Permanent Mounting of Unstained and Aceto-Orcein Stained Cells in the Water-Soluble Medium,Abopon

For cellular morphology, mammalian cells were grown on cover slips in Leigh ton tubes, fixed in 1% osmic acid vapor for 2 min, decolorized with 30% H₂O₂ in 5% ammonium oxalate solution (1:7) for 2 min, then washed thoroughly, and finally mounted in a water-soluble medium consisting of a saturated solution of Abopon in 0.2 M phosphate buffer, pH 7.0. For chromosomal analysis of similarly cultured cells, aceto-orcein preparations were made by conventional methods, with the following minor modifications: following pretreatment with colchicine and hypotonic expansion, the cells on the cover slips were fixed in acetic-alcohol (1:3), air dried, incubated at 37° C for 15 min in 2% orcein in 45% acetic acid, rinsed in 45% acetic acid, washed several times in distilled water, and finally mounted in Abopon mounting medium. Both kinds of preparations were allowed to harden for 24 hr before being handled. Such slides will keep for years at room temperature. Studies requiring frequent comparisons of cellular and chromosomal morphology of cultured cells can thus be extended over long periods of time.     

Avian Microchromosomes; As Shown by Prefixation Treatment with Colchicine, Squashing, and Haematoxylin Staining

Small pieces of bird tissue from developing feathers, embryos, embryonic gonads and adult testes are treated before fixation in a 0.25% solution of colchicine diluted with an equal volume of hypotonic Pannett and Compton solution containing KCI, 0.336 gm; CaCl₂, 0.160 gm; Na₂HP0₄. 12H₂O, 0.172 gm; and NaH₂PO₄. 4H₂O, 0.0172 gm per 1000 ml of double distilled water, for 20-30 min at 37° C. This material is subsequently centrifuged at 1000 rev/min for 1-3 min, fixed with acetic alcohol (1:3) and stained with either Gomori's haematoxylin or by Feulgen's procedure. Preparations are frozen in the freezing chamber of a refrigerator for the separation of the cover slips, then mounted in euparal after dehydration in alcohols. With this technique, unclumped and clear preparations of both macrochromosomes and microchromosomes are obtained regularly.     

A Phloxine-Azure-Hematoxylin Sequence for Differential Staining of Cells in Pancreatic Islets

Sections of 6 μ from tissues fixed in Susa or in Bouin's fluid (without acetic acid) and embedded in paraffin were attached to slides with Mayer's albumen, dried at 37 C for 12 hr, deparaffinized and hydrated. The sections fixed in Susa were transferred to a I₂-K₁ solution (1:2:300 ml of water); rinsed in water, decolorized in 5% Na₂S₂O₃; washed in running water, and rinsed in distilled water. Those fixed in Bouin's were transferred to 80% alcohol until decolorized, then rinsed in distilled water. All sections were stained in 1% aqueous phloxine, 10 min; rinsed in distilled water and transferred to 3% aqueous phosphotungstic acid, 1 min; rinsed in distilled water; stained 0.5 min in 0.05 azure II (Merck), washed in water; and finally, nuclear staining in Weigert's hematoxylin for 1 min was followed by a rinse in distilled water, rapid dehydration through alcohols, clearing in xylene and covering in balsam or a synthetic resin. In the completed stain, islet cells appear as follows: A cells, purple; B cells, weakly violet-blue; D cells, light blue with evident granules; exocrine cells, grayish blue with red granules.     

Preparation of Chromosomes of Tetrahymena Pyriformis for Photomicrography

Conjugating animals of the protozoan, Tetrahymena pryiformis, were affixed to cover slips by means of Nissenbaum's fluid, followed immediately by 1:3 acetic-alcohol for 18-24 hr. After fixation, the material was transferred through a descending alcohol series to water, then hydrolyzed in 1 N HCl, washed in water, followed by immersion in 45% acetic acid and subsequent mounting in aceto-carmine. Photomicrographs were made using a phase-contrast microscope and Microfile film. The schedule resulted in preparations with abundant material, adequate spacing of chromosomes in a single plane, and excellent differentiation of the chromosomes from the cytoplasm.     

A simple method of preparing permanent squash preparations using cellophane]

A technique to prepare permanent squashed preparations of cell nuclei and chromosomes is proposed. Fix a piece of material on the slide with acetic alcohol (1:3), macerate with a 45% acetic acid, cover with hydrophilic cellophane previously soaked in a 45% acetic acid and then with a cover slip and filter paper to squash finally as it is routinely performed. After that soak off the cover slip with alcohol, post-fix the squashed preparation together with cellophane in alcohol for 5-10 min, unstick the cellophane, pass the preparation through alcohol once again and dry it. The subsequent treatment of the squashed preparation depends on the purpose of investigation. The slide may be tinctured overlaid with photoemulsion for autoradiography, or processed by different ways.     

An Improved Pollen Grain Smear Method for Wheat

Anthers containing actively dividing pollen grains were treated 1 hour at 18-20° C. with 0.2% solution of colchicine, washed 1 hour in water, soaked in 0.002 M aqueous solution of 8-oxyquinoline at 10-14° C. for 1 hour, washed in water for 1 hour and then fixed in Carnoy's solution (alcohol, chloroform, acetic acid, 6:3:1) for 6 hours to overnight. They were washed successively in acetic-alcohol (1:1) 10-15 minutes, 70% alcohol 10-15 minutes and in water 30 minutes before hydrolysing them in bulk in 1 N HCl at 60° C. for 10-15 minutes. “Finally, they were stained in leuco-basic fuchsin for 15-30 minutes. Pollen grains were squeezed out of a stained anther in a small drop of egg albumen on a slide and the albumen smeared uniformly on the slide. The slide was dipped successively for a few seconds in glacial acetic acid and 45% acetic acid respectively. The smear was covered by a cover glass in a drop of aceto-carmine and pressed gently between folded filter papers. The cover glass was sealed with paraffin and stored overnight. To make the preparation permanent the paraffin was removed and the cover glass separated in a 1:1 mixture of acetic acid and n-butyl alcohol. The slide and the cover glass were then passed through n-butyl alcohol, 2 changes, and finally remounted in balsam.     

Preparation of Chromosomes of Tetrahymena Pyriformis for Photomicrography

Conjugating animals of the protozoan, Tetrahymena pryiformis, were affixed to cover slips by means of Nissenbaum's fluid, followed immediately by 1:3 acetic-alcohol for 18–24 hr. After fixation, the material was transferred through a descending alcohol series to water, then hydrolyzed in 1 N HCl, washed in water, followed by immersion in 45% acetic acid and subsequent mounting in aceto-carmine. Photomicrographs were made using a phase-contrast microscope and Microfile film. The schedule resulted in preparations with abundant material, adequate spacing of chromosomes in a single plane, and excellent differentiation of the chromosomes from the cytoplasm.     

Ag-staining of nucleolus organizer regions of chromosomes after A-,C-, G-, or R-banding procedures.

Metaphase chromosome preparations were made from leukocyte cultures of normal individuals. The cells were fixed in methanol:acetic acid (3:1 v/v), then dropped on cold, wet slides which were air-dried before storage at 4 degrees C. The slides were stained to identify the chromosomes by one of the following procedures: (1) Quinacrine. Slides were stained for 10 min in quinacrine mustard solution, rinsed in running tap water for 2 min, and mounted in Tris-maleat buffer, pH 5.6.     

The Peracetic-Orcein-Halmi Stain: A Stain for Connective Tissues

A selective stain useful for the study of connective tissues is described. The stain demonstrates elastic and oxytalan fibers as well as fibrils in mucous connective tissues previously undescribed. Reticular fibers are not stained. The stain may be used on sections that have been fresh frozen or fixed in formalin or ethanol. Sections are deparaffinized, washed in absolute ethanol, oxidized in peracetic acid 30 min, washed in running water, stained in Taenzer-Unna orcein 15 min, 37°C, differentiated in 70% ethanol, washed in running water, stained in Lillie-Mayer alum hematoxylin 4 min, blued in running water, and counterstained 20 sec in a modified Halmi mixture of 100 ml distilled water, 0.2 gm light green SF, 1.0 gm orange G, 0.5 gm phosphotungstic acid and 1.0 ml glacial acetic acid. Sections are rinsed briefly in 0.2% acetic acid in 95% ethanol, dehydrated and mounted.     

Simple Aids to Microscope Illumination

The following rapid but reliable method of making permanent preparations from temporary mounts has proved to be very useful.

Pollen mother-cell smears: Smeared anthers are treated hi the usual way with Belting's acetocarmine, except that the cover slip is left off. When correct differentiation is attained the stain is thoroly washed off with 50% acetic acid and the slide flooded with dioxan. This is followed by 2 changes of dioxan for 2 minutes each. A drop of Canada balsam dissolved in dioxan is added and a cover slip applied. In cases where a cover slip has been used at the acetocarmine stage it can be floated off in a staining jar of 50% acetic acid and dehydration with dioxan carried out as above.

Insect salivary gland chromosome smears: The glands are crushed under a cover slip in acetocarmine on a slide coated with dried egg albumen. After 20 minutes the area around the cover slip is flooded with 50% acetic acid and the cover slip floats loose so that it can be removed. The above described dioxan dehydrating procedure is then employed.

Squash preparations: Root tips are fixed in some suitable fixative and the Feulgen technic applied. The stained root tips can either be dehydrated by passing thru 3 changes of dioxan and mounting in dioxan-balsam where they are divided into small longitudinal sections by sharp needles, or they can be put immediately into a mixture of 1 part of 50% acetic acid to 1 part of corn syrup where shredding with needles is carried out. A cover slip is put on and separation of the cells completed by tamping or by applying pressure to the cover. This squash method is useful with anthers which are difficult to smear when in the early prophase stages of meiosis.     

Staining Cells on a Membrane Filter with Giemsa Stock and Tap Water

Cells grown on type AM 6 Alpha Metricel Gelman filter membranes are fixed 1-2 min with OsO₄ vapor, washed in distilled water, dehydrated 4 min in 95% and 4 min in absolute ethanol. They are then stained for 2 min in a 1:1 dilution of Giemsa stock solution and an approximately neutral, low mineral content tap water. The stain is removed from the filter by 1 min in 50%, 1 min in 95%, 1 min in absolute ethanol and 1 min in a mixture of absolute ethanol and xylene 1:1. The filter is finally cleared in xylene and mounted with a synthetic resin. This procedure gave polychrome staining on neonatal thymic cells from C57 black pedigreed mice and on human leucocytes, with little or no stain retained by the filter membrane.     

Permanent Preparations from Rapid Cytological Technics

The following rapid but reliable method of making permanent preparations from temporary mounts has proved to be very useful.

Pollen mother-cell smears: Smeared anthers are treated hi the usual way with Belting's acetocarmine, except that the cover slip is left off. When correct differentiation is attained the stain is thoroly washed off with 50% acetic acid and the slide flooded with dioxan. This is followed by 2 changes of dioxan for 2 minutes each. A drop of Canada balsam dissolved in dioxan is added and a cover slip applied. In cases where a cover slip has been used at the acetocarmine stage it can be floated off in a staining jar of 50% acetic acid and dehydration with dioxan carried out as above.

Insect salivary gland chromosome smears: The glands are crushed under a cover slip in acetocarmine on a slide coated with dried egg albumen. After 20 minutes the area around the cover slip is flooded with 50% acetic acid and the cover slip floats loose so that it can be removed. The above described dioxan dehydrating procedure is then employed.

Squash preparations: Root tips are fixed in some suitable fixative and the Feulgen technic applied. The stained root tips can either be dehydrated by passing thru 3 changes of dioxan and mounting in dioxan-balsam where they are divided into small longitudinal sections by sharp needles, or they can be put immediately into a mixture of 1 part of 50% acetic acid to 1 part of corn syrup where shredding with needles is carried out. A cover slip is put on and separation of the cells completed by tamping or by applying pressure to the cover. This squash method is useful with anthers which are difficult to smear when in the early prophase stages of meiosis.     

A Simplified Acid Hematein Test for Phospholipids

The acid hematein test of Baker was simplified by the following method: Tissues were fixed in either formal-calcium or formal-calcium-cadmium, frozen-sectioned in a cryostat, cbromatized with potassium dichromate, washed in water, stained in acid hematein solution, washed in water again, differentiated in borax-ferricyanide, dehydrated in alcohols, cleared in xylene, and finally mounted in balsam. The process requires only 2 days instead of the 6 days necessary for the original test of Baker. The technique stains phospholipid in mitochondria and in other cellular elements, and is recommended especially for mitochondria     

Chromosome Preparations from the Avian Allantoic Sac

An abundance of mitotic cells, a rapid and uniform response of cells to mitotic inhibition, ease in obtaining a monolayer of cells, clear and well-spread chromosomes make the allantois an ideal tissue for squash preparations. After a 45 min incubation of each embryo, still in the shell, with 0.02 ml of 0.05% Colcemid, 4-day avian embryos were treated in distilled water for 15 min or in 0.9% sodium citrate for 30-60 min and then fixed for at least 1 hr in 1:3 acetic-alcohol. Squash preparations were made after immersion of the allantois in 45% acetic acid for 5-10 mm. Phase contrast microscopy could be used, or permanent preparations made by freezing, air-drying and staining. Staining with Gram's iodine for 4 min followed by 1% crystal violet in 95% ethanol for 3 min is recommended. The allantois is well suited for use in biology laboratories to demonstrate avian chromosomes in different stages of mitosis.     

Squash Method for Meiosis in Ovules

The ordinary Feulgen or acetic-lacmoid squash tech-nic following fixation in freshly made Carnoy's fluid (alcohol, 6: chloroform, 3: glacial acetic acid, 1), provides an easy and reliable method of studying meiosis in ovules. After fixation for 1 day, the material was hardened in 95% ethyl alcohol for 1-2 days and taken to water by gradual hydration. For staining by the Feulgen method, the material was hydrolyzed 8-10 minutes in 1 N HO at 58-60°C., followed by staining in decolorized leuco basic fuchsin for 2 hours. The staining was intensified by transferring the material to water. After 15-20 minutes the water was replaced by 45% acetic acid. For staining by acetic-lacmoid, the ovules after fixation, hardening and hydration were transferred to standard acetic-lacmoid stain to which was added 1 drop of 1 N HCl to every 10 drops of stain. Gentle heat was applied till the stain started to give fumes. After allowing 20 minutes at room temperature the material was transferred to fresh acetic-lacmoid. Some 6-12 ovules were mounted either in a drop of 45% acetic acid or acetic-lacmoid, depending upon the Feulgen or acetic-lacmoid staining respectively. Gentle and repeated tapping over the cover glass by a blunt needle loosened the cells of integument and nucellus and finally left the megaspore mother cells undergoing meiosis, fully exposed to view. The process was carried out under constant observation using the low power of the microscope. The desired amount of flattening was brought about by light pressure over the cover glass and gentle heating. The preparations were made permanent by dehydrating in ethyl alcohol and mounting in Euparal.     

Sudan Black and Osmic Acid as Staining Agents for Testicular Interstitial Cells

Two standard cytological techniques have heen modified to stain specifically the interstitial cells of the testis. In Method 1, the tissue is fixed in Zenker-formol or Regaud's fluid for several hours or overnight and subsequently postchromed in 3% K₂Cr₂O₇ for 72 hr at 37°C. After paraffin embedding, sections are cut at 5μ, dewaxed, brought down to 70% alcohol and stained in an unfiltered saturated solution of Sudan black in 70% alcohol for 10-30 min. Sections are washed briefly in 70% alcohol to remove all excess dye, differentiated, if necessary, in 50% alcohol, downgraded to water and mounted in Farrants' medium or glycerol jelly. Interstitial cells: deep blue black; remainder of testicular tissue: light blue. Method 2 is essentially the Champy-Kull technique but specific staining for mitochondria is omitted and the sections are downgraded to water; then they are mounted in Farrants' medium or glycerol jelly without further treatment. In this way osmicated lipoids are preserved. Interstitial cells: conspicuous due to the variable number of black granules in their cytoplasm; the remainder of the tissue: yellow.     

Application of the Nauta-Gygax Technic for Degenerating Axons to Mounted Sections

Frozen sections of formalin-fixed brains containing lesions were mounted on slides that had been coated first with albumen-glycerol (1:1) then 4% gelatin and blotted. The slides were placed in formaldehyde vapor at 56° C for 40-60 min, washed, and stored (optional) in 10% formalin-saline. The staining technic was as follows: after washing, soak 30-40 min in 0.5% phosphomolybdic acid, rinse; put in 0.05% potassium permanganate 9-16 min (usually 12 min); decolorize in a 1:1 mixture of 1% hydroquinone and 1% oxalic acid; wash thoroughly; soak in 1.5% AgNO₃ at about 20° C for 25-35 min; rinse; put into an ammino-silver solution (4.5% AgNO₃, 20 ml; pure ethanol, 10 ml; ammonia, sp. gr. 0.880, 2.4 ml; 2.5% NaOH, 1 ml) for 1-2 min; reduce in acidified formalin (distilled water, 400 ml; pure ethanol, 45 ml; 1 % citric acid, 13.5 ml; 10% formalin, 13.5 ml) for 1-3 min; wash; dehydrate through ascending grades of alcohol, including absolute; coat with 0.5% collodion, allow to dry slightly and harden in absolute alcohol-chloroform (2:1); rehydrate and put into 1% Na₂S₂O₃ for 1 min; dehydrate and cover.     

Application and Evaluation of the Lead-Adenosine Triphosphatase Method in Skeletal Tissue

Application and evaluation of the lead-ATPase histochemical method in skeletal tissue has demonstrated an intracellular localization of enzyme activity. The skeletal tissue was demineralized for 72 hr in cold 10% aqueous EDTA adjusted to pH 7.2. Frozen sections were cut and placed on cold albumenized slides, oriented, thawed, dried in a cool air stream, and fixed for 10 min in cold (-2 to -3 C) 10% formalin buffered with Na-acetate and adjusted to pH 7.2. The sections were washed, treated with 10% EDTA for 20 min at room temperature, rewashed, and incubated for an optimal period of 30 min at 37 C. in the lead-ATP medium of Wachstein and Meisel. Following incubation the sections were washed, treated for 1 min with 1% (NH₄)₂S, rewashed, immersed for 30 min in 10% buffered formalin, dehydrated, cleared, and mounted. Evaluation of the substrate specificity suggests that other phosphatases associated with skeletal tissue do not complicate the ATPase reaction.     

Carbol Fuchsin as A Stain for Human Chromosomes

Cells derived from cultures of bone marrow or leucocytes were treated with hypotonic citrate solution, squashed in 45% acetic acid frozen with CO₂ to allow removal of the cover glass without disturbing the smear, and stained by the following schedule: absolute alcohol, 5 min; coat with 0.2% parlodion and air dry; 70% alcohol, 5 min; distilled water, 5 min; stain 2-5 min in a mixture of 45 ml of a 0.3% solution of basic fuchsin in 5% phenol, 6 ml of glacial acetic acid, and 6 ml of 37% formaldehyde. Differentiate and dehydrate in absolute alcohol, clear in xylene and cover. The stain is durable for several weeks if slides are stored in darkness when not in use. Results resemble those obtained by Feulgen or aceto-orcein methods.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.