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.     

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.     

Permanent-Type Mounting of Aceto-Iron-Haematoxylin Squashes in Corn Syrup

Preparations obtained by the aceto-iron-haematoxylin technique reported previously (Stain Tech., 37, 27-30, 1962) can be made relatively permanent either by ringing the cover slip with Karo corn syrup, or by mounting the squash in this syrup after separating slide and cover slip by the solid CO₂ freezing technique. In the latter procedure, both slide and cover slip may be placed briefly in 45% acetic acid for further differentiation of the stain, then recombined with a drop of syrup.     

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.     

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.     

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.     

Elimination of Material that Obscures Stained Chromosomes in Squashes of Vicia faba Root Tips

A procedure for elimination of cytoplasmic debris from Vicia faba root tip cells is: (1) a root tip previously fixed in 3:1 absolute alcohol-acetic acid and stained by the Feulgen method is placed on a slide and squashed in a small drop of water, (2) a cover slip is applied and the cells are flattened with a hand-operated lever device supplying 35 pounds pressure onto a 22 × 22 mm cover glass, (3) the slide is quick-frozen, the cover slip is removed, and the slide is dropped immediately into water, (4) the slide is cleared through an alcohol-xylene dehydration series and permanently mounted. The significant result of this procedure is the consistent presence of clear, flat cells showing excellent definition of chromosomes.     

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.     

Permanent Mounts Of Chromosomes After 8-Oxyquinoline and Squashing

Fresh young root tips or free-hand cross sections thereof were placed in 0.002 M 8-oxyquinoline (aq.) at 10-14^oC. for 3 hours. After rinsing in water 1-2 minutes, they were soaked in N HC1 at 55^oC. for 25 minutes, rinsed again and squashed under a cover glass on a dry slide. Slide and cover glass were separated by placing in 70% alcohol and allowed to remain therein at least 0.5 hour after separation. Both slide and cover glass were passed through 50% and 30% alcohol to water and stained by the Feulgen procedure (without further hydrolysis) or with crystal violet after mordanting in 1% chromic acid overnight and washing in running water 3-4 hours. Dehydration and mounting in balsam completed the process. The smear on the slide was covered with a clean cover glass and the cover glass, bearing stained material, mounted separately.     

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.     

An Acetocarmine Technic for Cucurbita

Staminate Cucurbita buds undergoing meiosis are fixed for 12-24 hours in a solution containing 3 parts of 95% alcohol, 1 part of acetocarmine to which iron acetate has been added, and a flake of rusted iron. After fixation the buds are washed in 95% alcohol and stored in 95% alcohol with the iron flake for 5-10 days. A stain containing 10 drops of 45% acetic acid, 10 drops of acetocarmine, and 10 drops of brown storage solution is prepared. A small piece of anther is placed in a drop of stain on a slide. At the moment the anther is macerated, the debris is removed, and when the cells turn grey to dark brown a cover slip is applied. The stain is differentiated by gentle heat and the cover slip is sealed with paraffin.     

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.     

Aceto-Iron-Haematoxylin-Chloral Hydrate for Chromosome Staining

Aceto-iron-haematoxylin can be used combined with the clearing agent chloral hydrate for the squash method. The stain is prepared by dissolving 2 gm of chloral hydrate in 5 ml of a stock solution of 4% haematoxylin and 1% iron alum in 45% acetic acid, which has been allowed to ripen for 24 hr to 1 wk. Heat must not be used to hasten solution. The material (fixed in 1:3 acetic-alcohol) is put on a slide, the fixative removed and a drop of stain added; if necessary the material is crushed before the cover slip is placed in position. The preparations are now carefully heated until a slight colour change occurs. Squashing needs more pressure than in other techniques. This stain gives best results in zoological and botanical material not requiring hydrolysis, e.g., leucocytes, ascites cells, and cells undergoing spermatogenesis and microsporogenesis. Well-spread and selectively stained mitotic and meiotic figures can be obtained.     

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.     

Squashing Under Scotch Tape No. 665 for Autoradio-Graphic and Permanent Histologic Preparations

Removal of the cover slip from squash preparations, for coating with auto-radiographic emulsion, or other purposes, is made easy if squashing is performed with a piece of Scotch double-coated adhesive tape No. 665, used as a cover slip. The material to be squashed is placed on a slide lightly coated with an adhesive consisting of 1% gelatin with 0.1% chrome alum added. The piece of tape is applied with the surface originally on the outside of the roll next to the specimen. Specimens should be soaked before squashing in aqueous 45% acetic acid, with or without added dye, such as carmine or orcein. After squashing, the tape is easily removed without damage to the cells. This allows autoradiographic emulsion to be applied, or, unstained material can be stained after squashing by technics suitable for microtome sections.     

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 New and Rapid Method for Making Permanent Aceto-Carmin Smears

Smear the pollen mother cells of a single anther from each flower bud on a clean dry slide, using a small scalpel. Flood the slide with Belling's acetocarmin and heat for a second over an alcohol flame. Examine under the microscope to determine the stage of microsporogenesis. If the stage is satisfactory, smear the remaining anthers in the same manner, but fix and stain them by immediate immersion, face downward, in a petri dish full of hot (steaming) acetocarmin for from 1 to 10 minutes. Then rapidly transfer thru the following mixtures: two parts 99% (glacial) acetic acid plus one part absolute ethyl alcohol; one part acetic acid plus two parts absolute alcohol; and finally one part acetic acid plus nine parts absolute alcohol. The slides are then to be dehydrated completely by 1 to 2 minutes immersion in pure absolute alcohol, and cleared 2 to 3 minutes in a mixture of xylene and absolute alcohol in equal parts. The preparations are then made permanent by mounting each with balsam and a cover glass. The whole process takes from 5 to 15 minutes and is particularly recommended for chromosome counts.     

Staining Germinating Pollen and Pollen Tubes

Germinating pollen on stigmas and pollen tubes in styles of Antirrhinum, Brassica, Oenothera, Raphanus, Rosa, solatium and Tagetes spp. were prepared for examination as follows: The styles were fixed in ethyl alcohol-acetic acid 3:1 for 1 hr, and hydrolyzed at 60°C for 5 to 60 min (depending on the species) in 45% acetic acid. The stigma with its attached strand(s) of stigmatoid tissue was then dissected out under a stereoscopic microscope, placed in a few drops of a staining solution made by dissolving 150 mg of safranin O and 20 mg of aniline blue in 25 ml of hot 45% acetic acid. After 5-15 min in this stain, the tissue was placed in a fresh drop of stain on a microscope slide and gently squashed under a cover glass. Because of a gradual precipitation of the aniline blue component, the stain had to be filtered regularly before use. However, a staining solution could be kept at room temperature for several weeks.     

Venetian Turpentine as an Aid in Squashing and Concomitant Production of Durable Chromosome Mounts

Root tips are hydrolyzed in 1 N HCl at 60 C for 10-12 min, Feulgen stained, and macerated in a minimal amount of propiono-carmine. A drop of Venetian turpentine mounting medium (Harleco brand was used) is mixed with the carmine stain, the cover slip applied, the tissue pressed gently while observed under a dissecting microscope to spread the chromosomes, and finally firmly squashed. High quality slides of over 1 yr durability are obtained which are well suited to morphological studies, photographing under oil, or routine screening in polyploidy studies. The carmine stain in conjunction with the Feulgen aids to give contrast to chromosomes which are difficult to stain (e.g. hops) but it may be omitted for other species.