Permanent Preparations from Rapid Cytological Technics

The following rapid but reliable method of making permanent preparations from temporary mounts has proved to be very useful.

Pollen mother-cell smears: Smeared anthers are treated hi the usual way with Belting's acetocarmine, except that the cover slip is left off. When correct differentiation is attained the stain is thoroly washed off with 50% acetic acid and the slide flooded with dioxan. This is followed by 2 changes of dioxan for 2 minutes each. A drop of Canada balsam dissolved in dioxan is added and a cover slip applied. In cases where a cover slip has been used at the acetocarmine stage it can be floated off in a staining jar of 50% acetic acid and dehydration with dioxan carried out as above.

Insect salivary gland chromosome smears: The glands are crushed under a cover slip in acetocarmine on a slide coated with dried egg albumen. After 20 minutes the area around the cover slip is flooded with 50% acetic acid and the cover slip floats loose so that it can be removed. The above described dioxan dehydrating procedure is then employed.

Squash preparations: Root tips are fixed in some suitable fixative and the Feulgen technic applied. The stained root tips can either be dehydrated by passing thru 3 changes of dioxan and mounting in dioxan-balsam where they are divided into small longitudinal sections by sharp needles, or they can be put immediately into a mixture of 1 part of 50% acetic acid to 1 part of corn syrup where shredding with needles is carried out. A cover slip is put on and separation of the cells completed by tamping or by applying pressure to the cover. This squash method is useful with anthers which are difficult to smear when in the early prophase stages of meiosis.     

Notes on Selling'S Green-Light Method for Critical Microscopy (Continued)

Kill root tips in 1 part glacial acetic acid to 3 parts absolute alcohol for 12 or more hours. Remove from killing fluid and place for 5 to 10 minutes in a solution consisting of 1 part 95% alcohol to 1 part concentrated HCl. Transfer to Carnoy's fluid for 5 minutes or longer. Cut a small piece (0.5 mm. or less) off the tip of the root and place on a clean slide in a small drop of iron-aceto-carmin stein. Press directly on the piece of root with a small flat scalpel; the cells will now separate and float free in the stain. Place cover slip over the drop of stain and apply gentle pressure. Heat carefully by passing the slide 3 or 4 times thru the flame of an alcohol lamp. Seal with heated mixture of 1 part Parowax to 1 part gum mastic. Make permanent by the McClintock permanent method.     

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.     

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.     

A Modification of the Cresyl Violet Technic for Staining Nerve Cells

Kill root tips in 1 part glacial acetic acid to 3 parba RB Solute alcohol for 12 or more hours. Remove from king fluid a d place for 5 to 10 minutes in a solution consisting of 1 part 95% alcohol to 1 part concentrated HC1. Transfer to Carnoy's fluid for 5 minutes or longer. Cut a small piece (0.5 mm. or less) off the tip of the root Press directly on the piece of root with a small fiat scalpel; the cells will now separate and float free in the stain. Place cover slip over the drop of stain and apply gentle pressure. Heat carefully by paseing the slide 3 or 4 times thru the flame of an alcohol lamp. Seal with heated mixture of 1 part Parowax to 1 part gum mastie. Make permanent by the McClintock permanent method. and place on a clean slide in a small drop of iron-ace-sinin.     

Staining Nuclei and Chromosomes in Protozoa

Technics for free-living forms such as Paramecium and for parasitic forms such as the opalinid ciliates are described.

Paramecium: Fix paramecia in hot Schaudinn's fluid containing 5% of glacial acetic acid for 5-15 minutes. (A hot water bath for maintaining the proper temperature of the fixative is described.) Dehydrate up to 83% alcohol. Mount the specimens on albuminized cover glasses. (A table for mounting animals on cover glasses is described.) Apply a thin layer of collodion to the cover glass to prevent the loss of the specimens during the subsequent handling. Pass through descending grades of alcohol to water. Mordant in 4% iron alum for 24 hours. Stain in 0.5% hematoxylin for 24 hours. Destain in saturated aqueous picric acid. Rinse in tap water, expose to ammonia vapor for a second, and then rinse again in tap water. Wash in running water for 1 hour. Dehydrate. Clear, then mount in damar.

Opalinid Ciliates: Make smears on cover glasses and fix them while wet. If the opalinids are to be subsequently stained in hematoxylin, fix in hot Schaudinn's fluid (containing 5% of glacial acetic acid) for 5-15 minutes. Pass through descending grades of alcohol to water. Mordant in iron alum for 24 hours. Stain in hematoxylin for 24 hours. Destain in saturated aqueous picric acid. For Feulgen reaction, fix in a modified weak Flemming's fluid for 1 hour. Wash in running water for 30 minutes. Hydrolyze. Leave 3 hours in fuchsin decolorized with H₂SO₃ (Feulgen formula). Wash in H₂SO₃, then in running water for 15 minutes. Dehydrate up to 95% alcohol. Counterstain with fast green FCF for 2 minutes. Dehydrate in absolute alcohol. Clear, then mount in damar.     

The Fixation of Tissues Vitally Stained with Trypan Blue

The following fixative is recommended for tissues vitally stained with trypan blue: Chloroform, 2 parts; absolute ethyl alcohol, 2 parts; glacial acetic acid, 1 part; mercuric chloride to the point of saturation.

The tissue should be fixed 1 to 2 hours; transferred to 95% ethyl alcohol for 12 hours; to absolute alcohol for 12 to 24 hours; to a mixture of absolute alcohol and xylol for 1/2 hour, and finally to xylol, before embedding in paraffin. Cedar oil may be used for clearing in the place of xylol; in that case the tissues should be transferred from absolute alcohol to a mixture of absolute alcohol and cedar oil for 24 hours before placing in cedar oil alone.

Various counterstains can be used; Mayer's carmalum is excellent.     

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.     

Marchi's Staining Method: II. Fixation

Following experimental lesions, spinal cords of cats and rabbits were fixed with acid, neutral, and alkaline solutions. Staining was limited to a chromate-osmic (Marchi's) solution and a chlorate-osmic solution. The following conclusions were drawn:

The presence of an acid in the fixative caused normal myelin sheaths to stain. This effect was reduced by washing tissues before staining, by adding acetic acid to the stain, or by employing a non-formalin fixative. It was, however, at no time entirely obviated.

A study was made of the granular deposits which occur in nearly all Marchi preparations and which are especially confusing if very light backgrounds are obtained.

The staining reactions of the granular deposits were very similar to those of degenerating myelin but some suppression of the granules was obtained by adding KCIO₃ to the formalin fixative.     

Preliminary Notes on Chromic Fixation in Alcoholic Media

In Note I are pointed out certain chemical difficulties which stand in the way of experiments on chromium fixation in alcoholic media; also a procedure is described by means of which these difficulties may be avoided in the preparation of a chrome-alcohol stock solution.

In Note II it is suggested that the properties of OsO₄ most important in the quick-killing effect conferred by this adjuvant upon aqueous chromic reagents are probably, in addition to its high toxicity, its volatility and its oxidant action. Volatile, toxic oxidants which do not attack alcohol are therefore discussed briefly, with reference especially to their use as quick-killing adjuvants in chrome-alcohol reagents. Iodine is noted as probably the most important inorganic possibility. Reasons are stated for considering the quinones the most promising of numerous possible organic compounds.

Results of fixations of Vicia faba root tips in nine variations of the proportions of acetic acid and iodine in a chrome-alcohol-aceticiodine combination are described. The chrome-alcohol-iodine reagents which proved best proportioned for V. faba root tips preserved some details of the chromosomes better than does Bouin's solution, but did not make certain details of the early prophase as clear as does Benda's modification of the Flemming reagent, according to a comparison with Sharp's figures of Bouin and Benda preparations of root tips of the same species.     

An Improved Technic for the Examination of Chromosomes in Root Tip Smears

Young, vigorous root tips are fixed in aceto-alcohol (1 part glacial acetic acid, 2 parts absolute alcohol) and are left in the fixative from 24 to 48 hours. If it is desired to store the material, the root tips can be transferred to 80% alcohol and be kept indefinitely. In preparing the smears, the root tips are placed on a slide and are sliced as thinly as possible with a sharp razor blade. Then the slices are smeared on the slide and immediately flooded with aceto-carmin, followed by a cover glass. Using absorbent paper and exerting considerable pressure, the excess aceto-carmin can be removed and the material flattened at the same time. Finally the slide is warmed gently to a point slightly below boiling. By sealing with gum mastic and paraffin, such preparations can be kept from 5 to 10 days.     

A Rapid Field Technique for Preparing Ant Chromosomes for Karyotypic Analysis

This technique for chromosomal preparation of ant tissues for karyotypic analysis is advantageous under field conditions because it reduces processing time and can be used under humid conditions. The cerebral ganglia from prepupae or early pupae are incubated 20 minutes in a hypotonic citrate solution, minced in a fixative solution of 3:3:4 glacial acetic acid: absolute methanol: distilled HOH, rinsed in a fixative solution of 1:1 glacial acetic acid: methanol followed by Carnoy's fixative, then immediately flame dried. The resulting metaphase chromosomes are well spaced and usually show banding characteristics.     

Prefixing with Paradichlorobenzene to Facilitate Chromosome Study

A technic was developed which resulted in preparations containing many mitotic divisions with chromosomes well fixed and stained, rod-shaped, and spread throughout the cell. This technic has given good results with guayule (Parthenium argentatum), Crepis, Allium, Pisum, Lycopersicon, Tradescantia, and other plants. Material is prefixed in a saturated solution of paradichlorobenzene for 1-4 hours, fixed in 65% acetic acid (or other suitable fixative) for 12-24 hours, hydrolyzed in 10% HCl for 10-30 minutes at 60° C, rinsed in water, transferred to a drop of 45% acetic acid on a slide, and smeared and stained in aceto-orcein. The preparation may be made permanent by separating slide and cover glass in 1 part glacial acetic acid to 1 part absolute alcohol, putting them in absolute alcohol, and then recombining them with a drop of euparol.     

A Simplified Stain for Hemoglobin in Tissues Or Smears Using Patent Blue

The following technic, based on the patent blue V hemoglobin reaction, is useful for identifying hemoglobin in tissue fixed in neutral formaldehyde solution and embedded in paraffin:

Stain the deparaffinized, hydrated sections 3 to 5 minutes in the working reagent, prepared by adding 2 ml. of glacial acetic acid and 1 ml. of 3% hydrogen peroxide to 10 ml. of the filtered stock solution (1 g. patent blue, 10 g. zinc powder, and 2 ml. glacial acetic acid). Counterstain 30 to 60 seconds in 1:1000 safranin solution in 1% acetic acid, rinse, dehydrate with alcohols, clear in xylene and mount in clarite. Total time required, 37 minutes.

Blood and tissue and smears may be stained, following fixation in methyl alcohol, by applying the working reagent as above.     

A Combined Hcl-Acetic-Alcohol Fixation and Hydrolysis Followed by Cresyl Violet Staining for Mosquito Chromosome Spreads

Mosquito tissues of cytogenetical importance were dissected out on a slide in 0.65% NaCl, under a dissecting microscope, and treated about 30 sec in a drop of 1:3 Carnoy's fixative diluted 1:19 with distilled water. Fixing and hydrolysis was done by a single step in a mixture consisting of: glacial acetic acid, 1; ethanol 96%, 3; HCl conc., 2; and distilled water, 2 (v/v) for 2-6 min at 20-25 C. The specimen was then rinsed with the acetic-alcohol fixative and covered in a drop of 1% cresyl violet in 50% acetic acid under a coverslip coated with Mayer's albumen. Washing was performed immediately by adding water dropwise to one side of the coverslip and drawing the fluid from the other side with absorbent paper. The preparation could be used either as a temporary slide or made into a durable mount. The DNA-containing bands of the giant polytenic chromosomes stained dark violet; interband regions, weakly stained or colourless against a clear background. Mitotic and meiotic figures in gonadal cells stained selectively dark violet or violet with a practically unstained cytoplasm.     

The Dry Preservation of Fixed Plant Material

A method for the dry-preservation of fixed plant material, root tips and buds, is described. The method seems to be advantageous on long expeditions and when material has to be sent away.

The material is transferred from the fixative to 70% alcohol (3 changes, 1/2 hour in the last). It is dried on blotting-paper. The dried material may be preserved a long time. Material kept dry for 4 1/2 years has proved to be quite satisfactory. Drying has been tried after fixation with CRAF-solutions (Webber and Randolph modifications) and fixatives containing osmic acid (Fleming-Benda and 2BD).

The dry material is swollen by keeping for 2 days in 10% alcohol. It is embedded in paraffin according to the usual method. A satisfactory staining has been obtained after these fixatives using iodine-gentian-violet and Feulgen stainings. In addition to chromosome counts dry material may be used for chromosome morphology studies.

Dried material fixed in aceto-alcohol (1:3) has not turned out to be specially suitable for squash preparations owing to the fragility of the chromosomes. If strong pressure is not applied, satisfactory results may, however, be obtained.     

Technic for Studying Chromosome Structure

The methods described are modifications of various technics for the study of spiral structure in chromosomes. They enable permanent preparations to be made with better fixation and allow the use of stains which give clear and more critical definition. The first method described involves the use of ammonium, hydroxide (880 vols.) fumes for the treatment of pollen mother cells before fixation. Anthers of Tradescantia are smeared on a slide and wet in a 3% cane sugar solution. The preparation is then immediately placed in a dish of fixative where it remains for two hours. The slide can then be washed, bleached and stained with gentian violet or hematoxylin. It was found that fumes of nitric acid, hydrochloric acid and glacial acetic acid gave similar results. For the second method, boiling water is used for pre-treatment. A smear is made on a slide and immersed in boiling water for five to ten seconds. The smear is then fixed and treated in the usual manner.     

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.     

Simple Methods for Mammalian Chromosomes

The ordinary Feulgen-squash technic, after acetic-alcohol fixation, provides a simple and reliable method of preparing many mammalian tissues for chromosome counts. Tissues best suited to the technic were the seminiferous tubules. Small pieces of tissue about 3-6 mm. long and 1-2 mm. wide were immersed in a freshly made fixative (composed of 3 parts absolute ethyl alcohol and 1 part glacial acetic acid) immediately after removal by biopsy or from a freshly killed animal. After fixation for 3-12 hours the tissue was stained by the standard Feulgen procedure after hydrolysis for 12 minutes in normal HCl at 60 °C A 1-2 mm. piece was then teased apart on a slide in 45% acetic acid with a pair of mounted needles, and the teased tissue was squashed between the slide and the cover slip. During squashing the pressure was applied by hand and was regulated so as to avoid any excessive scattering of the chromosomes. The preparations were made permanent by dehydrating and mounting in Euparal.     

A simple method of preparing permanent squash preparations using cellophane]

A technique to prepare permanent squashed preparations of cell nuclei and chromosomes is proposed. Fix a piece of material on the slide with acetic alcohol (1:3), macerate with a 45% acetic acid, cover with hydrophilic cellophane previously soaked in a 45% acetic acid and then with a cover slip and filter paper to squash finally as it is routinely performed. After that soak off the cover slip with alcohol, post-fix the squashed preparation together with cellophane in alcohol for 5-10 min, unstick the cellophane, pass the preparation through alcohol once again and dry it. The subsequent treatment of the squashed preparation depends on the purpose of investigation. The slide may be tinctured overlaid with photoemulsion for autoradiography, or processed by different ways.