A new technique for orcein banding with acid treatment

A rapid method for banding plant chromosomes using hydrochloric acid with orcein is suggested. The technique has been successful in both dioecious and monoecious families with short chromosomes. It involves pretreatment with an oxyquinoline-paradichlorobenzene mixture, fixation in acetic ethanol and treatment with hydrochloric acid at 60 C followed by staining with orcein. Stained chromosome bands and interbands are reproducible and species specific.     

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.     

A chromosome study of thirty Japanese heteropterans (Heteroptera)

The chromosomes of thirty Japanese heteropterans were studied in male or female germ cells and embryo cells in acetic orcein squashes or air-dried slides stained with Giemsa. This work revealed the chromosome formula and the sex-mechanism of Rhopalidae for the first time. New data on the chromosomes of the Acanthosomatidae, Alydidae, Belostomatidae, Cydnidae, Plataspidae and Urostylidae are presented.     

Meiosis Aspects and Nucleolar Activity in Triatoma vitticeps (Triatominae, Heteroptera)

Some aspects of both the nucleolar organizer activity and meiosis were studied in the testes of Triatoma vitticeps (Heteroptera, Triatominae). The techniques used included squashing followed by lacto-acetic orcein staining, silver-ion impregnation, fluorescent banding (CMA₃, Quinacrine mustard and DAPI) and fluorescent in situ hybridization (FISH). A close relationship between heterochromatin and nucleolus in testicular cells was observed. During meiosis, the silver-ion impregnation pattern varied. At metaphase plate, a small body appeared apart from the chromosomes. In the spermatids this small body was seen in preparations stained with orcein and silver- ion impregnation but not with fluorochromes or FISH. These characteristics combined suggest that these corpuscles represent a source of ribonucleoproteins (RNP) – RNA and specific nucleolar proteins. Silver-ion impregnation and (FISH) revealed nucleolar organizer activity in two metaphase sex chromosomes (X). These results indicate that, in these species, nucleolar organizer regions (NORs) are located in the sex chromosomes, X chromosomes were CMA₃⁺ and Y chromosome was DAPI⁺.     

The Use of Lacto-Propionic Orcein in Rapid Squash Methods for Chromosome Preparations

A solution of 2 gm of natural orcein dissolved in 100 ml of a mixture of equal parts of lactic and propionic acids, and diluted to 45% with water, proved more effective than other stain fixatives for meiotic preparations from fresh pollen mother cells. When used after 5 min fixation in modified Carnoy's fixative (alcohol, acetic acid, chloroform, formalin; 10:2:2:1) and 5 min maceration in 1 N HC1 at 60° C, the same stain proved the most suitable for the rapid preparation of root-tip chromosomes for counting and for studying detailed morphology.     

Removal of Yolk from Oyster Eggs by Soxhlet Extraction for Clear Chromosome Preparations

The mature, spawned eggs of the American Eastern oyster, Crassostrea virginica Gmelin, contain yolk which interferes with the preparation of chromosomes and nuclear groups for the study of meiosis, fertilization, and karyology. Most samples of eggs cannot be studied cytogenetically until the yolk is extracted. Simple and complete removal of all interfering yolky material can be efficiently accomplished, after Carnoy fixation in 3:1 alcohol-acetic acid, by extraction for 2 hr in a micro-Soxhlet apparatus with 1:1 mixture of chloroform and methyl alcohol. Standard orcein squashes can then be routinely made of these eggs hitherto considered refractory to clear staining of chromosomes. A thimble with a fritted glass end, pore size 40 μ, is used as a receptacle for the eggs in the Soxhlet apparatus. After extraction the eggs can readily be washed off the fritted glass with the aceto-orcein staining solution. If the eggs are to be stored for some time prior to squashing and staining, 45-60% acetic acid or Carnoy's alcohol-acetic acid 3:1 can be used for the storage fluid.     

The Use of Haematoxylin for Squash Preparations of Chromosomes

A simplified propionic-iron alum-haematoxylin stain for rapid squash preparations of chromosomes requires only two stock solutions: (A) 2% haematoxylin and (B) 0.5% iron alum, both in 50% propionic acid. For use, suitable volumes of A and B are mixed. With unripened solution A, equal volumes should be used and the stain is ready for use 1 day after mixing. As the haematoxylin ripens, progressively smaller amounts of B are required and the mixture may be used immediately. The stain gives excellent results when used in the same way that orcein and carmine are currently employed, with a wide range of animal and plant (including fungal) chromosomes, and with good nucleolar staining. It may be used either following acetic alcohol (1:3) fixation or as joint fixative and stain on unfixed material. In fungal material, where Lu's BAC fixative is recommended, the centrioles are also stained.     

Orcein,collastin and pseudo-elastica: a re-investigation of Unna's concepts

Summary Orcein has been recommended for identification of elastin. Since other traditional elastica stains proved to be unspecific, it was deemed of interest to determine the selectivity of orcein and to review pertinent literature.Orcein was employed as a textile dye in ancient Egypt and was used for dyeing of wool and silk until the early 20th century. It was introduced into histological technic in 1878 as a stain for cytoplasm. Unna recommended it for demonstration of elastic tissue in 1890 and retracted claims for its specifity in 1894 because orcein colored also certain collagen fibers. Unna suggested the term collastin for collagen fibers which share the affinity of elastin for acid orcein. Other orcein solutions were used as selective stains for collagen.In histochemical studies, the staining properties of resorcin-fuchsin and orcein were very similar; elastin and various collagen fibers were strongly colored. Unna's collastin is apparently identical with the pseudo-elastica described in sections stained with resorcin-fuchsin. Both dyes react with meshworks of fine fibers, embryonic, experimentally or pathologically altered collagens. It is suggested to use the term collastin, instead of pseudo-elastica, for collagenous fibers which bind the traditional elastica stains.     

A cytological demonstration of arrhenotoky in three mites of the family Phytoseiidae

Summary Chromosome numbers of Phytoseiulus persimilis. Amblyseius fallacis, and Typhlodromus caudiglans were determined in lacto-propionic orcein squashes of developing eggs. Eggs of all species had either four or eight chromosomes: this indicates arrhenotoky (2 n = 8). All chromosomes are acrocentric; one of P. persimilis and one of A. fallacis had a lighter staining area suggestive of heterochromatin.Contribution No. 93.     

Orcein, collastin and pseudo-elastica: a re-investigation of Unna's concepts.

Orcein has been recommended for identification of elastin. Since other traditional elastica stains proved to be unspecific, it was deemed of interest to determine the selectivity of orcein and to review pertinent literature. Orcein was employed as a textile dye in ancient Egypt and was used for dyeing of wool and silk until the early 20th century. It was introduced into histological technic in 1878 as a stain for cytoplasm. Unna recommended it for demonstration of elastic tissue in 1890 and retracted claims for its specifity in 1894 because orcein colored also certain collagen fibers. Unna suggested the term collastin for collagen fibers which share the affinity of elastin for acid orcein. Other orcein solutions were used as selective stains for collagen. In histochemical studies, the staining properties of resorcin-fuchsin and orcein were very similar; elastin and various collagen fibers were strongly colored. Unna's collastin is apparently identical with the pseudo-elastica described in sections stained with resorcin-fuchsin. Both dyes react with meshworks of fine fibers, embryonic, experimentally or pathologically altered collagens. It is suggested to use the term collastin, instead of pseudo-elastica, for collagenous fibers which bind the traditional elastica stains.     

Identification of the pattern of heterochromatin distribution in Passiflora species with C-banding

We tested four C-banding protocols to obtain heterochromatic bands in the passion fruit species Passiflora edulis and P. cacaoensis (Passifloraceae). Three of these protocols had been previously described. The three published protocols were not adequate to obtain C-bands in these species. An adapted protocol demonstrated heterochromatin distribution in metaphasic chromosomes of species of Passiflora for the first time. The differentiated coloration for C-bands was obtained with immersion of the slides in 99% ethanol, 45% acetic acid (additional step), 0.2 N hydrochloric acid, hydroxide of barium, 45% acetic acid, and 2X standard saline citrate at four different temperatures. The C-bands were observed in the satellites and in the telomere and centromere regions of all chromosomes, both in P. edulis and in P. cacaoensis.     

An Improved Method for Chromosome Counting in Maize

An improved method for counting chromosomes in maize (Zea mays L.) is presented. Application of cold treatment (5C, 24 hr), heat treatment (42 C, 5 min) and a second cold treatment (5C, 24 hr) to root tips before fixation increased the number of condensed and dispersed countable metaphase chromosome figures. Fixed root tips were prepared by the enzymatic maceration-air drying method and preparations were stained with acetic orcein. Under favorable conditions, one preparation with 50-100 countable chromosome figures could be obtained in diploid maize using this method. Conditions affecting the dispersion of the chromosomes are described. This technique is especially useful for determining the somatic chromosome number in triploid and tetraploid maize lines.     

Trypsin-Orcein Banding in Plant Chromosomes

A convenient and quick method using trypsin-orcein for handing plant chromosomes (O-banding) is suggested. The technique is directly applicable to meristematic tissues (e.g. root tip) and involves the treatment of root tip with 1-2% solution of trypsin either in buffer or in 05 N HCI for 5-10 minutes at 37 C or for 30-60 minuta near 0 C followed by staining with 1.5% acetic orcein: 1 N HCI (19:1). Dark staining bands are reproducible and species specific. These bands possibly represent specific DNA-protein-dye interaction.     

Trypsin-orcein banding in plant chromosomes.

A convenient and quick method using trypsin-orcein for banding plant chromosomes (O-banding) is suggested. The technique is directly applicable to meristematic tissues (e.g. root tips) and involves the treatment of root tips with 1-2% solution of trypsin either in buffer or in 0.5 N HCl for 5-10 minutes at 37 C or for 30-60 minutes near 0 C followed by staining with 1.5% acetic orcein: 1 N HCl (19:1). Dark staining bands are reproducible and species specific. These bands possibly represent specific DNA-protein-dye interaction.     

Observations which indicate heterochromatinization of an extra chromosome in the salivary gland cells of Drosophila hybrids

Salivary gland cells of Drosophila mulleri/D. arizonensis aneuploid male hybrids carrying 3 microchromosomes exhibited morphological features which indicate heterochromatinization of one of the small polytene chromosomes. The process apparently changes the chromosome surface producing a coating with a net-like structure and a strong affinity for lacto-acetic orcein. The possibility of a dosage compensatory mechanism operating to counteract the effect of the extra chromosome is discussed on the basis of previous data which indicated that the microchromosomes of these species have ribosomal cistrons and are controlled by regulatory mechanisms especially evident in hybrids.     

Handling Avian Chromosomes

The somatic and meiotic chromosomes of Galius gal-lus dom. (as well as of the rat, mouse and possibly other animals) may be adequately prepared for miscroscopic examination by the following smear technic: Pith the fowl of appropriate age through the palatal fissure (other killing technics may be substituted for other animals), remove testes, slice medianly into halves (or quarters if large) and fix in mixture of 2 parts absolute ethanol, 1 part propionic acid, 1 part chloroform. Pour off and replace fixative at the end of 12-24 hours, after which the tissue may be stained or stored under refrigeration. Fresh material can be stained in propionic-carmine (0.5 g. carmine boiled in 45% aqueous solution of propionic acid), but material stored for several months should be stained in a boiled mixture of 10 ml. vinyl acetate, 10 ml. tertiary butyl alcohol, 50 ml. propionic acid, 75 ml. H₂O, 0.25 g. carmine, 0.25 g. orcein. A method is described for preparing and making permanent such preparations.     

Synthetic Orcein as a Stain for Chick Embryo Cartilage Matrix

Chick embryo tissues fixed in Bouin's fluid, in 10% formol saline or in 10% formol saline with subsequent mordanting in saturated picric acid containing 3% HgCl₂, were examined as 5 μ paraffin sections after staining with 1% synthetic orcein in 80% ethanol containing 1% HCl (conc.). Orcein defined the young elastic fibres formed in the truncus arteriosus, aorta and other large arteries after the 5th day of embryonic development but also reacted with the matrix of cartilage in all parts of the skeleton from the 3rd day onward. It is thought that a glycoprotein or proteoglycan shared by these two tissues could account for their mutual affinity for orcein.     

The staining of Brunner's gland and other neutral mucins by carmine, hematoxylin and orcein in alkaline solutions.

Brunner's glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orcein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.     

Computer assisted karyotype analysis of Pyrrhopappus carolinianus

With the help of Computer Aided Karyotyping procedures, Ag-NOR staining and C-banding techniques, the karyotype of Pyrrhopappus carolinianus (Asteraceae, Lactuceae) has been studied. The species has 2n=12 chromosomes. Silver staining reveals that the two shortest pairs of chromosomes possess NOR's. On the basis of chromosome length and centromere position, only the longest chromosome pair and the satellite chromosomes can be identified. Two types of C-banding can be obtained, dependent on the temperature of the hydrochloric acid hydrolysis of the root tips. Hydrolysis at 60°C results exclusively in centromeric bands, whereas a treatment at room temperature reveals a pattern of intercalary bands. A computer assisted analysis of the intercalary banding pattern resulted in the construction of schematic representation of the average C-banding pattern. This banding pattern allows an easy identification of each of the chromosome pairs.     

Exploration of dye chemistry in Taenzer Unna Orcein type elastin staining

Summary Musso's demonstration of the amino- and hydroxyphenoxazone structure of synthetic orcein suggested trial of simpler mixtures and isolated pure dyes of the phenoxazine phenoxazone series. It was further thought that explorations of this sort might reveal which of the hitherto demonstrated 14 chromatographic components of orcein might take part in or even be chiefly responsible for the elective staining of elastin.Accordingly trials were made in a hydrochloric acid (0.12 N) 70% alcohol technic based on Taenzer's original method of a number of commercially available dyestuffs and indicators and a number of products were synthesized by variants of Musso's technic for resorcin blue: air oxidation of resorcinol in the presence of ammonia. Those tested are the following: Azolitmin, Lacmoid, Resorufin, Resorcin Blue (MLB), C.I. No. 51020, Gallocyanin C.I. 51030, Brilliant Cresyl Blue C.I. 51010, Nile Blue C.I. 51180, a Nile red preparation, Bernthsen's Methylene Violet; Azure A, Toluidine Blue C.I. 52040, several laboratory synthetic lots of Musso's Resorcin Blue in which oxidation was done with H₂O₂ and varying amounts of ammoni were used, and 2 batches in which resorcinol was partly or completely replaced by m-aminophenol.Successful to excellent elastin stains were achieved with Lacmoid, Resorcin Blue MLB, part of the Musso Resorcin Blue products and the two m-aminophenol oxilation products Elastin purple FP and Elastin Videt PR.From the failure of azolitmin and resorufin, both 7-hydroxy-2-phenoxazones and the success of resorcin blue (MLB) 7-N,N-dimethylamino-2-phenoxazone, it appears suggested that the aminophenoxazone structure may be a determining characteristic. The success with m-aminophenol substitution in the Musso air oxidation NH₃ synthesis tends to support this view.While I have had some success with the Victoria blues first used by Lustgarten, using other technics, these dyes and some other triphenylmethanes do not successfully take the place of orcein in acid alcohol staining methods. Rosanilin and pararosanilin do stain rodent elastica from acid aqueous and alcoholic solutions but adult human elastica does not so stain. We suspect a diphenamine Schiff base condensation with the known free aldehyde of rodent elastica. This is confirmed by more or less complete blockade of pararosanilin acid alcohol staining with p-toluidine in glacial acetic acid, 1 hr, hydroxylamine: sodium acetate: H₂O 102040 3 hr and 5% phenylhydrazine HCl 3 hr on dog, rat and guinea pig arteries. The hydroxylamine gave complete blocking, the other two reagents partial. Altogether about 50 dye samples were tested.Presented before the Histochemische Gesellschaft September 28, 1968.Supported by National Cancer Institute Research Grant C-4816, National Institutes of Health.