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.     

Use of Dimethylsulfoxide to Prevent Clumping during Feulgen Staining of Ceratocystis ulmi

The dimorphic fungus Ceratocystis ulmi is the causative agent of Dutch Elm Disease. As part of a study on the regulation of this developmental phenomenon, we attempted to stain the nuclei of cells growing vegetatively in the yeast phase by a modification of the Feulgen technique described by Gauger (1975). The cells were harvested by centrifugation, washed twice, and resuspended in 0.05 M phosphate buffer (pH 6.5). A small portion of this cell suspension was placed on a clean No. 2 glass coverslip (22 ± 22 mm) and allowed to air dry. The coverslip was flamed briefly to heat fix the cells whereupon they were fixed in glacial acetic acid: 95% ethanol (1:3 v/v) for one hour, hydrolyzed in 1 N hydrochloric acid at 60 C for 5 minutes, and stained for 30 minutes. The Feulgen stain was prepared according to Stevens (1974). Subsequently, the coverslip was rinsed briefly with distilled water and dehydrated for 30 seconds in 70% ethanol. After air drying, the coverslip was mounted on a glass microscope slide with Permount (Fisher Scientific Co.) and examined.     

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.     

Preparing Plant Root Tip Cells for Microscopical Examination

The technics generally used for preparing root tip cells for microscopical examination destroy mitochondria and other cytoplasmic particles and remove lipoidal material. Fixation in a bichromate solution followed by treatment with osmic acid preserves these granules through normal embedding procedures (cf. Zirkle, 1929; Newcomer, 1940); also fixation in neutral formalin and embedding in the water-soluble wax, Aquax, may completely preserve lipoidal matter, and thus the mitochondria. The separation of cells in squash preparations usually entails acid hydrolysis of the intercellular cement. Treatment for one hour with a 5% solution of a commercial pectinase powder in a 1% aqueous solution of peptone allows good separation of cells of bean root tips fixed in acetic-alcohol. By this method it has been demonstrated that the Feulgen hydrolysis removes cytoplasmic and nucleolar RNA. To preserve the mitochondria it is advisable to immerse the bean roots in a 5% aqueous solution of polyvinyl alcohol for 24 hours and to separate the cells with a 10% solution of pectinase in a 1% peptone solution. This procedure preserves the granules, leaves the nucleus optically homogeneous, and gives a result most closely approximating to that observed in living root tip cells.     

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.     

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.     

Permanent Stained Preparations of Thick Blood Films

Fixing thick films in alcoholic solution of dye after the usual staining-and-laking procedure preserves the appearance of parasites and blood elements very similar to that of the usual thick films (not fixed) for the diagnosis of malaria and relapsing fever.

Procedure recommended: Films are stained and laked for 15 minutes in diluted Giemsa—1 to 3 drops of stock solution (0.4 g. in 60 ml. equal parts absolute methyl alcohol and glycerin) per ml. distilled water; rinsed in water and allowed to dry. They are then immersed in, or flooded with, May-Griinwald's stain (0.5% in absolute methyl alcohol) for 30 seconds, rinsed in water and allowed to dry. Solutions of MacNeal's tetrachrome stain in methyl alcohol and glycerin may be substituted for Giemsa and a solution in methyl alcohol may be substituted for May-Griinwald. With slight modification of the procedure, both thick and thin films on the same slide may be stained together.

Films stained and fixed as described, and mounted in Diaphane, have shown no evidence of fading in 3 years.     

Cytochemical Staining of Sections from Plastic-Embedded Flagellates

Dinoflagellate chromosomes in sections of plastic-embedded cells were stained without removing the plastic. Azur B and Feulgen procedures were used to localise DNA. Azur B was used with Araldite or methacrylate sections by staining in 0.2% stain in 0.05 M citrate buffer at pH 4 for 1 hr at 50 C followed by rinsing in tertiary butyl alcohol to differentiate the chromosomes. Feulgen stain was used with Araldite sections by hydrolyzing in 1 N HCl at 60 C for 10 min, rinsing in water, staining for 24 hr, washing well, drying and covering. Fast green was used with methacrylate sections to stain proteins by flooding the slide with a 0.1% solution of stain in 0.06 M phosphate buffer at pH 8, allowing the stain to dry out at 40-50 C, washing well, drying and covering. Controls were carried out on material fixed in formalin and treated with nucleases or proteolytic enzymes prior to embedding, and staining.     

Peculiarities of cytochemical properties of cancer cells as revealed by study of deoxyribonucleoprotein susceptibility to Feulgen hydrolysis.

Chromatin of human squamous carcinoma cells reacts more intensively to short (1-2 min) acid hydrolysis in the Feulgen reaction and is, after such treatment, more intensively stained by Schiff reagent than chromatin of normal cells of the same origin. To reveal this difference in chromatin properties the use of a fluorescence variant of the Feulgen reaction is necessary because nuclei-binding of Schiff reagent after such short hydrolysis is so weak that the amount of the stain bound by means of absorption technique is hardly possible. The use of increased sensitivity of cancer cells chromatin to acid hydrolysis for cancer cytodiagnosis is suggested, especially for the diagnosis of so called diploid cancers for which detection on the basis of deoxyribonucleic acid content determination is impossible.     

Cytochemical Staining of Sections from Plastic-Embedded Flagellates

Dinoflagellate chromosomes in sections of plastic-embedded cells were stained without removing the plastic. Azur B and Feulgen procedures were used to localise DNA. Azur B was used with Araldite or methacrylate sections by staining in 0.2% stain in 0.05 M citrate buffer at pH 4 for 1 hr at 50 C followed by rinsing in tertiary butyl alcohol to differentiate the chromosomes. Feulgen stain was used with Araldite sections by hydrolyzing in 1 N HCl at 60 C for 10 min, rinsing in water, staining for 24 hr, washing well, drying and covering. Fast green was used with methacrylate sections to stain proteins by flooding the slide with a 0.1% solution of stain in 0.06 M phosphate buffer at pH 8, allowing the stain to dry out at 40-50 C, washing well, drying and covering. Controls were carried out on material fixed in formalin and treated with nucleases or proteolytic enzymes prior to embedding, and staining.     

Quantitative cytochemical aspects of a combined Feulgen-Naphthol Yellow S staining procedure for the simultaneous determination of nuclear and cytoplasmic proteins and DNA in mammalian cells

The simultaneous cytophotometric determination of nuclear and cytoplasmic proteins and DNA by means of a combined Feulgen-Naphthol Yellow S (NYS) staining procedure was investigated. According to this procedure Feulgen staining is performed prior to NYS staining. The following main results were obtained:

1. 1. After NYS staining alone, the amount of NYS bound to the cell was found to be closely correlated to the cellular dry mass. The correlation coefficient was 0.99 in ethanol-acetone fixed cells and 0.95 in formaldehyde-fixed cells. This close correlation was not significantly altered by the Feulgen staining procedure and was 0.92 in ethanol-acetone and 0.94 in formaldehyde-fixed cells. However, the absolute amount of NYS bound per unit dry mass was affected by the method of fixation and type of Feulgen hydrolysis.

2. 2. The cells lose material during the Feulgen procedure, particularly during the acid hydrolysis stage. The type of hydrolysis most suitable for the Feulgen procedure (5 N HCl, 22 °C, 60 min) resulted in a considerable loss of dry mass in ethanol-acetone fixed cells. This loss was smaller in formaldehyde-fixed cells (15%) and was in addition closely correlated (correlation coefficient 0.99) to the dry mass of the cells prior to hydrolysis. In formaldehyde-fixed cells the dry mass after the Feulgen procedure is thus a good measure of the true cellular dry mass of the fixed cells. This is further demonstrated by the close correlation between NYS binding to Feulgenstained cells and the dry mass of these cells prior to the Feulgen procedure (correlation coefficient 0.95).

3. 3. When using the combined Feulgen-NYS staining procedure under standardized conditions (formaldehyde fixation and acid hydrolysis in 5 N HCl, 22 °C, 60 min) a constant amount of NYS was found to be bound per unit dry weight to nuclear and cytoplasmic proteins in various types of mammalian cells with different proliferative activity.

4. 4. The Feulgen DNA determination was not found to be quantitatively affected by the subsequent NYS staining.

From the results of the present study it seems that, under standardized conditions, the combined Feulgen-NYS staining procedure can be used as a reliable quantitative method for the determination of nuclear and cytoplasmic proteins and DNA in mammalian cells.     

A Centrifugation Technique for Preparing Medium-Free Resinous Mounts of Feulgen-Stained Pollen Tubes

Air-dried pollen of Tradescantia paludosa and a colchicine doubled Allium ascalonium-fistulosum interspecific hybrid was sown in culture tubes on an inorganic salt-lactose liquid medium containing 0.02% colchicine. After a 16-20 hr incubation at 22 C, pollen tubes were harvested by centrifugation for 3-5 min at 1100-1400 rev/min and fixed with acetic-alcohol (1:3). Feulgen staining was carried out in the culture tubes with fluid changes made after the centrifugation following each step. Single drops of the final pollen-45% acetic acid suspension were flattened under silicone-treated coverglasses which were removed by the quick freeze technique prior to counterstaining with fast green, dehydration, and mounting in Diaphane or Canada balsam. Medium-free, Feulgen-stained, resin-mounted preparations of well-dispersed pollen tubes with arrested metaphases were obtained.     

Enzymatic Isolation of Archegonium of Pinus Yunnanensis Fr. and Observations on the Proembryo Development

The enzymatic isolation of megagametophyte from fixed and fresh of Pinus yunnanensis Fr. were macerated in a mixture solution of 3% pectinase and 3% cellulase at the 30℃ for 10–13 hours, and the archegonium has been isolated. Materials were stained with A-S, Feulgen and PAS, and following procedure was cleared in the lactophenol. The ventral canal and egg cell which from division of central cell, the functional sperm nucleus becomes pressed against the egg nucleus, and the new cytoplasm round the zygote nucleus were observated under the microscope of usual light. The four proembryonal nuclei divide simultaneously, resulting in eight nuclei arranged in two tiers of four cells each, and following development were formed wall. In the end, the proembryo of six- teen cells were formed. Two methods on enzymatic and dimethyl sulfoxide with potassium hydroxide to isolate the female sexual cells of gymnosperms have been compared, and several problems are discussed.     

Fast direct method of obtaining metaphase and prometaphase chromosomes from chorion biopsy cells and human embryos during the lst semester of pregnancy]

Samples of chorionic villi and embryonic tissues (brain, brain--sheaths) are thoroughly washed with Hank's solution, immediately subjected to hypotonic treatment (0.9% sodium citrate plus few drops of 0.01% colchicine) 37 degrees C, 30 min, prefixed 20 min with equal amount of standard fixative mixture, twice fixed in standard fixative solution (1 hour, -10 degrees C), hydrated with equal volume of distilled water (5-10 min), dried, macerated directly on the slide with 60% acetic acid. The cell suspension is then evenly spread on the slide surface, dried, postfixed and stained. The method provides sufficient amount of metaphase and prometaphase mitotic plates suitable for differentiating staining in 1.5-2 hours after sampling and might be recommended for routine chromosomal analysis in prenatal diagnosis of inherited diseases during early pregnancy.     

Collodion Membranes in Pollen Tube Technique

Membranes are formed by allowing a drop of collodion-acetone solution to come into contact with the surface of warm sugar solution in a petri dish. Pollen is germinated upon the smooth areas of the membrane when all traces of acetone have evaporated. Semipermanent preparations are made by isolating the pollinated area of the membrane, floating it onto a slide, and, after the removal of excess sugar solution, adding a drop of acetic-stain fixative, followed by an albumenized cover slip. The preparation can be made permanent by inverting a slide in a mixture of 1 part glacial acetic acid and 3 parts absolute alcohol, when the collodion membrane will dissolve and allow the cover slip and adhering grains to fall free. The cover slip is then passed through absolute alcohol (2 changes), xylene, and mounted in neutral mountant on a clean slide. By substituting a drop of the alcohol-acetic acid mixture in place of acetic-stain fixative, the grains adhering to the cover slip may be stained by the Feulgen method.     

An approach to the G-Banding Technique in Rye Chromosome

The G-banding technique has not yet been broken through in studying plant chromosomes. in this paper, we have described a new banding method in Secale cereale. The rye root tips were treated with actinomycin D (40-100 μg/ml) for two hours and with colchicine (0.01%) for 0.5 hour and then fixed with methanol-acetic acid (3:1). After cell wall degradation by cellulase and pectinase, the chromosome sample were made by a hypotonic and flame-drying method (hypotonic treatment→preparation of cell suspension→dropping suspension on slide flame-drying). Following an air-drying period of about a week, the slides were incubated in trypsin-EDTA solution (0.01–0.05%) at 30℃ for 10–15 sec. and subsequently stained with Giemsa. Lots of deep stained bands along the arms of many prophase and late prophase chromosomes were seen. The position of them was obviously different from that of the C-band and the number of them was approximately in proportion to the longitude of chromosomes. Such bands were not seen in metaphase chromosomes. We thought it preferable to use prophase chromosomes to probe G-banding technique in plant and this paper has proposed a possible way for studying G-banding technique in plant chromosome. We also discuss why metaphase chromosomes of plant do not show G-bands.     

Differential Staining Of Tissue In The Block With Picric Acid And The Feulgen Reaction

The authors have found a modification of the Feulgen reaction to be a satisfactory stain for tissue in the block.

Pieces of fresh mammalian tissue not thicker than 5 mm. are fixed for approximately 48 hours at 25° C. in a mixture of equal parts of 5% aqueous sulfosalicylic acid and saturated aqueous picric acid. They are washed for 30 minutes in three ten-minute changes of distilled water and placed in Feulgen's staining solution diluted to one-half strength with distilled water. The staining solution is allowed to act for 24 hours (2 to 3 mm. thick blocks) up to 48 hours for 5 mm. thickness. After staining, the specimens are transferred to a mixture of sodium bisulfite, 0.5 g. and N hydrochloric acid, 5 ml. in' 100 ml. of distilled water. Two changes of IS to 30 min. each in the acid sulfite are given and these are followed by dehydration through 50%, 70% and 95% alcohol. One to two hours are allowed for each change except the last 95%, in which the stained tissue is allowed to remain overnight. The dehydration is completed in two changes of absolute alcohol with subsequent clearing in xylene and embedding in paraffin. Sections may be cut 10 μ or other thickness desired, mounted on slides, paraffin removed, and covered in the usual manner. Nuclei stain reddish violet against a lemon yellow background when the stain is typical. Orange G, 200 mg. per 100 ml. may be added to the fixing fluid if a more polychromatic effect is desired.     

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.     

Carbol Fuchsin as A Stain for Human Chromosomes

Cells derived from cultures of bone marrow or leucocytes were treated with hypotonic citrate solution, squashed in 45% acetic acid frozen with CO₂ to allow removal of the cover glass without disturbing the smear, and stained by the following schedule: absolute alcohol, 5 min; coat with 0.2% parlodion and air dry; 70% alcohol, 5 min; distilled water, 5 min; stain 2-5 min in a mixture of 45 ml of a 0.3% solution of basic fuchsin in 5% phenol, 6 ml of glacial acetic acid, and 6 ml of 37% formaldehyde. Differentiate and dehydrate in absolute alcohol, clear in xylene and cover. The stain is durable for several weeks if slides are stored in darkness when not in use. Results resemble those obtained by Feulgen or aceto-orcein methods.     

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.