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.     

Leukocyte Culture and Chromosome Preparations from Adult Dog Blood

Plasma is obtained from dog blood after 3 hr settling in a syringe. Portions of the plasma (0.5-1.0 ml) are added to 4 ml of a medium consisting of 17 parts of BME Spinner, 3 parts of calf serum, 0.5 parts of glutamine, 0.5 parts of penicillin-streptomycin, and 0.1-1.0 parts of Scarlet Runner bean phytohemagglutinin. Colchicine, 0.1 ml of 10:1 stock solution, is added after 72 hr and incubation continued for 2 hr, then centrifuged 5 min at 700 rev/min. The supernatant is discarded, 3 ml of distilled water added, and the cell suspension centrifuged again. The supernatant is discarded and the fixative, consisting of 45% glacial acetic acid allowed to act for 0.5 hr. Acetic-orcein stains of smears were very satisfactory.     

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.     

Improved Preparation of Polyvinyl Alcohol-HgCl2 Fixative Used for Fecal Smears

Rather than dissolve polyvinyl alcohol (PVA) powder in a modified Schaudinn's fixative (containing glycerol) by prolonged heating and stirring, it was found better to coat the particles of PVA with glycerol first. PVA powder (Delkote brand), 5 gm, mixed with 1.5 ml of glycerol and added to 62.5 ml of distilled water, is allowed to soak 3-16 hr in a stoppered flask, then heated to 70-75 C in a water bath (with swirling) for about 10 min or until solution is complete. A separate solution of the other ingredients: 95% ethanol, 31 ml; HgCl₂ 4.5 gm; and glacial acetic acid, 5 ml, is then added to the heated PVA solution, and swirled in the stoppered flask until the mixture becomes clear. This fixative is easy to make, produces little or no sediment, does not thicken with age, and has a long shelf life. It is strongly adhesive and fixes protozoa as well or better than that made by the older method or purchased ready made.     

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.     

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.     

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.     

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.     

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.     

Aldehyde-Thionin: A Stain Having Similar Properties to Aldehyde-Fuchsin

A mixture containing thionin, 0.5 gm; paraldehyde, 7.5 ml; concentrated HCl, 1 ml; and 70% ethanol, 91.5 ml, when allowed to ripen for several days, produces a stain which, when applied to sections of tissue fixed in a Zenker-based fixative, resembles in its effects the aldehyde-fuchsin stain of Gomori, but presents certain advantages.     

Application of Methods for Mammalian Chromosome Spreads to the Cells of Cestodes

Tapeworm cells obtained by physical maceration between ground-glass surfaces are incubated for 3 hr in Hanks' balanced salt solution (BSS) supplemented with colchicine to a concentration of 10-⁴ M. After washing in BSS, the cells are incubated for 10 min in 1/4 strength BSS then centrifuged 10 min. Fixation of the intact button of cells (or alternatively, by squirting the cells directly into the fixative) in Carnoy's alcohol-chloroform-acetic acid (6;3:1) for 30 min follows, and cells, dispersed and washed in the fixative, are flattened by dropping the suspension on clean, water-wet slides which are then air-dried and stained with Giemsa diluted 1 ml;47 ml with distilled water to which 2 ml of buffer—M/15 KH₂PO₄, 32 ml, mixed with M/15 Na₂HPO₄, 68 ml—is added. After staining 15 min and washing in distilled water, slides are air-dried and mounted with resin. Well separated and well stained chromosomes have resulted.     

A simple method for the preparation of paraffin-embedded cell blocks from fine needle aspirates, effusions and brushings

A simple method for the preparation of paraffin-embedded cell blocks from cytologic specimens obtained by fine needle aspiration, by brushing or from effusions is described. The cells are fixed in suspension in 50% ethanol for one hour and pelleted by centrifugation in a 50-mL plastic tube. The fixative is removed, and the pellet is suspended in 3 mL of acetone for dehydration for ten minutes and thereafter repelleted. The acetone is then removed, and the cell pellet is dried at 60 degrees C for one hour. Melted paraffin is added onto the dry warmed cell mass and allowed to solidify at room temperature. A conical paraffin block with the cells in the top is obtained and can be handled as a routine tissue block.     

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.     

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 Colchicine, Hypotonic Citrate, Air Drying Sequence for Foetal Mammalian Chromosomes

Mice 14 days pregnant were given 0.3 ml of 0.025% Colcemid (Demecolcine, Ciba) and killed after 1 hr. The livers of the foetuses were removed and broken up in 0.1% Colcemid in phosphate-buffered 0.85% NaCl and left 1.5 hr. The cell suspension was then centrifuged, resuspended in 1% sodium citrate for 20 min, centrifuged and the cells fixed in acetic-alcohol (1:3) for 30 min at 4°C. The cells were then resuspended (twice) in 45% acetic acid and dropped onto warm slides. After drying, the cells were stained for 30 min with lactic-acetic-orcein and examined under oil-immersion without a cover slip. Good numbers of well-spread mitotic figures were obtained.     

Alcoholic Bouin fixation of insect nervous systems for Bodian silver staining. I. composition of 'aged' fixative

Alcoholic Bouin (Duboscq-Brasil) fixative being 'aged' at 60 C to improve tissue preservation and subsequent staining was sampled at various stages to determine its histological effectiveness and chemical composition. Histological performance was tested using ventral nerve cord ganglia of the cockroach Periplaneta americana and the locust Schistocerca gregaria. Chemical analysis was by ultraviolet spectroscopy, thin layer and gas-liquid chromatography, and mass spectrometry. Histological performance improved rapidly during the first 7-10 days and composition changed correspondingly. The rate of change then slowed as a more stable condition was approached. Fully aged solutions, after about 40 days, giving optimum fixation and staining, contained little more than half the amounts of the volatile components (formaldehyde, ethanol, and acetic acid) in the original mixture, together with ethyl acetate and a formal, diethoxymethane, as the principal reaction products, but picric acid content showed little change. Older ('overaged') solutions, fully aged and then kept at room temperature for 1-2 yr, gave poorer fixation and staining and contained still less of the original volatile constituents and correspondingly more of the reaction products. A 'simplified synthetic aged alcoholic Bouin' (15 ml 40% formaldehyde, 35 ml ethanol, 3.5 ml acetic acid, 5 ml ethyl acetate, 15 ml diethoxymethane, 0.46 g picric acid, and water to 100 ml) closely stimulated the performance of the fully aged orthodox fixative without the need for aging.     

Alcoholic Bouin Fixation of Insect Nervous Systems for Bodian Silver Staining. I. Composition of 'Aged' Fixative

Alcoholic Bouin (Duboscq-Brasil) fixative being 'aged' at 60 C to improve tissue preservation and subsequent staining was sampled at various stages to determine its histological effectiveness and chemical composition. Histological performance was tested using ventral nerve cord ganglia of the cockroach Periplaneta americana and the locust Schistocerca gregaria. Chemical analysis was by ultraviolet spectroscopy, thin layer and gas-liquid chromatography, and mass spectrometry. Histological performance improved rapidly during the first 7-10 days and composition changed correspondingly. The rate of change then slowed as a more stable condition was approached. Fully aged solutions, after about 40 days, giving optimum fixation and staining, contained little more than half the amounts of the volatile components (formaldehyde, ethanol, and acetic acid) in the original mixture, together with ethyl acetate and a formal, diethoxymethane, as the principal reaction products, but picric acid content showed little change. Older ('overaged') solutions, fully aged and then kept at room temperature for 1-2 yr, gave poorer fixation and staining and contained still less of the original volatile constituents and correspondingly more of the reaction products. A 'simplified synthetic aged alcoholic Bouin' (15 ml 40% formaldehyde, 35 ml ethanol, 3.5 ml acetic acid, 5 ml ethyl acetate, 15 ml diethoxymethane, 0.46 g picric acid, and water to 100 ml) closely simulated the performance of the fully aged orthodox fixative without the need for aging.     

A comparison of two methods of preparing cells or nuclei for high-resolution microspectrophotometry

Synopsis Suspensions of isolated mouse thymus cells were subjected to two preparative methods: either they were dropped through several mm of 9:1 v/v ethanol-acetic acid fixative, allowed to stand for 1 hr and then processed for staining; or they were fixed, passed through a graded ethanol series to 70% ethanol, centrifuged on to slides in a modified Shandon cytocentrifuge and then carried wet into the staining procedure. All preparations were stained by the Feulgen reaction and evaluated by high-resolution microspectrophotometry. While the two preparative procedures yielded similar results, there appeared to be less variability in the data obtained from the centrifuged cell populations.     

A Fixative for Use in Dissecting Insects

A fixative made of 5 ml of 40% formaldehyde, 2 1/2 ml of glacial acetic acid and 20 gm of chloral hydrate diluted to 100 ml with distilled water is useful for dissecting insects. The advantages of this fixative are that it hardens soft tissues without making them coalesce or become brittle, softens tracheae and the exoskeleton, causes little change in the dimensions of tissues, and it is an excellent preservative. Incisions in the exoskeleton and several sudden releases of vacuum aid in fixation. Details of methods of staining and dissecting are given.     

用生料制备功能性双歧醋生产工艺的实验室研究

目的将双歧杆菌、醋酸菌、酵母菌和粉碎的制醋原料及麸曲共同发酵,通过生料制醋的方法来制备功能性双歧醋。方法将粉碎的玉米与麸曲、酵母液、麸皮和水搅拌均匀,使其经过液态糖化和酒精发酵后,接入醋酸菌和双歧杆菌(二者比例为1∶1),同时加入辅料,进行醋酸发酵,当检测到醋酸酸度为5.0%~7.5%时,加入食盐终止发酵,经过过滤,除菌澄清得到功能性双歧醋。结果双歧醋的最终醋酸度为3.2%,外观红棕色,光泽度好,清澈透明,无沉淀和悬浮物。总菌数:醋酸菌为3.3×1011/m l,双歧杆菌为1.9×107/m l;活菌数:醋酸菌为1.7×1011/m l,双歧杆菌为6.8×106/m l;大肠菌群数3个/100 m l;致病菌:不得检出。结论双歧杆菌及其代谢物可以在双歧醋中存活,生料固态发酵制备双歧醋的方法可行。