Chromosome Preparations in the Field from Mammals Long After Death

Chromosome preparations of high quality can be obtained from bone marrow cells of small mammals that have been dead for 20 hr or longer. The bone marrow is rinsed out of the femurs with RPMI medium supplemented with 15% fetal calf serum. Add 0.05-0.1 ml of a 0.01 % colchicine solution to 5 ml of medium-cell suspension. After Vi-1 hr of colchicine treatment at 37 C the cells are spun down and the supernatant replaced by 5 ml of hypotonic (0.075 M) KC1. After 12 min in the hypotonic solution at 37 C the cells are fixed in methanokacetic acid 3:1. Air dried preparations are made after repeating the fixation procedure three times and the chromosomes are stained with Gietnsa, if required after prclieatment of the preparations for banding; e.g., GTG. Technical hints for field work are given. The technique has proven successful with several species of rodents and shrews.     

Chromosome Preparatons of Bone Marrow Cells without Prior In Vitro Culture or In Vivo Colchicine Administration

Bone marrow (about 0.5 ml) from au erythropoietic region is freed of blood clots by washing 1-3 min in 1 μg/ml colchicine solution (2-3 ml) and then soaking 1-2 hr at 20-30° C in a second change. For mammalian or avian marrows, the colchicine is made up in phosphate-buffered (pH 7) physiological NaCl solution; for amphibian, Ringer's A solution. Next the specimens are soaked about 20 min in a hypotonic solution as follows: for mammalian, 1% Na-citrate; for avian, a 1:4 dilution of the buffered NaCl solution by distilled water; and for amphibian, Ringer's A-distilled water, 1:1. Then they are heated in a mixture of 2% orcein in 45% acetic acid and 1 N HCl, 9:1. Immediately after heating, squash preparations are made with 2% acetic-orcein in the usual manner. An alternative method is to dissociate the marrow cells by agitating after colchicine treatment. Then, recovering the cells between changes by low-speed centrifugation, to carry out the hypotonic treatment and subsequent fixation in Carnoy's solution I (alcohol acetic, 3:1) before drying the cells onto slides from the fixative. After thorough drying the slides may be stained 10-20 min in acetic orcein, or by other suitable technics.     

A Colchicine, Hypotonic Citrate, Squash Sequence for Mammalian Chromosomes

The method was developed for bone marrow of mice but is applicable to other tissues and other species of small mammals. Mice are injected intraperitoneally with 0.5 ml of 0.025% colchicine solution and killed 1 hr afterwards. The femurs are dissected out rapidly, the epiphyses are removed, and the marrow is washed out of the shafts by warm hypotonic sodium citrate solution from a hypodermic syringe. Gentle aspiration of the marrow into and out of the syringe converts it into a fine suspension. The suspension is kept in the citrate solution at 37°C for 10 min. Connective tissue and bony spicules are removed by centrifuging through Nylon bolting cloth in a bacterial filtration tube, before fixing in acetic-alcohol (1:3) and staining by the standard Feulgen procedure. The cells are concentrated for each change of reagent by centrifuging slowly. The advantages of colchicine pretreatment and of working with cell suspensions are emphasized.     

A Colchicine,Hypotonic Citrate,Squash Sequence for Mammalian Chromosomes

The method was developed for bone marrow of mice but is applicable to other tissues and other species of small mammals. Mice are injected intraperitoneally with 0.5 ml of 0.025% colchicine solution and killed 1 hr afterwards. The femurs are dissected out rapidly, the epiphyses are removed, and the marrow is washed out of the shafts by warm hypotonic sodium citrate solution from a hypodermic syringe. Gentle aspiration of the marrow into and out of the syringe converts it into a fine suspension. The suspension is kept in the citrate solution at 37°C for 10 min. Connective tissue and bony spicules are removed by centrifuging through Nylon bolting cloth in a bacterial filtration tube, before fixing in acetic-alcohol (1:3) and staining by the standard Feulgen procedure. The cells are concentrated for each change of reagent by centrifuging slowly. The advantages of colchicine pretreatment and of working with cell suspensions are emphasized.     

Leukocytes Cultured from Small Inocula of Whole Blood and the Preparation of Metaphase Chromosomes by Treatment with Hypotonic KCL

Leukocytes were cultured from 0.2 ml of whole blood inoculated into 5 ml portions of a medium consisting of Eagle's basal amino acids and vitamins at double strength in Earle's balanced salt solution brought to pH 7.0 with 7.5% NaHCO₃, and containing additives: glutamine, 2 mM; penicillin, 100 units/ml; streptomycin, 100 μg/ml; phenol red, 7 μg/ml; fetal or newborn agammaglobulin bovine serum, 15%; phytohemagglutinin M, 2%; and U.S.P. heparin sodium, 20,000 units/liter. Cultures were incubated in closed 60 × 28 mm screw-cap vials, in a gas phase initially of room air, for 3 days at 37 C, with colchicine to make 0.2 μg/ml added for the final 3-5 hr. After incubation, the cells were separated from the medium by centrifugation, the medium replaced by 0.075 M KCI plus 16 U.S.P. units/ml of heparin sodium at 37 C, cells resuspended and allowed to incubate 10 min. Removal of the hypotonic KCI was followed by fixation in methanol-acetic acid, 3:1 (changed twice), spreading cells on slides by the air-drying method, and staining with 1% natural orcein (G. T. Gurr) in 60% acetic acid. Dehydration and covering completed the preparation. KCI, 0.075 M, has been used advantageously in the above way and for cells cultured by other means from skin and other organs of man and other mammals. Combined advantages of the method are: culture of leukocytes from small volumes of whole blood, with very few failures to obtain mitotic cells; a medium which can be stored frozen in culture vials, and which in a simpler form is usable for long term culture of other cell types; and the use of KCI for hypotonic treatment.     

Leucocyte Culture and Chromosome Preparations from Pig Blood

Blood was drawn into heparinized tubes from any large vein and allowed to settle 2-3 hr at 3-5 C. The cell sample consisting of 1 ml drawn from the buffy coat and 2 ml from the plasma was planted in the following medium: Medium 199 (Difco), 10 ml; penicillin G sodium, 1000 USP units; dihydrostreptomycin, 1 mg; and Bacto-PHA-M (Difco), 0.2 ml. Incubation, with twice daily shaking, was at 37 C for 68-70 hr; colchicine to give 4 μ ml was then added and incubation continued for 3-4 hr. The bulk of the medium was removed by centrifugation, the cells washed once in Hanks' salt solution, centrifuged, and all but 0.5 ml of the fluid decanted; 1.5 ml of distilled water at 37 C was added, the cell suspension incubated at 37 C 5-15 min, followed by centrifugation and fixation in methanolacetic acid 3:1 (3 changes) as usual. Spreads were made by applying 4-5 drops of cell suspension to ice-cold slides and burning off the fixative. Giemsa stain was used. The method has proved very satisfactory for determining chromosome numbers in the domestic pig. This number, as determined in 690 cells from Poland China and Duroc gilts and crosses of these breeds was 38 in 611 (88.6%) of the cells.     

Permanent Mounting of Unstained and Aceto-Orcein Stained Cells in the Water-Soluble Medium, Abopon

For cellular morphology, mammalian cells were grown on cover slips in Leigh ton tubes, fixed in 1% osmic acid vapor for 2 min, decolorized with 30% H₂O₂ in 5% ammonium oxalate solution (1:7) for 2 min, then washed thoroughly, and finally mounted in a water-soluble medium consisting of a saturated solution of Abopon in 0.2 M phosphate buffer, pH 7.0. For chromosomal analysis of similarly cultured cells, aceto-orcein preparations were made by conventional methods, with the following minor modifications: following pretreatment with colchicine and hypotonic expansion, the cells on the cover slips were fixed in acetic-alcohol (1:3), air dried, incubated at 37° C for 15 min in 2% orcein in 45% acetic acid, rinsed in 45% acetic acid, washed several times in distilled water, and finally mounted in Abopon mounting medium. Both kinds of preparations were allowed to harden for 24 hr before being handled. Such slides will keep for years at room temperature. Studies requiring frequent comparisons of cellular and chromosomal morphology of cultured cells can thus be extended over long periods of time.     

Permanent Mounting of Unstained and Aceto-Orcein Stained Cells in the Water-Soluble Medium,Abopon

For cellular morphology, mammalian cells were grown on cover slips in Leigh ton tubes, fixed in 1% osmic acid vapor for 2 min, decolorized with 30% H₂O₂ in 5% ammonium oxalate solution (1:7) for 2 min, then washed thoroughly, and finally mounted in a water-soluble medium consisting of a saturated solution of Abopon in 0.2 M phosphate buffer, pH 7.0. For chromosomal analysis of similarly cultured cells, aceto-orcein preparations were made by conventional methods, with the following minor modifications: following pretreatment with colchicine and hypotonic expansion, the cells on the cover slips were fixed in acetic-alcohol (1:3), air dried, incubated at 37° C for 15 min in 2% orcein in 45% acetic acid, rinsed in 45% acetic acid, washed several times in distilled water, and finally mounted in Abopon mounting medium. Both kinds of preparations were allowed to harden for 24 hr before being handled. Such slides will keep for years at room temperature. Studies requiring frequent comparisons of cellular and chromosomal morphology of cultured cells can thus be extended over long periods of time.     

Avian Microchromosomes; As Shown by Prefixation Treatment with Colchicine, Squashing, and Haematoxylin Staining

Small pieces of bird tissue from developing feathers, embryos, embryonic gonads and adult testes are treated before fixation in a 0.25% solution of colchicine diluted with an equal volume of hypotonic Pannett and Compton solution containing KCI, 0.336 gm; CaCl₂, 0.160 gm; Na₂HP0₄. 12H₂O, 0.172 gm; and NaH₂PO₄. 4H₂O, 0.0172 gm per 1000 ml of double distilled water, for 20-30 min at 37° C. This material is subsequently centrifuged at 1000 rev/min for 1-3 min, fixed with acetic alcohol (1:3) and stained with either Gomori's haematoxylin or by Feulgen's procedure. Preparations are frozen in the freezing chamber of a refrigerator for the separation of the cover slips, then mounted in euparal after dehydration in alcohols. With this technique, unclumped and clear preparations of both macrochromosomes and microchromosomes are obtained regularly.     

Direct and Repeatable Bone Marrow Chromosome Preparations from Living Mice

Using a 27 gauge hypodermic needle, bone marrow is aspirated from a lumbar vertebra into 0.1 ml of Hanks' salt solution. The aspirate is kept well mixed in 1% sodium citrate for 15 min, centrifuged, and the cell pellet fixed for 30 min in Clarke's 3:1 ethanol-acetic fixative. After removal of the fixative the cells are suspended in 0.05-0.1 ml of 60% acetic acid, centrifuged and resuspended in 0.03 ml of this fixative. Chromosome preparations are made by spreading the suspension on a slide heated to 60 C.     

Culture and Slide Preparation of Leukocytes from Blood of IMACACA

Leukocyte-rich plasma is recovered from monkey blood by centrifugation. Portions of the plasma are planted in a medium consisting of 70% NCTC 109, 10% F 10, 15% fetal bovine serum, 5% human cord serum, and phytohemagglutinin (0.1 ml/5.0 ml of medium). Incubation is 48-72 hr, followed by colchicine 24 hr, hypotonic treatment with water 1-1 1/4 hr, and fixation in absolute methanol-glacial acetic acid (3:1). Permanent spreads are made by ejecting drops of suspension onto iced slides and flame drying. Staining is done in cresyl violet acetate. Because of some inconsistencies encountered in the spreading of monkey cells, alternate methods have been suggested for trial until one is found that works uniformly for the technician.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

Obtaining Suspensions of Animal Cells in Metaphase from Cultures Propagated on Glass

Cells of some strains continuously cultivated in vitro have a pronounced tendency to lose their attachment to glass during mitosis. If division of these cells is arrested in meta-phase by treatment with colchicine, many of the cells are set free and float into the medium. Advantage has been taken of this property to collect large numbers of suspended cells in c-metaphase. By applying standard procedures of staining with orcein and manual squashing to these cells, preparations are obtained that are useful for critical chromosome analysis. The procedure consists in treatment of cultures with colchicine (final concentration 0.1 μg/ml) for 18-24 hr; agitation in hypotonic medium (quarter-strength Tyrode's or CMRL-1066) for 5 min; addition of 1:3 acetic-alcohol for 5-10 min; removal and centrifugation of the resulting fluid mixture from the culture; and resuspension of the sedimented cells in a small volume of the fluid. Advantages of the method are: large numbers of metaphase plates can be examined on a single slide, the chromosomes are contracted and well separated, and cytoplasmic boundaries are preserved.     

Silver Protein Staining of Nerve Fibers in Celloidin Sections

Celloidin sections from formalin-fixed brain and spinal cord of primates are stored in 70% alcohol after cutting, soaked in 2% pyridine in 50% alcohol for 6-8 hr at 37 C, and transferred to 1% concentrated NH₄OH in 50% alcohol 15-18 hr at 20-25 C. After washing and flattening, the sections are transferred to 1% silver protein solution containing 30 ml of 0.2 M H₃BO₃/100 ml. Impregnation is accomplished in 50 ml screw-top jars, 50 mm in diameter, which are filled to a depth of 35 mm, and have 1 gm of copper foil, 0.002 inch thick added. The foil is folded in loose accordion-fashion, pierced and threaded, cleaned in 5% HNO₃, rinsed in distilled water, and suspended in the solution just above the sections by fastening the thread to the jar lid. The sections are impregnated for 24 hr at 37 C, rinsed in distilled water, reduced in a solution of 5% Na₂SO₃ and 1% hydroquinone for 10 min, washed in distilled water and toned in 0.2% gold chloride for 5 min. After rinsing in distilled water, the sections are transferred to 1% oxalic acid for 45-60 sec, washed in distilled water and placed in 5% Na₂S₂O₃ for 5 min. Sections are then washed, dehydrated to 95% alcohol, cleared in terpineol, followed by 3 changes in xylene, and mounted.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

An Air-Drying Technique for Flattening Chromosomes in Mammalian Cells Grown In Vitro

The method described was developed to facilitate the analysis of chromosome complements in cells freshly isolated from monkey kidney cortex and grown on glass, and in “altered” monkey cells grown on glass or in suspension. Cells were treated with hypotonic solution (quarter-strength Tyrode or diluted medium) for 30 min, or with colchicine in a final concentration of 25 μg/ml (.0025%) for 12-18 hr followed by hypotonic salt solution for 5 min, then fixed in acetic alcohol (1:3) for 5 min. With cells centrifuged from suspended cultures, addition of fixative had to be gradual. Directly after fixation, films of cells on slides were air dried completely. This produces a more uniform and complete flattening of cells than can be achieved by manual pressure; yet, fragmentation of chromosome complements does not occur. Fixed and air dried slides may be stored for days without deterioration or they may be stained immediately in 2% natural orcein (G. T. Gurr, London) in 50% acetic acid. Preparations can be made permanent by a dry ice schedule, without loss, shrinkage, or distortion of cells.     

Simplified Culture and Chromosome Preparations of Primate Leukocytes

Plasma recovered from 1 ml of primate peripheral blood by centrifugation is planted in a medium consisting of 80% TC-199 and 20% fetal bovine serum, to which 0.125 ml of phytohaemagglutinin/5 ml is added. The pH is adjusted to 7 with 10% NaHCO₂. The mixture is incubated 68 hr, Colcemide to give 1 μg/ml is added, and incubation continued for 4 hr. Following centrifugal separation, the cells are given a hypotonic treatment with 0.75% sodium citrate for 15 min, then centrifuged again and fixed in 3:1 methanol-glacial acetic acid, 3 changes. Tiny drops of the cell suspension are placed on a slide, spread by blowing, and air dried. The preparations are stained with 15% Giemsa solution in methyl alcohol. The method has been successfully used in 256 specimens from 25 different species.     

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.     

A Copper Sulfate-Silver Nitrate Method for Nerve Fibers of Planarians

For staining in toto, planarians are fixed in a mixture of 10 ml of commercial formalin, 45 ml of 95% ethanol and 2 ml of glacial acetic acid. After treatment with 70% ethanol 3-10 days, they are washed in distilled water and immersed in 10% CuSO₄. 5H₂O for 3 hr at 50° C, transferred without washing to 1% AgNO₃ for 1.0-1.5 hr at 50° C; and then developed in: 10 ml of 1% pyrogallol, 100 ml of 56% ethanol and 1 ml of 0.2% nitric acid. Gold toning, 5% Na₂S₂O₃ and dehydration follow as usual. For staining sections, material is fixed in the same fixative, embedded in paraffin and sectioned at 10 μ. After bringing sections to water, they are immersed in 20% CuSO₄. 5H₂O for 48 hr at 37° C; then rinsed briefly in distilled water and placed in 7% AgNO₃ for 24 hr at 37° C. They are washed briefly in distilled water and reduced in: hydroquincne, 1 gm; Na₂SO₃, 5 gm and distilled water 100 ml. Gold toning, followed by 5% Na₂S₂O₃ and dehydration completes the process. Any counterstaining may follow.     

An Improved Chemical Substitute for Fetal Calf Serum for the Micronucleus Test

The routine use of the micronucleus test in the mutagenicity evaluation of xe-nobiotics is limited by high cost and limited availability of fetal calf serum. On the other hand, there are disadvantages, such as hypotonic damage and clumping of cells, associated with the use of mineral medium substitutes for fetal calf serum. Alternatively, we recommend a chemical medium containing Hanks' buffered salt solution, 1% (w/v) bovine serum albumin, and 0.15% (w/v) ethylenediaminetetraacetic acid, final pH 7.4, to preserve morphology, density and homogeneity of bone marrow cells. Mast cell granules are efficiently removed from rat bone marrow preparations by washing twice with this medium. The morphological preservation of cells is further enhanced by fixation with 70 % (v/v) ethanol for 5 min. The proposed medium provides a cost-effective and convenient substitute for fetal calf serum with substantially improved quality of bone marrow preparations for the micronucleus test.