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.     

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.     

Permanent Smears of Leaf Tips for the Study of Chromosomes

Young leaf tips are soaked in a saturated aqueous esculin (aesculine) solution at 10-12° C for 15 min to 24 hr and fixed in acetic-alcohol, 1:1. The materials are then stained in a mixture of 2% aceto-orcein and 12V HCl (9:1), 3-4 sec over a flame followed by 30 min or longer at 30° C and then smeared in 1% aceto-orcein. Preparations are made permanent by loosening the cover glass in tertiary butyl alcohol and mounting directly in Canada balsam.     

Enhanced Differentiation of Zaprionus Chromosomes by Prestaining Acid Hydrolysis

Two variations of orcein staining have been adapted to salivary gland chromosomes of Zaprionus. Method I: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, stained with 2.5% orcein in 60% acetic add for 15-20 min, and squashed in 60% acetic acid. Method II: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, transferred to a saturated solution of carmine in 45% acetic acid for 1 min, then to a mixture of 50 ml of 1% orcein in concentrated lactic acid and 50 ml of 30% acetic add for 5 min. They are squashed in the same mixture. The unproved differentiation of chromosomes from cytoplasm is attributed to the removal of cytoplasmic ribonucleic add by add hydrolysis.     

Staining Tissue of the Central Nervous System with Luxol Fast Blue and Neutral Red

The tissue is fixed in 10% neutral saline formalin for 1 day to 3 wk depending on the size of the block, dehydrated and embedded in paraffin. The sections are stained at 57° C for 2 hr, then at 22° C for 30 min, in a 0.0125% solution of Luxol fast blue in 95% alcohol acidified by 0.1% acetic acid. They are differentiated in a solution consisting of: Li₂CO₃, 5.0 gm; LiOH-H₂O, 0.01 gm; and distilled water, 1 liter at 0-1° C, followed by 70% alcohol, and then treated with 0.2% NaHSO₃. They are soaked 1 min in an acetic acid-sodium acetate buffer 0.1 N, pH 5.6, then stained with 0.03% buffered aqueous neutral red. Sections are washed in distilled water, 1 sec, then treated with the following solution: CuSO₄·5H₂O, 0.5 gm; CrK(SO₄)₂·12H₂O, 0.5 gm; 10% acetic acid, 3 ml; and distilled water, 250 ml. Dehydration, clearing and covering complete the process. Myelin sheaths are stained bright blue; meninges and the adventitia of blood vessels are blue; red blood cells are green. Nissl material is stained brilliant red; axon hillocks, axis cylinders, ependyma, nuclei and some cytoplasm of neuroglia, media and endothelium of blood vessels are pink.     

Staining of fresh, Undecalcified, thin Bone Sections

Fresh, undecalcified bone sections can be reproducibly and reliably stained by any of the following procedures: (A) Basic fuchsin, 1% in 30% alcohol, 48 hr, 22°C. (B) AgNO₃, 0.033 M, 48 hr, 22°C; washing 48 hr in a large volume of distilled water; exposure to light to develop the color. (C) Metallic sulfides (Co⁺⁺, Pb⁺⁺, Hg⁺⁺, Cu⁺⁺): the nitrate of the metal, 0.033 M, 48 hr, 22°C; then Na₂S, 0.033 M, 48 hr, 22° C. (D) Alizarin Red S, 0.1% solution in distilled water, 48 hr, 22°C; differentiated 48 hr at 22°C in weakly alkaline water, pH about 8. (E) KMnO₄: boiling 8-10 min in a 0.1 N, solution. With the exception of D the surface stain must be ground off the section for microscopic examination of its interior. Stain concentration, time and temperature can be altered to suit specific needs.     

Histological Localization of Microelectrode Placement in Brain by Ferrocyanide and Silver Staining

After recordings had been taken from a microelectrode used for mapping nerve impulses, a current of 100 μa from the positive pole of a direct current generator was run through the electrode for 5 sec while it was still in place. On terminating the experiment, in which the use of several electrodes was possible, 50-75 ml of a 1:1 mixture of 4% potassium ferrocyanide and 4% acetic acid was injected into each common carotid artery, and the brain left in situ for 0.5 hr. It was then removed and the electrode-bearing part fixed 5-6 hr in a 1:1 mixture of 40% formalin and 95% ethyl alcohol at 55 °C. This specimen was washed in running water 5-10 min, the electrodes removed and frozen sections of 40-80 μ cut and placed in 95% alcohol. Sections were stained 5-10 min at 25-30°C in 10% silver nitrate solution in 75-80% alcohol acidified by 3-4 drops of glacial acetic acid per 50 ml, washed 4-5 sec in each of 2 baths of 95% alcohol, and reduced while being agitated constantly in a 2% solution of pyrogallol and 6-7% formalin in 75-80% alcohol. Washing in 95% alcohol, clearing in clove oil or methyl salicylate followed by xylene and mounting in synthetic resin or balsam completed the process. Sites of electrolysis at the tips of electrodes (under magnification) were blue before silver staining and black after staining. Axons stained brown to black on a yellow background.     

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.     

Isopsoralene and its Use in Karyotype Analysis

Root-tips and leaf tips of different plants were kept for 1-2 hr at 12-14° C in saturated aqueous isopsoralene solution and stained in a mixture of 1 N HCl and 2% acetoorcein (1:9 by volume) by heating for 3-4 sec over a flame. They were then squashed in 1% aceto-orcein under a cover glass, the excess being blotted and the cover sealed. Preparations could be kept for about 15 days. A good chromosome morphology was secured. Isopsoralene was found to be suitable in cases where other coumarin derivatives as well as paradichloro-benzene failed to yield suitable results.     

Fuchsin staining with naoh clearing for lignified elements of whole plants or plants organs

Three methods that are adapted to the various consistencies of plants are as follows: 1. Samples are placed for 10-14 hr at 60° C in a 1% aqueous solution of basic fuchsin, to which 10 gm of solid NaOH per 100 ml are added. 2. Samples when taken out of 95% alcohol are placed in a 1% solution of basic fuchsin in 95% alcohol for 24 hr; after washing in water, they are placed in a 15% solution of NaOH at 60° C until cleared. 3. Samples are placed in a 15% aqueous solution of NaOH at 60° C until cleared, then for 24 hr at 60° C in 15% NaOH containing basic fuchsin. After being stained and cleared by one of these three methods, the samples are rinsed in water, dehydrated and then passed into a mixture of absolute alcohol and concentrated HC1 (3:1) for 1-15 min, rinsed in absolute alcohol, cleared in xylene and mounted in Canada balsam. The lignified tissues appear red; the others, transparent.     

Staining and Mounting Helminths

The following procedure is recommended: Fix ces-todes and trematodes (while held flat between glass slides) 0.5-2.0 hr. in the following mixture: formalin, 15; acetic acid (gl.), 5; glycerol, 10; 95% ethyl alcohol, 24; distilled H₂O, 46; all proportions by volume. After freeing them from the slides, wash thoroughly in running water and stain immediately thereafter. Stock staining solution: ferric ammonium alum (violet cryst.), 2 g.; distilled H₂O (cold) 100 ml.; after solution, add 2 ml. concentrated H₂SO₄, bring to a boil; add 1 g. coelestin blue B (Nat. Aniline), boil 3-5 min.; cool and add 10 ml. absolute methyl alcohol and 10 ml. glycerol. Dilute 1 vol. with 3 vol. distilled H20 for use. Stain 5-30 min., depending on size of specimens. Wash with 2 changes 0.5 hr. each of distilled H₂O, then 50% isopropyl alcohol 12-16 hr., 50% isopropyl alcohol 2 hr., followed by graded isopropyl alcohol for dehydration. Ether: ethyl alcohol (equal parts), 1 hr., is followed by embedding in celloidin in a sheet just thick enough to cover the specimens. Trim embedded specimens and dehydrate with isopropyl alcohol, 80%, 90% and absolute. Clear in beechwood creosote. Mount in balsam with cover glasses that overlap the edges of the celloidin 1-2 mm. While drying at 37°C, refill edges of mount with fresh balsam as needed. When dry, remove excess balsam and ring the edges with ordinary gloss enamel paint.     

Water-Pretreatment Squash Technic: A New and Simple Practical Method for the Chromosome Study of Animals

To simplify the staining of animal chromosomes (especially in insect testes) the authors have borrowed (with necessary modifications) the squash technic of plant cytology. The method has four steps: (1) Water pretreatment. This step requires only about 5-10 minutes either in water at room temperature or in water kept at about 38°C. in a water bath. (2) Fixation. Ordinarily only 5 minutes in 10-15% aqueous solution of glacial acetic acid is necessary. (3) Staining. The fixed tissue is rinsed in two or three changes of distilled water and then placed in a solution of basic fuchsin: either 1% in 30% ethyl alcohol, or 0.2-0.4% in 5-10% lactic acid. In the former solution the staining period should be about 2 minutes: in the latter, 5-20 minutes. The time is not critical. (4) Squashing. The material is rinsed in several changes of distilled water, placed on a clean slide and squashed under a cover glass. Such preparations last 4-5 weeks, and a technic is described for removing the cover glass in order to mount in Euparal and to make them permanent. The authors list various species of vertebrates as well as invertebrates in which the technic has given good chromosome staining, as shown by illustrations.     

Colchicine-Feulgen Leaf Smears

The colchicine-Feulgen leaf smear has many advantages. It (1) prevents spindle formation, (2) allows the chromosomes to be widely spread in the cell, (3) straightens the chromatids, (4) allows the constrictions to become very noticeable, (5) increases the number of chromosome plates by preventing anaphase, (6) facilitates smearing, and (7) stains only the chromosomes. (8) Young leaves are generally easily obtainable, while roots of the proper sort are to be had only under very special conditions. (9) Mitoses are frequently more numerous in young leaves than in roots. The schedule for specimens of Phlox involves a 1 to 2 hour pretreat-ment of young leaves in a 0.2% colchicine solution, fixation in Sem-men's Carnoy (3 vol. absolute alcohol, 1 vol. glacial acetic, 1 vol. chloroform), hydrolysis for 25 minutes in 10% HC1 at 58° C., staining in decolorized fuchsia, smearing in 45% acetic acid, and running the material on slide and cover glass thru acetic alcohols (1:1, 1:3, 1:9), absolute alcohol, xylol, and balsam.     

Staining the Nucleus in Yeasts

Yeast cells kept young by repeated subculturing were centrifuged, washed twice in distilled water and smeared on slides coated with a little egg albumen. The cells were treated with 0.002 M 8-hydroxyquinoline for 1 hr, fixed first in OsO, vapour for 30 sec and then in chloroform for 30 sec. The slides were passed through descending grades of alcohol, washed in distilled water, then immersed in 0.17 M NaCl solution for 2 hr. at 57°C. They were again washed in distilled water and later hydrolysed in 1 N HCl at 60°C for 5-7 min. This was followed by washing in distilled water and in buffer. The slides were then kept for 3 hr in Giemsa stain comprising 96 ml of phosphate buffer of pH 7.0 and 4 ml of the stain. After dehydration, mounting was done in balsam. Nuclei were brightly stained and well differentiated; centrosomes were clear, and the process of nuclear division and movement to daughter cells could be studied. Pretreatment with 8-hydroxyquinoline increased the viscosity of the cytoplasm, while NaCl treatment and acid hydrolysis led to the complete removal of ribonucleic acid and basophilic material. A selective staining of chromatin was thus achieved. Structures resembling chromosomes could be seen when fixed and stained cells were squashed, soon after the removal of the slides from the stain, under a cover glass by applying uniform pressure with a rubber stopper. Fixation in osmic acid vapor and chloroform followed by acid hydrolysis and staining in leucobasic fuchsin also helps to obtain bright staining of the nucleus; however, the preparations are inferior to those obtained after 8-hydroxyquinoline, NaCl treatment and Giemsa staining.     

Aceto-Orcein and Feulgen Stains for Anatomy and Cytology of Trematodes

Dicrocoelium dendriticum and Fasciola hepatica were killed in the extended condition without anesthetization by dropping them into 40% acetic acid or into aceto-orcein. By using aceto-orcein (La Cour, 1941), killing, fixing and staining were accomplished simultaneously: staining time 24 hr or more. Whole mounts were made by dehydrating, clearing and mounting in Canada balsam, or testes or the upper part of the uterus could be removed for squash preparations after as long as 2 mo in the fixing and staining fluid. For Feulgen staining, living specimens were placed in 40% acetic acid for 10—15 min and then transferred to either Gilson's fluid, for sections, or to acetic-ethanol (1:3) for squashes. Hydrolysis was either by 10% perchloric acid at 25°C for 12 hr or in 12V HCl at 60° for 10 min. The time for Feulgen staining (De Tomasi, 1936) was 1.5-4.0 hr. Squashes were made from testes and uterus in the same manner as after aceto-orcein or sections obtained after embedding in paraffin.     

A Pollen Grain Squash Technique for Saccharum and Related Genera

Anthers collected between 9 and 10 AM were treated for 1 hr at 26-28 C with a 0.5% solution of colchicine, washed for 2-4 min in water, placed in 0.002 M 8-hydroxyquinoline for 1 hr, washed in water for 10 min and fixed in: methanol, 60 ml; chloroform, 30 ml; distilled water, 20 ml; picric acid, 1 gm and mercuric chloride 1 gm, for 24 hr. After washing they were hydrolysed in 1 N HCl for 15 min at 60 C, stained in leuco basic fuchsin for 30 min, then smeared on a slide in a drop of acetocarmine. The slides were sealed, stored overnight, the paraffin was removed, and the slide passed through a 1:1 mixture of n-butyl alcohol and acetic acid, then through pure n-butyl alcohol and mounted in Canada balsam. The significant features of this procedure are: (1) use of chromosomes in the haploid condition for karyotype analysis, (2) better exaggeration of constrictions for easier interpretation of chromosome types and (3) good spreading in plants with a large chromosome number.     

Cold and Chemical Pretreatments to aid Chromosome Counts in a Grass Leaf Squash Technique Incorporating Hot Pectinase Maceration

Leaf buds, comprising the basal 3-5 mm of the youngest leaves attached to short stems, were dissected out of fast-growing young tillers of certain grasses, including Festuca, Lolium and Phalaris spp. and various hybrids. They were kept overnight in distilled water at 0-2 C, treated in a mixture of equal parts by volume of saturated aqueous solutions of 5,7-dibromo-8-hydroxyquinoline containing a surfactant (Tween 80), and 1-bromo-naphthalene for 3-4 hr at 0-2 C, and fixed in Newcomer's fluid. The rinsed samples were hydrolysed in 1 N HCl for 8 min at 60 C and Feulgen stained for 1 hr. After rinsing, the buds were macerated in a filtered 3% solution of Pectinol 100-D (Rohm and Haas) in 0.1 M acetate buffer at pH 4.5 for 10 min at 60 C. Squashes were made in 45% acetic acid. The combined cold and chemical pretreatments resulted in strongly contracted, easily counted metaphase chromosomes, while intact cells with full chromosome complements were more readily retained during squashing after enzyme maceration at 60 C than at room temperature.     

Squash Method for Meiosis in Ovules

The ordinary Feulgen or acetic-lacmoid squash tech-nic following fixation in freshly made Carnoy's fluid (alcohol, 6: chloroform, 3: glacial acetic acid, 1), provides an easy and reliable method of studying meiosis in ovules. After fixation for 1 day, the material was hardened in 95% ethyl alcohol for 1-2 days and taken to water by gradual hydration. For staining by the Feulgen method, the material was hydrolyzed 8-10 minutes in 1 N HO at 58-60°C., followed by staining in decolorized leuco basic fuchsin for 2 hours. The staining was intensified by transferring the material to water. After 15-20 minutes the water was replaced by 45% acetic acid. For staining by acetic-lacmoid, the ovules after fixation, hardening and hydration were transferred to standard acetic-lacmoid stain to which was added 1 drop of 1 N HCl to every 10 drops of stain. Gentle heat was applied till the stain started to give fumes. After allowing 20 minutes at room temperature the material was transferred to fresh acetic-lacmoid. Some 6-12 ovules were mounted either in a drop of 45% acetic acid or acetic-lacmoid, depending upon the Feulgen or acetic-lacmoid staining respectively. Gentle and repeated tapping over the cover glass by a blunt needle loosened the cells of integument and nucellus and finally left the megaspore mother cells undergoing meiosis, fully exposed to view. The process was carried out under constant observation using the low power of the microscope. The desired amount of flattening was brought about by light pressure over the cover glass and gentle heating. The preparations were made permanent by dehydrating in ethyl alcohol and mounting in Euparal.     

A Paradichlorobenzene, Saponin, Iron Mordant Sequence in the Staining of Meiotic Chromosomes of Ipomea

For the meiotic study of Ipomea spp., flower buds were stripped of the calyx and corolla and soaked in saturated aqueous paradichlorobenzene at about 28° C for 3 hr, transferred to acetic-alcohol (1:3) for 6 hr, then into 1% saponin solution and left overnight. They were mordanted in 1:3 acetic-alcohol saturated with ferric oxide for 24 hr and stained in a mixture of 1% aceto-carmine and 2% aceto-orcein with 1 N HCl in the proportion of 9:9:1. The preparations were mounted in 1% aceto-carmine for temporary use and made permanent by dehydration through the n-butanol schedule. The pollen mother cells had clear cytoplasm with deeply stained chromosomes.     

A Colchicine, Hypotonic Citrate, Air Drying Sequence for Foetal Mammalian Chromosomes

Mice 14 days pregnant were given 0.3 ml of 0.025% Colcemid (Demecolcine, Ciba) and killed after 1 hr. The livers of the foetuses were removed and broken up in 0.1% Colcemid in phosphate-buffered 0.85% NaCl and left 1.5 hr. The cell suspension was then centrifuged, resuspended in 1% sodium citrate for 20 min, centrifuged and the cells fixed in acetic-alcohol (1:3) for 30 min at 4°C. The cells were then resuspended (twice) in 45% acetic acid and dropped onto warm slides. After drying, the cells were stained for 30 min with lactic-acetic-orcein and examined under oil-immersion without a cover slip. Good numbers of well-spread mitotic figures were obtained.