A Chrome-Alum Preparation for Delicate and Difficult Fixations

A chrome-alum fixative is recommended as a reagent for general use. The basic formula is: C.P. chrome-alum, 3 g.; 40% formaldehyde, 30 ml.; glacial acetic acid, 2 ml.; distilled water, 238 ml. This fixative permits easy sectioning of yolk-rich amphibian embryos. It can be used to make permanent slides of Euglena showing the flagellum. It is a satisfactory fixative for insect larvae and fixes sharply the slime droplets of Planarians. Fixation should not exceed two hours or the material being fixed will swell. Rinses of 70% alcohol or water may follow the fluid. The fluid keeps well, does not harden tissues and gives good cytological detail.     

A Method for Cytogenetic and Cytological Examination of Small Shelled Larvae of Bivalves and Other Zooplankton

Studies on chromosomes and nuclei of very small bivalve larvae have been impeded by the veliger shell. It has been determined that the alcohokacetic acid fixative commonly used in cytogenetic techniques can be made to act as a decalcifying agent upon repeated heating. In addition, transfer of formalin fixed shelled specimens, routinely used as marine bioassay organisms, into ethyl alcohohacetic acid (3:1) fixative also yields clear cells for cytological examination of decalcified but otherwise intact oyster larvae and other zoo-plankton. Identification of cell type, such as germ-line primordia, in, for example, reproductive and ploidy level studies, and observations on the presence of bacteria can be made from the preparations. Material can be examined up to at least a year after preservation. The method is evaluated and its modifications are discussed.     

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.     

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.     

Cytogenetic Analysis of Bone Marrow and Peripheral Blood Samples Stored in Fixative for Several Years

We have developed a method which improves the spreading of chromosomes and permits banding analysis of cytogenetic samples of bone marrow and unstimulated peripheral blood which have been stored in fixative for up to 15 years. Metaphase cells had been harvested as usual and stored in fixative (acetic acid:methanol 1:3) at -15 C. The procedure includes 4-5 changes of fixative (acetic acid:ethanol 1:1). Next, cells are dropped onto a chilled, wet slide. The back of the slide is then rinsed with 70% ethanol and dried by ignition. C-, G-, Q-, or R-banding patterns can now be obtained with these specimens. The procedure is useful for reinvestigation of cytogenetic samples that were obtained prior to the development of banding techniques.     

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 Rapid Field Technique for Preparing Ant Chromosomes for Karyotypic Analysis

This technique for chromosomal preparation of ant tissues for karyotypic analysis is advantageous under field conditions because it reduces processing time and can be used under humid conditions. The cerebral ganglia from prepupae or early pupae are incubated 20 minutes in a hypotonic citrate solution, minced in a fixative solution of 3:3:4 glacial acetic acid: absolute methanol: distilled HOH, rinsed in a fixative solution of 1:1 glacial acetic acid: methanol followed by Carnoy's fixative, then immediately flame dried. The resulting metaphase chromosomes are well spaced and usually show banding characteristics.     

The Fixation of Tissues Vitally Stained with Trypan Blue

The following fixative is recommended for tissues vitally stained with trypan blue: Chloroform, 2 parts; absolute ethyl alcohol, 2 parts; glacial acetic acid, 1 part; mercuric chloride to the point of saturation.

The tissue should be fixed 1 to 2 hours; transferred to 95% ethyl alcohol for 12 hours; to absolute alcohol for 12 to 24 hours; to a mixture of absolute alcohol and xylol for 1/2 hour, and finally to xylol, before embedding in paraffin. Cedar oil may be used for clearing in the place of xylol; in that case the tissues should be transferred from absolute alcohol to a mixture of absolute alcohol and cedar oil for 24 hours before placing in cedar oil alone.

Various counterstains can be used; Mayer's carmalum is excellent.     

Theory and practice of combining coagulant fixation and microwave histoprocessing

The German, F. Blum, introduced formalin as a fixative in 1893. Formalin rapidly became popular for hardening and preserving gross human and animal specimens. As a result, microscopy for diagnostic pathology by combining paraffin embedding and formalin fixation was developed. Alcohol-based fixatives have coagulation of proteins as their main preservative effect. Because there is no cross-linking, immunostaining is not compromised, and DNA and RNA is not damaged. Ethyl alcohol was used by Dutch scientists of the 18th century, but was replaced by the cheaper formalin. Addition of low molecular weight polyethylene glycol (PEG) optimized the coagulant fixative, Kryofix. The polyethylene glycol prevents excessive hardening and enhances the speed of coagulation of proteins. Kryofix was used on a large scale for skin biopsies in Leiden between 1987 and 2001. DNA preservation by the formulated coagulant fixative, BoonFix, is related to the concentration of ethyl alcohol, PEG and acetic acid. BoonFix has been used since 2004 in Leiden for over 40,000 diagnostic skin biopsies and more than 100,000 cervical samples. A literature review and three decades of experience with coagulant, formalin-free fixatives in pathology suggest that when health authorities realize that formalin invalidates expensive tests, it might eventually be eliminated legislatively from diagnostic pathology. Finally, coagulant fixation is optimal for microwave histoprocessing where ethyl alcohol is followed by isopropanol.     

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.     

Collodion Membranes in Pollen Tube Technique

Membranes are formed by allowing a drop of collodion-acetone solution to come into contact with the surface of warm sugar solution in a petri dish. Pollen is germinated upon the smooth areas of the membrane when all traces of acetone have evaporated. Semipermanent preparations are made by isolating the pollinated area of the membrane, floating it onto a slide, and, after the removal of excess sugar solution, adding a drop of acetic-stain fixative, followed by an albumenized cover slip. The preparation can be made permanent by inverting a slide in a mixture of 1 part glacial acetic acid and 3 parts absolute alcohol, when the collodion membrane will dissolve and allow the cover slip and adhering grains to fall free. The cover slip is then passed through absolute alcohol (2 changes), xylene, and mounted in neutral mountant on a clean slide. By substituting a drop of the alcohol-acetic acid mixture in place of acetic-stain fixative, the grains adhering to the cover slip may be stained by the Feulgen method.     

Prefixing with Paradichlorobenzene to Facilitate Chromosome Study

A technic was developed which resulted in preparations containing many mitotic divisions with chromosomes well fixed and stained, rod-shaped, and spread throughout the cell. This technic has given good results with guayule (Parthenium argentatum), Crepis, Allium, Pisum, Lycopersicon, Tradescantia, and other plants. Material is prefixed in a saturated solution of paradichlorobenzene for 1-4 hours, fixed in 65% acetic acid (or other suitable fixative) for 12-24 hours, hydrolyzed in 10% HCl for 10-30 minutes at 60° C, rinsed in water, transferred to a drop of 45% acetic acid on a slide, and smeared and stained in aceto-orcein. The preparation may be made permanent by separating slide and cover glass in 1 part glacial acetic acid to 1 part absolute alcohol, putting them in absolute alcohol, and then recombining them with a drop of euparol.     

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.     

An Acetic Acid Dissociation, Air-Drying Technique for Insect Chromosomes, With Aceto-Lactic Orcein Staining

The method differs from mammalian techniques for somatic chromosomes in that it uses very small amounts of material. Drosophila melanogaster and an ant, Dorymyrmex sp., are used as examples. Pretreatment with 0.05% Colcemid in insect Ringer solution is applied to mature Drosophila larvae for 5 hr, by feeding, but Dorymyrmex prepupae require puncture and a 15 hr exposure of the puncture to the solution. Organs are removed under 1% sodium citrate, tansferred to fresh citrate for 10-20 min, than fixed in acetic-methanol, 1:3, for 30 min. Transfer to a drop of 60% acetic acid on a clean warmed slide dissociates the cells, which are spread by adding a small drop of fixative and tilting the slide in all directions. After immersion in acetic ethanol, 1:3, for 4 hr, rinsing in the stain solvent and draining the slides then have 2-3 drops of aceto-lactic orcein placed on each, coverslips added, and warmed (at about 50 C) for about 12 hr or until staining is sufficient. They can then either be treated as semipermanent or made permanent by allowing the coverslips to slide off in acetic-ethanol, dehydrating, and mounting in Euparal, or a synthetic resin.     

Whole Mounts of Small Embryos Attached Directly to Glass Slides

Suspensions of sea urchin embryos spread over the surface of glass slides were attached to the glass by a rapid coagulation of the surface with alcohol. This was done either by dipping the glass slides into absolute alcohol or by a short exposure to alcohol vapor. Thereafter the slides were immediately transferred to the fixative. A suitable procedure includes fixation with Carnoy's fluid (alcohol, chloroform, acetic acid; 6:3:1) and staining with Gomori's hematoxylin after acid hydrolysis.     

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.     

A Combined Hcl-Acetic-Alcohol Fixation and Hydrolysis Followed by Cresyl Violet Staining for Mosquito Chromosome Spreads

Mosquito tissues of cytogenetical importance were dissected out on a slide in 0.65% NaCl, under a dissecting microscope, and treated about 30 sec in a drop of 1:3 Carnoy's fixative diluted 1:19 with distilled water. Fixing and hydrolysis was done by a single step in a mixture consisting of: glacial acetic acid, 1; ethanol 96%, 3; HCl conc., 2; and distilled water, 2 (v/v) for 2-6 min at 20-25 C. The specimen was then rinsed with the acetic-alcohol fixative and covered in a drop of 1% cresyl violet in 50% acetic acid under a coverslip coated with Mayer's albumen. Washing was performed immediately by adding water dropwise to one side of the coverslip and drawing the fluid from the other side with absorbent paper. The preparation could be used either as a temporary slide or made into a durable mount. The DNA-containing bands of the giant polytenic chromosomes stained dark violet; interband regions, weakly stained or colourless against a clear background. Mitotic and meiotic figures in gonadal cells stained selectively dark violet or violet with a practically unstained cytoplasm.     

An Air-Drying Procedure for Mammalian Male Meiotic Chromosomes, Following Softening in Gluconic Acid and Cell Separation by An Ethanol-Acetic Mixture

A I cm³ sample of tubules from testes is placed in 5 ml of 0.7% Na-citrate for 20-30 min, then 5 ml of glacial acetic acid is added, mixed well, and allowed to stand for 30 min. The mixture is centrifuged, the supernatant removed, and 3 ml of 3 M gluconic acid is mixed with the tissue and allowed to act for 3 hr. The gluconic acid is removed with a pipette and the tissue is suspended in 5 ml of a freshly made 1:1 absolute ethanol-glacial acetic acid mixture. The tissue is drawn into and discharged from a syringe several times through an 18, a 20, and finally a 22 gauge needle to separate and suspend the cells. The cells are centrifuged and resuspended several times in fresh fixative to remove the gluconic acid. Finally, the cells are suspended in sufficient fixative to give a smear of suitable density, and air-dried preparations are made, or the suspension may be stored at 0-5 C for several days. The cells can be stained by any of the usual stains for chromosomes. This technique results in the improved spreading produced by the air-drying technique and permits recovery of all stages of meiosis and mitosis present.     

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.