Aceto-Iron-Haematoxylin-Chloral Hydrate for Chromosome Staining

Aceto-iron-haematoxylin can be used combined with the clearing agent chloral hydrate for the squash method. The stain is prepared by dissolving 2 gm of chloral hydrate in 5 ml of a stock solution of 4% haematoxylin and 1% iron alum in 45% acetic acid, which has been allowed to ripen for 24 hr to 1 wk. Heat must not be used to hasten solution. The material (fixed in 1:3 acetic-alcohol) is put on a slide, the fixative removed and a drop of stain added; if necessary the material is crushed before the cover slip is placed in position. The preparations are now carefully heated until a slight colour change occurs. Squashing needs more pressure than in other techniques. This stain gives best results in zoological and botanical material not requiring hydrolysis, e.g., leucocytes, ascites cells, and cells undergoing spermatogenesis and microsporogenesis. Well-spread and selectively stained mitotic and meiotic figures can be obtained.     

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.     

Acetic Acid as a Diluent and Dehydrant in the Preparation of Whole, Stained Helminths

Aqueous 45% acetic acid can be used successfully as a diluent for Ehrlich's haematoxylin and for Horen's trichrome stain (chromotrope 2 R, 0.6 gm; phosphotungstic acid, 0.7 gm; glacial acetic acid, 1.0 ml; water, 100 ml). Glacial acetic acid is used for dehydration of the stained helminths, and followed by a glacial acetic acid-methyl salicylate series for clearing. The whole process can be completed within 1 hr, from fixation to the cleared specimen, with helminths up to 5 mm in length. A satisfactory fixative for Monogenea, Digenea and Acanthocephala is: 85% ethanol, 85; formalin (40% HCHO), 10; and glacial acetic acid, 5—parts by volume. For Cestoda, 5% aqueous formalin is preferable because they are hardened excessively by the alcoholic fixative.     

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.     

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.     

A Schedule for Chromosome Counts M Some Plants with Small Chromosomes

A schedule is given which produced excellent results for somatic chromosome counts on some British species of Galium, These species present great difficulties owing to the small size of the chromosomes (ca. 1.5μ long), the large somatic numbers (up to 96) and the slenderness of the root tips. Special features of the schedule, which is the result of much experiment with various technics, are: fixation with Belling's Navashin type fixative, which was quite the best tried; the modification of Randolph's card mount method to overcome the difficulty of the small diameter of the root tips by mounting together a number from the same plant, so that they can be embedded and sectioned almost as easily as much larger root tips; staining with dilute (0.1%) crystal violet, the most critical stain used, after mordanting with 1% aqueous chromic acid to intensify the stain in the small chromosomes; and the addition of an extra stage of differentiation in absolute alcohol diluted with xylol to remove strands of stain, which are often left in the cytoplasm between the chromosomes, since clove oil, usually the last differentiating fluid used, differs from alcohol in removing crystal violet more rapidly from the chromosomes than from the cytoplasm. It is suggested that this schedule will be valuable in chromosome counts of other plants where similar difficulties arise.     

Aceto-Iron-Haematoxylin for Staining Chromosomes in Squashes of Plant Material

Haematoxylin can be used successfully in the acetic squash technic if adequate mordanting is provided, (a) in the stain—composed of 4% haematoxylin and 1% iron alum in 45% acetic acid—and (b) in a step that combines additional fixation, mordanting and maceration in a 1:1 HCl-alcohol mixture, to which is added chrome alum, iron alum and iodic acid: 0.1 gm of each to 6 ml of HCl-alcohol. The material is usually given a preliminary fixation in 1:3 acetic alcohol, then macerated, fixed and mordanted in the acidified alum-HIO₃ step for 10 min, transferred to Carney's fluid (6:3:1) for 10-20 min, squashed in a drop of stain and gently heated. In some species, the preliminary fixation may be omitted. The method yields intensely and selectively stained chromatin. To secure consistently good results, the stain can be diluted with 45% acetic acid, and the iodic acid omitted for some plant materials.     

Chlorazol Black E as A Stain for Root-Tip Chromosomes

A simple method of shining root-tip chromosomes which gives excellent results in photomicrography is described. The schedule is as follows: run paraffin sections of root tips killed in Bouin's fixative down to water; stain for approximately two hours in 1% aqueous chlorazol black E; wash in water; run up to xylene.     

The Dry Preservation of Fixed Plant Material

A method for the dry-preservation of fixed plant material, root tips and buds, is described. The method seems to be advantageous on long expeditions and when material has to be sent away.

The material is transferred from the fixative to 70% alcohol (3 changes, 1/2 hour in the last). It is dried on blotting-paper. The dried material may be preserved a long time. Material kept dry for 4 1/2 years has proved to be quite satisfactory. Drying has been tried after fixation with CRAF-solutions (Webber and Randolph modifications) and fixatives containing osmic acid (Fleming-Benda and 2BD).

The dry material is swollen by keeping for 2 days in 10% alcohol. It is embedded in paraffin according to the usual method. A satisfactory staining has been obtained after these fixatives using iodine-gentian-violet and Feulgen stainings. In addition to chromosome counts dry material may be used for chromosome morphology studies.

Dried material fixed in aceto-alcohol (1:3) has not turned out to be specially suitable for squash preparations owing to the fragility of the chromosomes. If strong pressure is not applied, satisfactory results may, however, be obtained.     

Insect Embryo Chromosome Techniques

The whole-mount method for studying chromosomes of insect eggs is used; the eggs are caused to adhere to cover glasses, which are handled in racks especially designed for carrying large numbers. A basic and helpful change in the usual technique after fixation and before staining involves extraction of the material 1 hr to overnight with a 1:1 methanol-chloroform mixture to remove plasmal-reactive substances. Either leucobasic fuchsin or sulfonated azure A after acid hydrolysis may be used satisfactorily to stain the chromosomes.     

An ethanol-acetic acid-formol saline fixative for routine use with special application to the fixation of non-perfused rat lung

An ethanol-acetic acid-formol saline fixative (40 : 5 : 10 : 45 v/v) has been developed which gives good results with non-perfused rat lung and which may be used routinely for the fixation of a wide range of rat tissues. The special qualities of the fixative include good penetration, good fixation of nuclei and mitotic chromosomes, and little shrinkage after paraffin embedding. The fixative is also easy to use and has a flexible fixation period (nominally 48 h). Although several fixative mixtures containing alcohol, acetic acid and formalin have previously been reported, none are identical to the present mixture, which was developed independently and systematically in accordance with specific listed requirements.     

Chromosome Preparations from Mouse Embryos During Early Organogenesis: Dissociation After Fixation, Followed by Air Drying

Embryos are put into 1% sodium citrate at 37 C; 7- and 8-day specimens requiring about 20 min. With increasing age, the duration of treatment is increased up to 50 min. Handling is facilitated by keeping specimens in a small glass vessel for observation under a binocular microscope, and by changing fluids with a fine-tipped pipette. Fixation in ethanol-acetic acid 3:l for 2-3 hr is uncritical, as material may be stored in the fixative overnight at 4 C. Staining in toto with 2% orcein in 50% acetic acid follows, requiring 0.5-1 hr (storage in this solution up to 2 wk at 4 C is permssible). After staining, specimens are subjected to cellular dissociation in a mixture of glacial acetic and 50% lactic acid, the action of which is controlled by the duration of treatment and by increasing the ratio of lactic to acetic from 1:Z (younger embryos) to 3:2 (older embryos). Only 1-3 drops of the dissociating fluid is used for each embryo, to favor concentration of the free-floating cells. Since the time required varies from several minutes to nearly an hour, the most favorable degree of dissociation can best be judged by the cloudiness produced in the dissociating fluid. A small drop not exceeding 2 mm in diameter, of the cell suspension, is placed on a slide and followed immediately by a normal-sized drop of fresh 3:1 ethanol-acetic. After drying, the chromosomes are stained with lactic-acetic-orcein or other suitable stain. The method gives satisfactory results with embryos from the 7th to 11th day of pregnancy.     

Acetic Acid Methods for Chromosome Studies at Prophase and Metaphase in Meristems

Monobromobenzene and monobromonaphthalene proved easier to handle than paradichlorobenzene and acenaphthene, and at least as effective for inhibiting spindle formation, for shortening and straightening chromosomes to permit accurate counts and size comparisons. For prophase studies, methyl alcohol pretreatment was found effective in revealing centromeres, heterochromatin, and knobs. The following schedules were found simple and effective.

For metaphase chromosomes: (1) Remove root tip from germinating seed and place in saturated aqueous monobromonaphthalene for three hours. (2) Pour solution from vial and replace with a mixture of 70 volumes 95% ethanol and 30 volumes glacial acetic acid. Leave in fixative at least two days. (3) Remove opaque tip from root and macerate in a drop of acetic-orcein. Cover, heat to near boiling, flatten by pressing slide, cover down, onto a thick blotter.

For prophase chromosomes: (1) Remove root tip from germinating seed and place in 3% aqueous methanol for three hours. (2) Pour solution from vial and replace with a mixture of 65 volumes methanol, 5 of chloroform, and 30 of glacial acetic acid. Leave in fixative at least two days. (3) Remove opaque tip from root and macerate in a drop of acetic lacmoid. Cover, heat to near boiling, flatten by pressing slide, cover down, onto a thick blotter.     

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.     

Fixation and Staining of the Bacterial Nucleus

An examination of some early methods in bacterial cytology shows that technics for demonstrating the nucleus of the Eubacteriales were available for at least twenty years before the current era of investigation, which began in 1942. Although the bacterial nucleus reacts in many respects like the chromatin elements of higher plants, it shows certain peculiarities in its staining reactions. For example, hematoxylin, methyl green, and several preparations used to stain chromosomes, apparently do not exhibit the same affinity for the bacterial nucleus that they do for chromosomes in higher plants. Not only is the selection of a fixative important in nuclear studies, but also the manner in which the fixation is obtained. For example, when bacteria are fixed and processed in a completely wet state it is generally impossible to stain their nuclei. None of the special fixatives studied revealed any unusual organization in the bacterial cell or exhibited any advantage over the fixatives now in common use by bacteriologists. In view of the properties of osmium tetroxide vapor, particularly its relative lack of interference with positive nuclear staining, there can be little doubt of its superiority as a nuclear fixative. It appears that the basophilic material removed from the bacterial cell by hydrochloric acid is not only in the cytoplasm, but that a very significant amount of it is in close contact with the cell wall.     

混合甲醛固定液固定大肠癌淋巴结标本的最佳免疫组化效果研究

目的:探讨混合甲醛固定液固定大肠癌淋巴结标本的最佳免疫组化效果。方法:采用不同pH值(6.0、7.0、8.0)的混合甲醛固定液对39枚大肠癌淋巴结标本进行不同时间(6 h、6 h-12 h、1 d-7 d)的固定处理。以细胞角蛋白20(CK20)为目标抗原,运用OIympusdp 70图像采集分析仪抽选出混合甲醛固定液最佳免疫组化染色的pH值及固定时间。结果:经pH值为7.0混合甲醛固定液处理后,阳性率为92.31%,高于经pH值为6.0、8.0的混合甲醛固定液处理后的76.92%、74.36%,且经pH值为7.0、8.0处理后的阳性率比较有统计差异(P0.05)。混合甲醛固定液的固定时间在6 h-12 h时的阳性率为94.87%,高于固定时间为6 h、1 d-7 d处理的30.77%、76.92%(P0.05)。结论:对于大肠癌淋巴结标本,以CK20为目标抗原,选择pH值为7.0的混合甲醛固定液固定6 h-12 h能够得到质量较佳的免疫组化染色效果。     

Niagara Blue 4B As A Fast Simple Stain for the Adenohypophysis

For differentiation of cells of the adenohypophysis, the Niagara blue 4B method requires no special preliminary fixative nor very fresh tissue, and requires no more time than routine hematoxylin-eosin (H-E) staining. The method requires fixation in 10% formalin. After processing to paraffin wax, deparaffinise and hydrate the sections and stain in 1% aqueous Niagara blue 4B solution for 2 min. Stain afterwards with hematoxylin for 1 min then differentiate, wash, dehydrate, clear and mount. This method can be used also for staining old HE slides by removing the covers, applying the Niagara blue 4B and restaining with eosin. The Niagara blue 4B combined with H-E gives the best and most colorful result. This method allows special staining of the adenohypophysis from human post-mortem material to become routine.     

Possible uses of Dioxan in Botanical Microtechnic

Dioxan has been well established as an advantageous dehydrating agent for plant tissues. It dehydrates equally well after fixatives containing formalin, acetic acid, chromic acid, chromates, mercuric chloride, osmic acid, and alcohol. Better infiltration of paraffin after dehydration may be obtained by passing the material thru (1) a cold bath composed of 30 cc. of dioxan, 5 cc. of xylol and 20 cc. of melted soft paraffin and, (2) a warm bath of 50 cc. of dioxan, 50 cc. of paraffin, and 10 cc. of xylol. Transfer from (2) to soft paraffin. A dioxan fixative consisting of dioxan 50 cc., formalin 6 cc., acetic acid 5 cc., water 50 cc. was devised for delicate subjects. The fixed material is transferred directly into dioxan and mounted in dioxan-diaphane or dioxan-balsam. Very delicate objects require dioxan dilution of the balsam and slow concentration of the mounting medium by evaporation.

Entire plant parts or epidermal peelings are fixed in any desired fixative, washed if necessary, transferred to dioxan and mounted in diluted dioxan-balsam or diaphane. Dioxan may be used to mount hyalin objects whose refractive indexes approach those of balsam in media of higher index than balsam. It may be used in place of alcohol in finishing parafin sections, and since it exhibits different stain solubilities than alcohol it offers an important new tool in obtaining and maintaining stain balances.     

Chlorazol Black E As An Aceto-Carmine Auxiliary Stain

In making chromosome counts on plants and plant parts treated with colchicine it was found that in cases where aceto-carmine alone is not satisfactory—as in axillary buds of apple, pear, plum, peach, apricot, and cherry—the following method was effective : Dissect out the meristematic parts of the axillary bud under a binocular (or cut free-hand sections) and transfer the dissected tissue immediately to a solution of 3 volumes alcohol to 1 volume acetic acid for killing and fixing. Let the fixative act at least 10 minutes; a longer time, 12-24 hours, improves the staining quality. Wash in at least 3 changes of 70% alcohol to remove most of the acid. Stain for 5-25 minutes in 1% chlorazol black E² in 70% alcohol. Rinse in 3 changes of 70% alcohol to remove excess stain. Transfer the material to a slide, cover with a drop of aceto-carmine, and if necessary, dissect further under a binocular. Cover with cover glass, heat, flatten and seal, or run Zirkle's fluid under the cover for permanent mounting. For smears of sporocytes, chlorazol black E may also be employed alone, or in combination with aceto-carmine, if a dark purple nuclear stain is desired.     

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.