Gallocyanin as a Nuclear Stain in Autoradiography

In autoradiography, staining sections with gallocyanin and counterstaining with metanil yellow produces clear autoradiograms and avoids the staining of the emulsion encountered by using hematoxylin. Gallocyanin is a gradually progressive stain and by appropriate timing the intensity of staining is readily controlled. It is unnecessary to subject the plates to differentiating solutions.     

Gallocyanin as A Nuclear Stain

Gallocyanin has been used successfully as a nuclear stain. Sections are cut by the freezing method of either fixed or unfixed tissue. The tissues are warmed (not exceeding 70°C.) for 2-4 minutes in the gallocyanin solution. A counterstain may be used if desired. The most effective are Biebrich scarlet, phloxine, or eosin Y. The sections are then dehydrated and mounted in clarite. The nuclear pattern is clearly demonstrated and the sections are permanent.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4′,6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

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.     

Trypan Blue as a Nuclear Stain for Plant Material

Although McWhorter¹ and later Rich² mentioned that trypan blue stained the nuclei of plant cells, their procedures were concerned with the demonstration of virus inclusions. The following method was developed and is presented with the emphasis on nuclear staining. The present author hopes that others will try it for comparison with the popular aceto-carmine and aceto-orcein methods.     

Characterization and Separation of Some of the Coordination Compounds of Chromium and Aluminon

Chromic nitrate and aluminon (ATA) react to form highly colored lakes. Examination of these lakes by the Vosburgh and Cooper method has led to the identification of a pigment [(ATA)Cr(H₂O)_x] which is insoluble in water and absolute alcohol, a deep red anionic component [(ATA)_BCr(H₂O)_x]^-y, and a purplish-red cationic component [(ATA)Cr₄(H₂O)_x]^+y. These components of the lake have been isolated and separated in the dry form. The anionic compound is apparently a simple coordination compound and can be used as a cytoplasmic stain while the cationic component is a chelate and can be used as a rather selective nuclear stain. In addition to these components, a number of other components were also found. Not only the ratio between chromium and aluminon but also the concentration of the reactants influences the formation of these different components. The amount of pigment formed is maximal with a molar ratio of 1 and a concentration of 10^-2 M or 10^-1 M. The anionic component is maximal with a molar ratio of 1 Cr to 6 aluminon at the 10^-1 M concentration, the cationic component is maximal with a molar ratio of 4 chromium to 1 aluminon at the 10^-1 M concentration. None of these are formed at the 10^-4 M concentration but only a deep redpurple soluble compound at the 1 : 1 ratio, which was not further investigated.     

Phosphotungstic Acid-Chromic Acid as a Selective Electron-Dense Stain for Plasma Membranes of Plant Cells

A mixture consisting of 1% phosphotungstic acid (PTA) in 10% chromic acid (CrO₃) selectively stains the plasma membrane of plant cells. Whole tissue or pelleted cell fractions are prepared for electron microscopy using conventional methods including glutaraldehyde fixation and OsO₄ postfixation, dehydration in acetone and embedding in Epon. To stain the plasma membrane, thin sections are transferred with a plastic loop to the surface of a 1% aqueous solution of periodic acid for 30 min for destaining. Following transfer through 5 distilled water rinses, the sections are exposed to the PTA-CrO₃ mixture for 5 min, rinsed and mounted on grids for viewing with the electron microscope. The selectivity of the stain is retained in homogenates and serves to identify the plant plasma membrane in cell fractions.     

Crystal Violet as A Nuclear Stain for Gaffkya Tetragena and Other Bacteria

A method is given to stain the nucleus of Gaffkya tetragena and other bacteria. Only water solutions of crystal violet, mercuric chloride, and nigrosin are used. While the application of heat (50°C. for 20 seconds) is not absolutely necessary, it facilitates the decolorizing process. Some of the nuclei of cells from old cultures were found in various stages of division. The nucleus of a mature cell divides, and each daughter nucleus undergoes a second division usually in a plane at right angles to that of the first division. The nuclear division indicates cell development in units of four cells each. The method was found reliable for demonstrating the nucleus at various periods and stages of growth.     

Luxol Fast Blue as a Selective Stain for Alpha Cells in the Human Pituitary

Human pituitaries fixed in Bouin's fluid or 10% formalin were stained by the PAS, Masson trichrome and luxol fast blue methods. By comparing adjacent sections stained by these 3 methods it was found that the alpha cells which are PAS negative, but stained red by the Masson trichrome method, were intensely stained by luxol fast blue. The beta cells which are stained blue by the PAS and Masson methods were not stained by luxol fast blue. Similar observations were made on a series of pituitaries from 8 other mammalian species. It is concluded that luxol fast blue is a selective stain for alpha cells in the mammalian pituitary.     

Oxazine Dyes. I. Celestine Blue B with Iron as a Nuclear Stain

It is suggested that celestine blue B can stain as a colloidal dispersion, the nuclear specificity of which is controlled by the pH. The staining solution is prepared by adding 0.5 ml of concentrated H₂SO₄ to 1 gm of celestine blue B and dissolving the resultant granular mass in 100 ml of 2.5% ferric alum containing 14 ml of glycerol. Sections of amphibian, avian, and mammalian tissue placed for 1 min in this solution and then rinsed in water show as sharp nuclear staining as that usually produced by hematoxylin. A wide variety of fixatives is permissible. Overstaining is not possible within reasonable limits of exposure and no differentiation nor bluing is required. Both the staining solution and stained slides are stable.     

Hematoxylin as a Pituitary Stain

The accurate picture of the acidophil granules of the anterior pituitary which is provided by iron hematoxylin can be combined with the differential staining of the basophils by either the periodic acid-Schiff (PAS) or combined aldehyde-fuchsin-PAS procedures. To accomplish this the two stages of the iron hematoxylin technique are separated so that mordanting in iron alum precedes and application of hematoxylin follows the basophil procedures.     

Isohematein as a Biological Stain

The possible use of isohematein as a biological stain is considered. Certain characteristics of the dye are discussed in relation to staining technic. A preliminary series of experiments is described. The stomach of the frog, skeletal muscle of the frog, and spinal cord of the cat were used as representative tissues. The dye has greater tinctorial power than hematoxylin (hematein) but it is not so selective for nuclei. The results at hand indicate that the dye may have some value as a differential stain for nerve cell bodies. Fibrillae in smooth muscle cells and cross striatums in skeletal muscle were also brought out.     

The Site of Action of the Crystal Violet Nuclear Stain

Enzymatic treatment of bacterial cells prior to staining revealed that the crystal violet nuclear stain reacts with protein components of the nucleus as contrasted to the desoxyribonucleic acid specificity of some nuclear stains.     

Rhodanile Blue; a Rapid and Selective Stain for Heinz Bodies

Equal volumes of heparinized or EDTA-treated blood and a 0.5% solution of rhodanile blue (E. Gurr, Michrome No. 1156) in 1% NaCl were mixed and allowed to stand for 2 rain. Thin smears were then prepared, air dried and examined under oil. Heinz bodies stained deep purple and contrasted well with the yellow-orange to blue-green cytoplasm. Durable mounts could be made by applying a cover glass with a resinous mediiun to the dry smear (D. P. X. was used). The reticular material in reticulocytes did not stain in 2 min but could be stained by allowing the stain to act 5 min.     

Gomori's Hematoxylin as a cHromosome Stain

The chromic hematoxylin of Gomori (1941) can be used as an excellent chromosome stain after hydrolysis of the tissue in warm 1-N hydrochloric acid. The hydrolysis must be accurately timed for different material as in the case of the Feulgen reaction. The staining of sections can be performed at room temperature and requires about 15 minutes. For pieces of tissue and whole preparations, it is recommended to stain at 60°C. for 40 minutes. Sections stained at room temperature can be differentiated in 1% hydrochloric acid alcohol for one minute and can be counterstained with phloxine according to Gomori's formula. Whole preparations or sections stained at 60°C. must be differentiated in 45% acetic acid for half an hour or more. Tissue pieces may, after staining, be squashed and examined in the acetic acid, but the preparations can also be made permanent. The blue-black stain is very selective and has the advantage of giving high contrast, and it is nonfading, and insoluble in water and other common reagents. It proved definitely superior to other chromosome stains for difficult material such as planarians, rabbit blastocysts, and cleavage stages of sea urchins. Though both the procedure and the result of this method show some similarity to the Feulgen reaction nothing can be said with certainty about its chemical basis.     

The Use of Sudan Black B as a Bacterial Fat Stain

Sudan black B was introduced as a specific fat stain for the detection of lipids in tissue sections by L. Lison in 1934. Saturated solutions of Sudan black B in 70% alcohol or in ethylene glycol stain the fat bodies of bacteria a deep blue-black color, and this dye is recommended as superior to the other Sudans.

The method used in staining the bacteria was to suspend a loopful of the cells in a drop of the stain solution and to prepare flat wet mounts. The organisms giving positive fat tests with Sudan black B included Bacillus cereus, Bacillus mycoides, Azotobacter beijerinckii, Rhizobium leguminosarum, Mycobacterium avium, Mycobacterium leprae, Oospora lactis, Bacillus tumescens, water spirilla, and fungi.