A Smear Technic for the Cucurbitaceae

It is very difficult to make satisfactory smear preparations of species in the Cucurbitaceae by ordinary methods because, (a) the differentiation between chromosomes and cytoplasm is poor, (b) the pollen mother cells are held together in tissue-like masses, (c) the chromosomes are comparatively small and numerous. Special procedures have been devised to overcome these difficulties. Staminate buds selected at the proper stage of maturity are fixed for a period of 12 to 24 hours in a mixture composed of 3 parts of 100% ethyl alcohol and 1 part acetocarmine to which a small quantity of iron acetate has been added. After prefixation the material is rinsed in several changes of 100% alcohol. It can then be transferred directly to the slide for smearing, or hydrated to 70% alcohol for storage. For smearing the anthers are dissected out, the extraneous flower parts discarded, and the storage fluid removed. A drop of acetocarmine diluted to one-half strength with 45% glacial acetic acid is added, and the anthers are macerated into small pieces and smeared. The anther debris is removed, and the cover slip added. It is necessary to carry out the above operations with a low power binocular microscope. After heating the cover slip can be sealed with Pyseal for temporary storage.     

A Controllable Carmine Technic for Plants with Small Chromosomes

Anthers of small chromosome plants (Antirrhinum, Brassica, Capsicum etc.) were fixed 12 hours or longer at 0-3° C. in: ferric acetate in glacial acetic acid (sat. soln.), 1 part; absolute alcohol, 3 parts. They were transferred to: ferric acetate (sat. soln.) in 45% acetic acid, 3 parts; 45% acetic acid, 5 parts; 1% formalin (aq.), 2 parts, and allowed to remain 5-15 minutes at room temperature for mordanting. The amount of iron introduced into the specimens was controllable by the time in the mordanting fluid. After rinsing the specimen in 45% acetic acid and macerating in a drop of Belling's acetocarmine on a slide, a cover slip was applied followed by warming and pressing with blotting paper to flatten the pollen mother cells and expel excess stain. Preparations stored temporarily by sealing the edges of the cover slip with rubber solution were best made permanent by removing the cover slip after 1-2 days, dehydrating and mounting in euparal.     

A Controllable Carmine Technic for Plants with Small Chromosomes

Anthers of small chromosome plants (Antirrhinum, Brassica, Capsicum etc.) were fixed 12 hours or longer at 0–3° C. in: ferric acetate in glacial acetic acid (sat. soln.), 1 part; absolute alcohol, 3 parts. They were transferred to: ferric acetate (sat. soln.) in 45% acetic acid, 3 parts; 45% acetic acid, 5 parts; 1% formalin (aq.), 2 parts, and allowed to remain 5–15 minutes at room temperature for mordanting. The amount of iron introduced into the specimens was controllable by the time in the mordanting fluid. After rinsing the specimen in 45% acetic acid and macerating in a drop of Belling's acetocarmine on a slide, a cover slip was applied followed by warming and pressing with blotting paper to flatten the pollen mother cells and expel excess stain. Preparations stored temporarily by sealing the edges of the cover slip with rubber solution were best made permanent by removing the cover slip after 1–2 days, dehydrating and mounting in euparal.     

A Procedure for Staining Pollen Nuclei When Obscured by Cytoplasmic Inclusions

The number and shape of pollen nuclei in cytologically refractive plants can be determined from anthers fixed in FAA, preserved in 70% ethanol, treated 10-30 min with 50% HCl in 95% ethanol, and stained with iron acetocarmine saturated with chloral hydrate, 1-16 hr. Pollen is then pressed from the anther into a drop of plain acetocarmine, warmed, examined and permanently mounted in Venetian turpentine medium. This procedure clears the cytoplasm of many opaque obstructions and frequently separates the exine from the cytoplast. It may be used with flowering material which has been stored several years at room temperature.     

The Use of Bismarck Brown Y in some new Stain Schedules

The staining quality of Bismarck brown Y may be improved and sterility maintained by adding 5% phenol to a 1% aqueous solution. Use the phenolic Bismarck brown in combination with iron alum hematoxylin except for stripped epidermis in the following procedures:

Stem and Root Schedule: Mordant sections from water in 4% iron alum for 10 minutes. Rinse in distilled water and stain in 0.5% aqueous hematoxylin for 1 minute or until darkly stained. Rinse in distilled water and destain in 2% iron alum until a gray color appears. Rinse thoroly in distilled water and intensify hematoxylin by transferring sections to 0.5% aqueous lithium carbonate until the desired black color appears. Rinse thoroly in distilled water and stain for 1-5 minutes in phenolic Bismarck brown. Rinse in distilled water, dehydrate successively in 30, 50, 70, 95 and 100% alcohol. Clear in methyl salicylate for 5 minutes, then to xylene for 3-5 minutes, and mount in balsam.

Middle Lamellae in Wood: Destain more thoroly in 2% iron alum than for the general stem and root schedule, and intensify in lithium carbonate for a longer period (about 1 hour).

White Potato Tuber Sections: Modify above schedule by reducing time of destaining in 2% iron alum to about 30-60 seconds and intensify hematoxylin until starch grains appear bluish in color. Stain in phenolic Bismarck brown for 1-2 minutes.

Wheat Grain Sections: Fix grain for sectioning when in “dough” stage. Use schedule the same as for potato tuber except for reducing time of staining in phenolic Bismarck brown to about 45 seconds.

Tradescantia zebrina Epidermis: Strip epidermis from leaf while submerged in water. Fix in 100% alcohol 10 minutes, pass thru 95, 70, 50, 30, and 10% alcohol to water. Stain in phenolic Bismarck brown for 10-20 minutes. Dehydrate, clear in methyl salicylate and mount in balsam.     

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.     

Sudan Black B And Acetocarmine as a Combination Stain

Epidermis stripped from either fresh or fixed plant organs, or sections of paraffin-embedded or fresh material are placed on a slide and covered with a drop or two of iron-acetocarmine. The stain is intensified by warming the slide over a flame. After a few minutes a drop or two of a saturated solution of Sudan black B in 45% acetic acid is added and a cover slip applied. The preparations cannot be made permanent, but last a few weeks if sealed with a compound such as gum mastic-paraffin, or if the combined stain is drained off and a drop of Karo syrup is added before the cover slip is applied. The acetocarmine produces its usual staining effects, i.e., nuclei dark red and some components of the cytoplasm of certain cells a less intense red. The Sudan black B colors lipid structures an intense blue.     

Simple Aids to Microscope Illumination

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.     

A Method for Making Aceto-Carmin Smears Permanent

Anthers are collected and placed in a solution of 1 part acetic acid to 3 parts of absolute alcohol. The contents of the anther are squeezed out on a slide in a drop of Belling's iron-aceto-carmin solution and a cover glass placed over the drop. Care should be taken to remove all anther walls and flower parts. Heat the slide over an alcohol flame for a second, repeating 4 or 5 times. Place the slide in a petri dish filled with a 10% solution of acetic acid. When the cover glass has risen away from the slide gently remove the cover glass and place in a Coplin jar containing equal parts of alcohol and acetic acid. Likewise, place the slide in this solution. Run both cover and slide thru the following solutions: 1 part acetic acid to 3 parts absolute alcohol, 1 part acetic acid to 9 parts absolute alcohol, absolute alcohol and finally equal parts of absolute alcohol and xylol. Recombine the cover and slide in xylol-balsam directly from this solution.     

A Method for Making Aceto-Carmin Smears Permanent

Anthers are collected and placed in a solution of 1 part acetic acid to 3 parts of absolute alcohol. The contents of the anther are squeezed out on a slide in a drop of Belling's iron-aceto-carmin solution and a cover glass placed over the drop. Care should be taken to remove all anther walls and flower parts. Heat the slide over an alcohol flame for a second, repeating 4 or 5 times. Place the slide in a petri dish filled with a 10% solution of acetic acid. When the cover glass has risen away from the slide gently remove the cover glass and place in a Coplin jar containing equal parts of alcohol and acetic acid. Likewise, place the slide in this solution. Run both cover and slide thru the following solutions: 1 part acetic acid to 3 parts absolute alcohol, 1 part acetic acid to 9 parts absolute alcohol, absolute alcohol and finally equal parts of absolute alcohol and xylol. Recombine the cover and slide in xylol-balsam directly from this solution.     

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 Temporary Stain for Paramecium and Other Ciliate Protozoa

A new temporary stain for the demonstration of nuclear and cytoplasmic structures in Paramecium and other protozoan ciliates during both vegetative reproduction and meiotic reorganization consists of a mixture of 10.5 parts of acetocarmine, 4.5 parts of 45% acetic acid, 2 parts of 1 N HCl and 1 part of 1% solution of fast green FCF in 95% alcohol. This stain replaces the acetocarmine and acidified methyl green nuclear stains commonly employed and has the following advantages: (1) it affords simultaneous differential stainability of nucleus and cytoplasm (brown-red and green to grey-green, respectively); (2) it provides differential stainability of newly developing macronuclei (homogeneous pale green), fragments of the old macronucleus (brown-red), and food vacuoles (granular, bright blue-green); and (3) it results in a delicate and more transparent stain which affords greater clarity of internal structure. Proportions may be shifted slightly to achieve the optimum results for any particular organism. Concentrations of the acids employed may be diluted in instances where organisms tend to be easily distorted by fixation.     

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.     

Cleared Whole Mounts of Shoot Apices of Angiosperms for Topographic Study

Cleared and stained whole mounts of stem apices of two Labiates and of Phaseolus plumule giving a three-dimensional picture of the apical structure have been prepared as follows. Fix the buds in formalin-acetic acid-50% alcohol (5:5:90) for 24 hr or longer and then dissect under a binocular microscope to leave only the youngest leaves surrounding the apex. Wash for several minutes in distilled water and then clear the material in a 5% solution of sodium hydroxide at approximately 40° C for 24-48 hr. Wash thoroughly in several changes of distilled water, transfer to a solution of 1% tannic acid and 0.5% sodium salicylate for up to a minute. Wash briefly in distilled water and stain in a 1.5% solution of ferric chloride until blue-black. Wash in distilled water and dehydrate through 50%, 70%, 85%, 95% and 2 changes of absolute ethyl alcohol. If the xylem is not stained well, counter-stain for a few seconds in a 0.5% solution of safranin O in a 1:1 mixture of xylene and absolute alcohol and wash out the excess stain in the same mixture. Clear in 2 changes of xylene and place on a glass slide in thick Canada balsam. Orient with needles under low magnification and cover.     

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.     

A Urea Silver Nitrate Method for Nerve Fibers and Nerve Endings

A silver nitrate stain for nerve fibers and endings applicable to paraffin sections on the slide utilizes the properties of urea to accelerate the procedure and improve the specificity of the stain. After removal of the paraffin the sections are run through absolute, 95% and 80% alcohol and placed for 60-90 minutes at 50-60°C. in: 1% aqueous silver nitrate, 100 ml.; urea, 20-30 g.; 1g. mercuric cyanide and 1 g. picric acid in 100 ml. of distilled water, 1-3 drops. After the silver bath they are rinsed quickly in 2 changes of distilled water and reduced for 3-5 minutes at 25-30°C. in: water, 100 ml.; sodium sulfite, anhydrous, 10g.; hydroquinone, 1-2g.; urea, 20-30g. They are then washed thoroughly in 4-5 changes of distilled water, passed through graded alcohols into 80% alcohol and examined under the microscope. If nerve fibers are not distinct, the sections are returned to the same urea-silver-nitrate bath for 10-15 minutes, rinsed, reduced, washed and dehydrated as before. This process may be repeated until staining is adequate; then they are dehydrated, cleared, and mounted.

Nerve fibers show a color range from brown to black; nerve cells from yellow to brown; and the background, depending on the type of tissue and its fixation, from yellow to light brown.     

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.     

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.     

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.     

Staining Plant Tissues with Chlorazol Black E and Pianese III-B

The staining schedule was developed for a study of the mycorrhizae of red pine, Pinus resinosa Ait. From 70% alcohol, sections are stained in a saturated solution of chlorazol black E in 70% alcohol, 10-30 min; free dye removed by washing in 95% alcohol; stained 18-24 hr in Pianese III-b; rinsed in 95% alcohol, acidified by the addition of 2 ml of saturated aqueous picric acid per 100 ml, 3-4 changes or until the last change is pale yellow or light green; and rinsing in 95% alcohol to remove the acid. If the acid fuchsin is too intense, a cautious differentiation with 95% alcohol containing 1-3% of a 0.1 N solution of NaOH is made. If too much chlorazol black is removed, the effect can be compensated by overstaining with this dye at the beginning of the process. Sections are dehydrated, cleared, and covered in the usual manner. This stain has applications to plant tissues generally, and is particularly effective for meristematic tissues. It shows details of cytoplasmic structures and gives sharp delineation of primary cell walls.