Silver Staining for Nucleolar Organizing Regions of Vertebrate Chromosomes

Refinements to a simple, one-step silver staining technique for nucleolar organizing regions are described. These include fixation of silver stained material with sodium thiosulfate and standardization of silver development conditions for different groups of vertebrates. The central advantages to the method are that it is rapid, reliable, simple, and inexpensive. Additional benefits include (i) consistent and uniform silver staining of nucleolar organizing regions, (ii) few reduced silver deposits elsewhere on the chromosomes or on the slides, (iii) generally unaltered chromosome morphology after silver treatment, and (iv) relative permanence of Permounted preparations. The method works equally well on chromosomes made from cell cultures and from solid tissues of live specimens.     

黑麦染色体的银染研究

在对黑麦染色体银染过程的盐酸解离条件进行探索的同时,对黑麦染色体的银染正反应区进行了研究,首次发现经短时间空气干燥(4-24h)的黑麦染色体制片,随着盐酸解离强度的递增,分别出现了核仁组织区(NOR)、NOR和端粒以及NOR和着丝点的银染正反应,就此现象讨论了端粒和着丝点的银染机理。     

黑麦染色体的银染研究

在对黑麦染色体银染过程的盐酸解离条件进行探索的同时,对黑麦染色体的银染正反应区进行了研究,首次发现经短时间空气干燥（4～24 h）的黑麦染色体制片,随着盐酸解离强度的递增,分别出现了核仁组织区(NOR)、NOR和端粒以及NOR和着丝点的银染正反应,就此现象讨论了端粒和着丝点的银染机理。     

A Differential Staining Technique for Simultaneous Visualization of Mitotic Spindle and Chromosomes in Mammalian Cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanobacetic acid solution containing 4 mM MgCl₂ and 1.5 mM CaCl₂ at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

Double Fluorescent Staining for the Separate Demonstration of Chromosomes and Microtubules in Mitotic Cells In Vitro

A simple fluorescent method for double staining of mitotic cells using a rhodamine B indirect immunofluorescent method for tubulin and the DNA-specific fluorescent dye Hoechst 33258 for nuclei and chromosomes is described. This procedure enables one through the use of appropriate excitation filters to view at will either chromosomes and nuclei or tubulin within the same cell.     

甜瓜有丝分裂染色体制片技术及核型分析

以萌发种子根为材料,研究预处理取样时间和预处理药剂对甜瓜染色体制片的影响.结果表明,上午8:00左右为最佳预处理取样时间,可以观察到近14%的中期分裂相;在4种药剂预处理活体根尖中,以添加对二氯苯(饱和)的放线菌酮(40 m g.L-1)水溶液的效果最佳.用此方法对厚皮和薄皮两种类型甜瓜进行核型分析,结果发现:两类甜瓜核型相似,都具有一对随体,核型均为2A型,核型公式均为2n=2x=24=14m+10st(2SAT).但两类甜瓜的染色体总长、平均长度以及随体在染色体上的分布都不相同.     

Actinomycin Deffects on Termite Chromosomes: Induction of High Resolution Banding Patterns with silver Staining in Mitotic Stages

The treatment of termite male spermatogonia with actinomycin D induces highly elongated and finely banded late prophase and prometaphase chromosomes as evidenced by the silver staining method. Actinomycin D suppresses the silver staining of nucleolar organizing regions in prometaphase and reduces it in metaphase chromosomes.     

Staining Nuclei and Chromosomes in Protozoa

Technics for free-living forms such as Paramecium and for parasitic forms such as the opalinid ciliates are described.

Paramecium: Fix paramecia in hot Schaudinn's fluid containing 5% of glacial acetic acid for 5-15 minutes. (A hot water bath for maintaining the proper temperature of the fixative is described.) Dehydrate up to 83% alcohol. Mount the specimens on albuminized cover glasses. (A table for mounting animals on cover glasses is described.) Apply a thin layer of collodion to the cover glass to prevent the loss of the specimens during the subsequent handling. Pass through descending grades of alcohol to water. Mordant in 4% iron alum for 24 hours. Stain in 0.5% hematoxylin for 24 hours. Destain in saturated aqueous picric acid. Rinse in tap water, expose to ammonia vapor for a second, and then rinse again in tap water. Wash in running water for 1 hour. Dehydrate. Clear, then mount in damar.

Opalinid Ciliates: Make smears on cover glasses and fix them while wet. If the opalinids are to be subsequently stained in hematoxylin, fix in hot Schaudinn's fluid (containing 5% of glacial acetic acid) for 5-15 minutes. Pass through descending grades of alcohol to water. Mordant in iron alum for 24 hours. Stain in hematoxylin for 24 hours. Destain in saturated aqueous picric acid. For Feulgen reaction, fix in a modified weak Flemming's fluid for 1 hour. Wash in running water for 30 minutes. Hydrolyze. Leave 3 hours in fuchsin decolorized with H₂SO₃ (Feulgen formula). Wash in H₂SO₃, then in running water for 15 minutes. Dehydrate up to 95% alcohol. Counterstain with fast green FCF for 2 minutes. Dehydrate in absolute alcohol. Clear, then mount in damar.     

A Technique for the Simultaneous Staining of Both Nucleolar Organizer Regions and Kinetochores of Human Chromosomes with Silver

Pretreatment of human metaphase chromosomes with NaOH at a pH of 8.5, followed by staining with silver nitrate, differentially stains both the nucleolar organism regions on the 10 acrocentric chromosomes as well as the kinetochore centers on all 46 chromosomes.     

A Feulgen-Carmine Technic for Staining Fungus Chromosomes

Apothecia of Pyronema confluens Tul. were stained by the Feulgen reaction and then squashed and mounted in propiono-carmine. Exceptionally clear differentiation of the chromosomes was obtained by this method. Snail stomach cytase was used as an aid in flattening the asci and a simple method for its extraction is described.     

Silver Staining in Clinical Cytogenetics

Silver staining of human chromosomes at prometaphase or metaphase identifies variants in the stalk (nucleolar organizing) regions of acrocentric chromosomes (Nos. 13, 14, 15, 21, 22). Variants are defined by size, number, and morphology of silver staining areas. They are heritable polymorphisms and have not been associated with clinical abnormalities. However, these variants are useful in clinical cytogenetics, specifically in studies attempting to determine 1) whether genetic material has been gained or lost in chromosomal rearrangements, 2) the origin of chromosomal aberrations, 3) the origin of cells in tissue culture, 4) the chromosomal location of single genes, 5) clonal origin of tumors, 6) the zygosity of twins, and 7) paternity. Some chromosomal aberrations require silver staining for their definition. Because loss of the stalk regions per se is apparently not deleterious, demonstration that chromosomal breaks occurred within this region without concomitant loss or gain of genetic material essential for normal human development provides basis for a good prognosis for the individual with the chromosomal rearrangement resulting from such breakage. The principle underlying most of the other applications is to determine whether variants being compared are identical or dissimilar, and to make inferences from these results (e.g., variants in monozygotic twins should all be identical, whereas in dizygotic twins they are as similar as in any pair of sibs). Silver staining is a valuable technique for special questions in clinical analysis.     

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes ¹. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions ^2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes ^{4, 5, 6}. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti ⁷ and Cx. quinquefasciatus ⁸, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates ⁹. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti ¹⁰, the accumulation of multiple chromosomal rearrangements in cell line chromosomes ¹¹ makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4^th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol ¹² is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.     

The Physical Chemistry of Silver Staining

It has been shown that silver deposition plays a part in the silver staining process. From this it has been concluded that the rate of reduction of silver within and on histological structures is an important factor.

Some factors controlling the rate of reduction, such as the adsorption of silver hydroxide and ammonia, the affinity of silver for proteins, and the protective power of the gel structures have been pointed out.

Some simple applications of the ideas to silver staining have been given and two technics described, one making use of piperidine instead of ammonia, the other carrying out the reduction in the presence of the silver solution to facilitate deposition.     

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.     

Rapid Staining of Nuclei and Chromosomes in Root Tips

Feulgen reagent quickly heated to and maintained at 60 C just before immersion of plant material, basic fuchsin in acid alcohol at room temperature, and pinacyanol at room temperature will stain hydrolyzed root tip nuclei and chromosomes in one minute or less. This technic, coupled with fast fixation, can be utilized when uncertainties exist as to when to begin sampling plant meristem cells for mitoses or when time does not allow for standard fixation and Schiff staining.