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.     

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.     

A Method for Staining Certain Bacteria and Antherozoids

A modification of Loeffler's method for staining the flagella of bacteria was employed in staining large forms of bacteria and antherozoids. The bacteria or the antherozoids are killed and fixed in a drop of water on a slide and set aside to dry, before the next step is undertaken. The slide is treated for a period of time, varying from about ten minutes to several hours, in a practically saturated solution of tannic acid. After the slide is thoroly rinsed in water, it is stained with either a single stain or a combination of stains. The slide is then dehydrated with absolute alcohol, cleared, with clove oil, and completed in the usual manner.

The body of the bacterium and that of the antherozoid are well differentiated and the cilia are distinctly brought out by means of the method herein described.

The technic is of especial value in staining the antherozoids of mosses, liverworts, and ferns.     

A Quick-Freeze Method for Making Smear Slides Permanent

Many types of smear slides can be made permanent rapidly and effectively by substituting for the usual dehydration series a single-step process of freezing the slide on a block of dry ice, placing it immediately in 95% or absolute alcohol, and then mounting it. Advantages of the technic are its speed, the ease of separation of cover slip from slide with a minimum loss of cells, and the superiority of the resulting permanent slides.     

The Germicidal Effect of Staining Solutions

Gentian violet, crystal violet and carbol fuchsin applied to cover slip preparations for one minute will destroy the majority of non-spore-forming bacteria and yeasts, tho they can not be relied upon to do this consistently and in all cases.

The Gram staining procedure is more effective and non-spore-formers were never found to survive this process.

Methylene blue stains exert very little if any germicidal power and most organisms survived them readily. India ink was totally ineffective.

Several species of yeasts and yeast-like molds were killed in every instance by the Gram stain, gentian violet, crystal violet and carbol fuchsin, but survived both Loeffler's methylene blue and a plain aqueous solution of methylene blue.     

Recent Advances in Microtechnic. I. Methods of Studying the Development of the Male Gametophyte in Angiosperms

Among methods used for a study of nuclear details in the development of pollen grains, the following were found to be very satisfactory: (1) warming the entire grains in aceto-carmine and then clearing with chloral hydrate; (2) making smear preparations stained with crystal-violet-iodine or iron alum hematoxylin. For paraffin sections, a counterstain with dilute alcoholic erythrosin is often very useful after the usual iron hematoxylin technic.

A method of making cultures of pollen tubes on slides coated with thin films of sugar agar is described in detail. The tubes can be fixed by immersing the slide in formol-acetic-alcohol and then stained by any desired schedule. Iron alum hematoxylin was found to be the most satisfactory, but the Feulgen reaction is very valuable in such cases where the nuclei are obscured by the density of the pollen tube cytoplasm. Living pollen tubes can be kept under observation by dissolving a small quantity of neutral red or other vital stain in the sugar agar before it is spread on the slide.

For studying stages in fertilization or gametogenesis, styles should be fixed and sectioned only after a preliminary study with iodine-chloral-hydrate or safranin-anilin-blue or aceto-carmine. Once the extent to which pollen tubes grow in a given time in the stylar tissues has been determined, it is possible to fix material with some knowledge of what it is going to show.

Some other methods, that have not been tried by the authors but appear to be valuable, are also briefly described.     

The Smear Technic in Plant Cytology

The following method of making permanent smears of pollen mother cells is in general use and gives excellent results. Determine the stage of meiosis from aceto-carmin mounts. Smear the pollen mother cells on a dry slide. Fix in Navaschin's or a modified Flemming's solution from 1 to 2 hours. Wash in 10 to 20% alcohol from 15 to 30 minutes. Stain in 1% aqueous crystal violet from 1 to 5 minutes. Rinse in water and pass thru 30 to 50% alcohol, about 15 to 20 seconds in each. Transfer to 80% alcohol containing 1% iodine and 1% potassium iodide for 30 seconds. Destain with absolute alcohol, followed by clove oil. xylol, balsam and cover.

Permanent smears for chromosome counts can be quickly made by smearing pollen mother cells on a dry slide, fix and stain with aceto-carmin, dehydrate with mixtures of absolute alcohol and acetic acid, follow with xylol, balsam, and cover.     

Staining Methods For Studying Ingredient Distribution In Baked Cakes

A method is described for preparing cake crumb for sectioning and staining. Previous to embedding, the fat was stained and fixed by exposing small blocks of cake to the fumes from a 5%, freshly-prepared, aqueous solution of osmic acid (OsO₄). This was followed by dehydration in ethyl alcohol and tertiary butyl alcohol, removal of air under vacuum and infiltration with paraffin.

Sections were cut 20 and 9Op thick and mounted with water.

Wax was removed by immersion in xylene. The sections were rehydrated in a series of ethyl alcohol dilutions, from concentrated to dilute, then transferred to distilled water.

Protein was then stained pink by immersion of the slides in an acidified 0.04% water solution of eosin Y, or starch was stained blue with a dilute aqueous solution of iodine. Ten grams iodine and 10 g. KI were dissolved in 25 ml. distilled water. This stock solution was diluted for use one to two hundred times.

The relationship between protein and starch was demonstrated by staining the sections with eosin, differentiating in 50% alcohol and staining with iodine.

When slides of cake crumb were prepared in this way, the fat was stained black, the protein bright pink and the starch granules a dark blue.     

A Note on the Staining of the Skeleton of Cleared Specimens with Alizarin Red S

A selective, progressive method for staining the skeleton in cleared specimens, developed with rat material.

Fix in 95% alcohol for at least 48 to 96 hrs. Even longer fixation is desirable. Then place in a 1% solution of KOH until the bones are clearly visible through the surrounding tissues. Transfer directly to a dilute solution of alizarin in KOH, one part alizarin to 10,000 parts of 1% KOH. Allow the stain to act until the desired intensity is attained. Fresh stain may be added if necessary.

Complete the clearing process, (1) in Mall's solution, water 79 parts, glycerine 20 parts and KOH 1 part; (2) in increased concentrations of glycerine. Store in pure glycerine.

The success of the method depends on obtaining the proper degree of clearing before staining. If the specimen is insufficiently cleared, a general staining of all tissues usually occurs.     

Permanent-Type Mounting of Aceto-Iron-Haematoxylin Squashes in Corn Syrup

Preparations obtained by the aceto-iron-haematoxylin technique reported previously (Stain Tech., 37, 27-30, 1962) can be made relatively permanent either by ringing the cover slip with Karo corn syrup, or by mounting the squash in this syrup after separating slide and cover slip by the solid CO₂ freezing technique. In the latter procedure, both slide and cover slip may be placed briefly in 45% acetic acid for further differentiation of the stain, then recombined with a drop of syrup.     

Specific Staining of Nucleolar Substance with Aceto-Carmine

A method is described for staining nucleoli intensely by treating tissues with formaldehyde, hydrolysing in normal HC1 at 60°C. and staining with aceto-carmine. With correct hydrolysis time, chromosomes and cytoplasm are almost colorless.

Formaldehyde increases the acidity of cell parts, especially the nucleolus, presumably by neutralizing the basic protein groups, and increases the resistance to hydrolysis, perhaps by protecting the phospholipoprotein complexes which are most abundant in the nucleolus.

Hydrolysis reduces the acidity of cell parts, chiefly by removal of nucleic acids.

Aceto-carmine stains cell structures which are weakly acid in character (about pH 4-5) probably by precipitating as large dye aggregates.

The technic appears to be highly specific for nucleoli and related cell bodies.     

Initial and Persisting Staining power of Solutions of Iron-Hematoxylin Lake

A study was made of factors affecting the initial staining power and the stability of iron-hematoxylin lake solutions. The findings were applied to the preparation of a superior hematoxylin staining solution. This is made up as follows: in 50 ml. water dissolve, in order, 1.0 g. ferric ammonium sulfate [FeNE₄ (SO₄)₂⋅ 12H₂O], 0.8 ml. sulfuric acid, 50 ml. 95% ethyl alcohol, 0.5 g. hematoxylin. Filter the solution to remove the insoluble, white crust of the ferric ammonium sulfate. The solution stains well ten minutes after it has been made. Peak performance is attained within 5 hours, and is maintained for 4 to 8 weeks. Staining time is 3 to 30 minutes. Excess stain can be rinsed off the slide and section by immersion in water, after which destaining, if necessary, can be accomplished with a solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.18 ml. sulfuric acid. The slides may or may not be placed next in a neutralizing solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.5 g. sodium bicarbonate. They may then be passed through 50 ml. water, 50 ml. 95% ethyl alcohol on the way to alcoholic counterstaining solutions, or through water leading to aqueous counterstains.

The nuclear stain produced is black, intense and very sharp and has proved to be consistently excellent on a variety of animal and human tissues following a number of different fixatives.     

Some Synthetic Resins IN Combined Fixing, Staining and Mounting Media

Many of the recently devised plasticizers and resins can be utilized to advantage in cytological technics. Some of them have solubilities which enable us to incorporate them in such fixing and staining solutions as aceto-carmine and propiorric-carmine. They are non-volatile, do not alter the fixation images of the fluids with which they are mixed, and serve as mounting media as the volatile components evaporate. Thus it is possible to make a permanent slide in a single operation. These newer compounds are better adapted for this technic than are the natural balsams which have been used previously, as their greater tolerance for water provides a much greater margin of safety. Procedures are described for the utilization of (1) Rezyl 7020, a water-soluble resin (now, unfortunately, not available), which dries to form a water insoluble film, (2) Amberol 750 and (3) Bakelite BR-7160, two alcohol soluble resins, more miscible in solutions containing water than are the natural balsams. Formaldehyde can be included in the aceto-carmine and propionic-carmine fluids with the result that more nuclear detail is preserved. Lacto-gelatin has some valuable properties as a mounting medium and can be used when the specimen is stained with orcein. Carmine, which gives a permanent stain in Rezyl 7020, Amberol 750 and Bakelite BR-7160 fades in lacto-gelatin.     

Notes on Technic

This report details a whole mount technique which allows rapid assessment of meiotic stages in large numbers of mammalian oocytes using an aceto-orcein stain.

Previous studies of mammalian oocyte meiosis have been hampered by the use of time-consuming staining techniques. Temporary preparations have been produced using squashes of oocytes with fixative and stain drawn under the coverslip by diffusion (Austin 1961). An air-drying technique requires less attention by the technician and allows permanent preparations to be made (Tarkowski 1966). However, these methods require that each slide be treated individually. The improvements outlined below permit many slides to be processed together, allowing surveys of larger numbers of oocytes than before.     

Basic Fuchsin as A Nuclear Stain

Five distinct nuclear stains and staining procedures which utilize basic fuchsin as the dye have been studied, compared and tested on a Feulgen-weak fungus, Blastomyces dermatitidis, and other fungi.

Aqueous basic fuchsin has been shown to be an excellent, though impermanent, stain with which to study the nuclei of this and other fungi. The conditions under which formaldehyde acts as a mordant for basic fuchsin and produces a permanent nuclear stain have been established.

Comparison of crystal violet and basic fuchsin suggests that the mordanting action of the aldehyde operates through the para-amino groups of the dye. Certain other basic dyes were not mordanted by formaldehyde.

Gentle acid hydrolysis of the tissues has been found to be essential both to the specificity of the dye as a nuclear stain and to the mordanting effect of the aldehyde.

The possible relationship of these observations to the Feulgen reaction is discussed. A protocol for the method developed is presented.     

Histological Processing in Autoradiography: Loss of Radioactivity

Histological methods suitable for use in autoradiographic technics are described. An investigation has been carried out on the amount of activity lost from rat and human tissues during fixation and dehydration. Losses in the processing fluids varied from 25% to 90% of the initial activity for radioactive phosphorus and 4% to 20% for radioactive iodine in various fixatives.

The care necessary in handling sections if distribution of total activity is being studied is emphasized and floating on absolute alcohol is suggested as an alternative to warm mercury. Various procedures for staining sections before application of photographic emulsion and after developing are discussed. Ehrlich's hematoxylin applied regressively has given good results and eosin has been used successfully as a counterstain. Orth's lithium carmine is resistant to photographic developer and also Feulgen's stain counterstained with fast green can be used before covering the slides with photographic emulsion.     

Polyvinyl Alcohol with Cadmium Iodide and Fructose as an Aqueous Mounting Medium

An aqueous histologic and cytologic mounting medium containing polyvinyl alcohol, cadmium iodide and fructose is described. It is used in the preparation of permanent slides and may be applied to sections stained and rinsed in water or in any concentration of ethyl alcohol. The medium forms a hard, tough, quick-drying film which adheres well to the slide and cover glass. It is nonfluorescent, clear, optically homogeneous, and isotropic; it does not admit bubbles under the cover glass or crystallize on drying. It prevents or minimizes the bleeding of many stains from the sections; a list of 60 dyes, mostly basic, is given. The fresh medium has a pH of 4.4. The refractive index of the fresh medium is n_D²⁰ 1.4674; the dried film is n_D 1.6020 which may be lowered to n_D 1.5150 by decreasing the cadmium iodide in the formula. The viscosity at 25°C. is 7,299 centipoises. The medium has the following composition: distilled water, 40 g.; cadmium iodide, 34 g.; polyvinyl alcohol (Elvanol 51-05, du Pont's low viscosity grade) 18 g.; fructose, 8 g. The medium is prepared as follows: wash the polyvinyl alcohol with absolute methyl alcohol; dry and then grind in a mortar; dissolve the cadmium iodide in the water; add the polyvinyl alcohol while stirring with a speed-controlled motor-driven mixer; heat to 75°C. on a water bath with continuous stirring until dissolved; remove from the water bath and add the fructose while stirring; replace water lost by evaporation. The medium is ready for use when the foam has dispersed after standing.     

The Surface Staining of Embedded Tissues

The staining procedure is based on the theory that the freshly cut surface of embedded material will absorb stain only in the exposed tissue elements, provided that the embedding compound itself will not absorb the staining fluid. Concentrated stains are used for short intervals to insure minimum penetration. For paraffin embedded materials: (1) Cut block, preferably on microtome, to the desired tissue surface. (2) Rinse in absolute alcohol. (3) Float face down in stain. (Ripe, concentrated alum hematoxylin—Galigher's formula recommended—will stain in 10 to IS minutes. Heidenhain's iron hematoxylin works exceptionally well in some cases.) Mordant 20% alum 5 to 10 minutes, briefly rinse, and stain comparable 5 to 10 minutes in 1 to 1.5% hematoxylin. (4) Allow to become blue in tap water (for hematoxylin stains). (5) Counter-stain if desired. (6) Dehydrate in absolute alcohol for not more than 10 minutes. (7) Dry for 15 to 20 minutes. (8) Trim block to 2-3 mm. and mount between two cover glasses by use of microflame. Attach mount to slide with balsam. For celloidin embedded materials: (1) Dehydrate block with 90% alcohol, phenol-toluene, finally pure toluene. (2) Rinse cut surface with 90% alcohol, then apply stain. (3) Wash, after hematoxylin stains, counterstain if desired. (4) Dehydrate surface, 90% alcohol, phenol toluene, pure toluene, and mount in medium dissolved in toluene.

Possible applications of surface staining technic are suggested and illustrated.     

The “Dispersion Staining” Method for the Selective Coloration of Tissue

A method has been developed for the selective coloration of fixed tissue without the use of dyes. Microtome sections of formalin-fixed material are mounted under a cover glass in a mixture of two liquids such as diethylene glycol monobutyl ether with cin-namaldehyde and examined with the dark-field microscope. The refractive index of the liquid used for mounting must be of high dispersion and equal or close to the index of the specimen.

Tissue elements, dependent on their refractive index, whether slightly lower, the same as, or slightly above the mounting medium appear colored in shades of blue, red or yellow.

The optical principles involved in this optical dispersion method are similar to those involved in the production of colored light by the Christiansen filter.