material

Numerous mitotic plates of contracted and well-spread chromosomes may be obtained from root tips of plants set on melting ice or snow overnight at room temperature. After 1:3 acetic-alcohol fixation for 0.5 to 3 hours the material is mordanted in a mixture of 7 parts of alcohol plus 21/2 parts of 3% ferric ammonium sulfate for 3 hours to overnight. This solution may be used as storage fluid for flower buds. Deep chromosome coloration without precipitates is secured by staining in a few drops of aceto-carmine for 10-15 minutes after which the tissues are softened by heating in aceto-carmine diluted with 3 parts of 45% acetic acid.     

A Mordanting Fixation for Intense Staining of Small Chromosomes

A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)₃ per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)₃ in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.     

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.     

A Mordanting Fixation for Intense Staining of Small Chromosomes

A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)₃ per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)₃ in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.     

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.     

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.     

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.     

The Use of Haematoxylin for Squash Preparations of Chromosomes

A simplified propionic-iron alum-haematoxylin stain for rapid squash preparations of chromosomes requires only two stock solutions: (A) 2% haematoxylin and (B) 0.5% iron alum, both in 50% propionic acid. For use, suitable volumes of A and B are mixed. With unripened solution A, equal volumes should be used and the stain is ready for use 1 day after mixing. As the haematoxylin ripens, progressively smaller amounts of B are required and the mixture may be used immediately. The stain gives excellent results when used in the same way that orcein and carmine are currently employed, with a wide range of animal and plant (including fungal) chromosomes, and with good nucleolar staining. It may be used either following acetic alcohol (1:3) fixation or as joint fixative and stain on unfixed material. In fungal material, where Lu's BAC fixative is recommended, the centrioles are also stained.     

Suggested Counterstains for Davenport Reduced Silver Preparations of Peripheral Nerves

—Peripheral nerves which have been fixed in a mixture of formaldehyde and acetic acid and stained according to the method of Davenport can be successfully counterstained for demonstration of myelin sheaths and stroma. After mounted sections have been silvered, reduced and toned, the coating of nitrocellulose is removed by passing thru two changes of acetone. Following brief washes in 100,95,85 and 75% alcohols they are stained in an acidified aqueous solution of azo carmine for 30 to 60 minutes. Excess azo carmine is extracted with anilin alcohol followed by acetic alcohol after which the sections are mordanted for 15 to 60 minutes in a 5% aqueous solution of phosphotungstic acid. Without washing they are transferred to a stain mixture of either anilin blue and orange G (acidified) or light green and orange G (acidified) where they remain from 1 to 5 hours. After destaining in 95% alcohol and dehydration in absolute alcohol the sections are mounted in dammar. Result: axons stain black; sheath and fibroblast nuclei, red; myelin sheaths, orange; and connective tissue, blue or green. When the counterstains are applied to ganglia, cytological details of individual cells are demonstrated.     

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.     

A Microspectrophotometric Analysis of Staining with Carmine, Orcein and Carmine-Orcein

Microspectrophotometric measurements of carmine, orcein and carmine-orcein were made in solutions, in air-dried films and in stained adult and embryonic tissues of the domestic chicken. For individual stains only minor differences were found between dried dye and stained tissue. The absorption curve for carmine in solution showed a single peak at 490 mμ but was bimodal at about 530 and 570 mμ in dry films and stained tissue. Orcein showed a single broad peak at 510 mμ in solution; in dry films and stained tissue a broadening of the absorption curve in the red wavelengths was observed. The dye mixture carmine-orcein in solution showed a single peak at 500 mμ, but in tissue the spectral absorptions closely resembled carmine. With alum-like carmine, spectral changes due to the addition of iron were not detected. The results indicate that nuclear staining with carmine-orcein is due mainly to the carmine component of the mixture. Interpretation of spectral shifts indicates that acew-carmine is a metachromatic stain while aceto-orcein is mainly an ortho-chromatic stain, although some metachromasy is evident.     

Brilliant Cresyl Blue as A Stain for Chromosome Smear Preparations

Brilliant cresyl blue is substituted for carmine in the acetocarmine technic. A mixture of one part of 1% toluidine blue and three parts 0.75% brilliant cresyl blue dissolved in 45% acetic acid gives good results. Propionic acid may be used instead of acetic acid for more rapid penetration.     

An Improved Pollen Grain Smear Method for Wheat

Anthers containing actively dividing pollen grains were treated 1 hour at 18-20° C. with 0.2% solution of colchicine, washed 1 hour in water, soaked in 0.002 M aqueous solution of 8-oxyquinoline at 10-14° C. for 1 hour, washed in water for 1 hour and then fixed in Carnoy's solution (alcohol, chloroform, acetic acid, 6:3:1) for 6 hours to overnight. They were washed successively in acetic-alcohol (1:1) 10-15 minutes, 70% alcohol 10-15 minutes and in water 30 minutes before hydrolysing them in bulk in 1 N HCl at 60° C. for 10-15 minutes. “Finally, they were stained in leuco-basic fuchsin for 15-30 minutes. Pollen grains were squeezed out of a stained anther in a small drop of egg albumen on a slide and the albumen smeared uniformly on the slide. The slide was dipped successively for a few seconds in glacial acetic acid and 45% acetic acid respectively. The smear was covered by a cover glass in a drop of aceto-carmine and pressed gently between folded filter papers. The cover glass was sealed with paraffin and stored overnight. To make the preparation permanent the paraffin was removed and the cover glass separated in a 1:1 mixture of acetic acid and n-butyl alcohol. The slide and the cover glass were then passed through n-butyl alcohol, 2 changes, and finally remounted in balsam.     

Recent Advances in Microtechnic. I. Methods of Studying the Development of the Male Gametophyte in Angiosperms

Among methods used for a study of nuclear details in the development of pollen grains, the following were found to be very satisfactory: (1) warming the entire grains in aceto-carmine and then clearing with chloral hydrate; (2) making smear preparations stained with crystal-violet-iodine or iron alum hematoxylin. For paraffin sections, a counterstain with dilute alcoholic erythrosin is often very useful after the usual iron hematoxylin technic.

A method of making cultures of pollen tubes on slides coated with thin films of sugar agar is described in detail. The tubes can be fixed by immersing the slide in formol-acetic-alcohol and then stained by any desired schedule. Iron alum hematoxylin was found to be the most satisfactory, but the Feulgen reaction is very valuable in such cases where the nuclei are obscured by the density of the pollen tube cytoplasm. Living pollen tubes can be kept under observation by dissolving a small quantity of neutral red or other vital stain in the sugar agar before it is spread on the slide.

For studying stages in fertilization or gametogenesis, styles should be fixed and sectioned only after a preliminary study with iodine-chloral-hydrate or safranin-anilin-blue or aceto-carmine. Once the extent to which pollen tubes grow in a given time in the stylar tissues has been determined, it is possible to fix material with some knowledge of what it is going to show.

Some other methods, that have not been tried by the authors but appear to be valuable, are also briefly described.     

Control of the Ferric Ion Concentration in Iron-Aceto-Carmine Staining

The Fe⁺⁺⁺ concentration is controlled by adjusting the FeCl₃ normality of the iron-aceto-carmine staining solutions. Two stock mordant solutions are prepared by dissolving ferric chloride (FeCl₃·6H₂O; m. w. =270.31) in 45% glacial acetic acid, the normality of one being N/1, and of the other N/10. By combining aceto-carmine (preferably prepared from Merck's carmine No. 40 N. F., or from the Coleman and Bell product) and one or the other of the stock mordant solutions, a series of iron-aceto-carmine solutions is made up, each solution being of different normality for FeCl₃, depending on the proportions combined. Trial series: N/50, N/100, N/500, N/1000 and 0 N.

Tissue (spermatogenic) from one specimen is fixed in Carnoy-2, divided equally among the five iron-aceto-carmine solutions for staining, then squashed, dehydrated and mounted as usual. Subsequently the trial series may be retained or adjusted. Advantages of the method: 1) discloses quickly the optimum stain for a particular tissue type; 2) automatically gives an optimum stain to cells in different maturational stages; 3) results are reproducible in subsequent operations.

Tables and equations are provided for a number of other normalities and quantities of stain.