An Acetocarmine Technic for Cucurbita

Staminate Cucurbita buds undergoing meiosis are fixed for 12-24 hours in a solution containing 3 parts of 95% alcohol, 1 part of acetocarmine to which iron acetate has been added, and a flake of rusted iron. After fixation the buds are washed in 95% alcohol and stored in 95% alcohol with the iron flake for 5-10 days. A stain containing 10 drops of 45% acetic acid, 10 drops of acetocarmine, and 10 drops of brown storage solution is prepared. A small piece of anther is placed in a drop of stain on a slide. At the moment the anther is macerated, the debris is removed, and when the cells turn grey to dark brown a cover slip is applied. The stain is differentiated by gentle heat and the cover slip is sealed with paraffin.     

A Temporary Stain for Paramecium and Other Ciliate Protozoa

A new temporary stain for the demonstration of nuclear and cytoplasmic structures in Paramecium and other protozoan ciliates during both vegetative reproduction and meiotic reorganization consists of a mixture of 10.5 parts of acetocarmine, 4.5 parts of 45% acetic acid, 2 parts of 1 N HCl and 1 part of 1% solution of fast green FCF in 95% alcohol. This stain replaces the acetocarmine and acidified methyl green nuclear stains commonly employed and has the following advantages: (1) it affords simultaneous differential stainability of nucleus and cytoplasm (brown-red and green to grey-green, respectively); (2) it provides differential stainability of newly developing macronuclei (homogeneous pale green), fragments of the old macronucleus (brown-red), and food vacuoles (granular, bright blue-green); and (3) it results in a delicate and more transparent stain which affords greater clarity of internal structure. Proportions may be shifted slightly to achieve the optimum results for any particular organism. Concentrations of the acids employed may be diluted in instances where organisms tend to be easily distorted by fixation.     

A new method of image formation using cathodoluminescence in the SEM: CL absorption by superficial stains

The distribution of superficial histological stains on tooth samples can be demonstrated in the scanning electron microscope (SEM) due to the absorption by the stain of the auto-cathodoluminescence (light) arising from the underlying substrate. Practical procedures by which this has been achieved are described.     

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.     

Luxol Fast Blue as a Stain for Mallory's Alcoholic Hyaline

Luxol fast blue MBS (du Pont), which has frequently been used as a stain for phospholipids, stains Mallory's “alcoholic” hyaline a deep purplish blue. The stain is stable and provides histological appearances far superior to other methods. It is used on paraffin sections of tissue fixed in formalin or formalin-sublimate as a 0.1% solution in 90% alcohol at 60°C for 8 hr. Differentiation is made with 0.05% Li₂CO₃ and a red counterstain applied.     

Staining Mitochondria in Fixed Blood Smears

Wet blood smears are placed immediately in Helly's fluid for 24 hr, transferred directly to a saturated solution of potassium dichromate for 48 hr and washed in running water for 2-4 hr. The slides are then treated with iodine and sodium thiosulf ate and washed several hours or overnight. Excess water is removed by blotting the slide around the smear, Altmann's aniline fuchsin is placed on the smear and the slide is heated over a spirit lamp until white fumes appear. After the slide cools the stain is poured off and the excess removed by washing with distilled water. Methyl green (1% aqueous) is dropped on the smear and left for approximately 30 sec. It is then passed rapidly through 2 changes of absolute ethanol and into xylene, from which it is mounted in Permount. This stains mitochondria, red blood corpuscles and specific granules of eosinophilic granulocytes red on a green background.     

Histochemical detection of thiosulphate sulphurtransferase (rhodanese) activity

Summary The distribution of superficial histological stains on tooth samples can be demonstrated in the scanning electron microscope (SEM) due to the absorption by the stain of the auto-cathodoluminescence (light) arising from the underlying substrate. Practical procedures by which this has been achieved are described.     

Histochemical Differentiation of Anterior Pituitary Cell Types by Contrasting Azocoupling and Mucoprotein Stains

Azo compounds couple with aromatic amino-acid nuclei in the cytoplasmic proteins of human anterior pituitary acidophile and basophile cells. In acidophile cells the reaction appears to be due primarily to tyrosine, but also in part to histidine. Procedures are given for the use of naphthanil diazo blue B (tetrazotized di-ortho-anisidine), with or without a second coupling agent (H acid; 8-amino-1-naphthol-3, 6-disulfonic acid), to demonstrate acidophile cells, in contrast to mucoprotein stains (periodic acid oxidation followed by leukofuchsin or leukothionin) for the cytoplasm of basophile cells. Evans blue can also be used to give a contrasting color to basophile cells.     

Histochemical Differentiation of Anterior Pituitary Cell Types by Contrasting Azocoupling and Mucoprotein Stains

Azo compounds couple with aromatic amino-acid nuclei in the cytoplasmic proteins of human anterior pituitary acidophile and basophile cells. In acidophile cells the reaction appears to be due primarily to tyrosine, but also in part to histidine. Procedures are given for the use of naphthanil diazo blue B (tetrazotized di-ortho-anisidine), with or without a second coupling agent (H acid; 8-amino-1-naphthol-3, 6-disulfonic acid), to demonstrate acidophile cells, in contrast to mucoprotein stains (periodic acid oxidation followed by leukofuchsin or leukothionin) for the cytoplasm of basophile cells. Evans blue can also be used to give a contrasting color to basophile cells.     

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.     

A Preliminary Survey of Occurrence of Nuclear Extrusion in Various Higher Plants with DAPI (Flu-orescent) Stain

DAP (4,6.Diamidino-2-phenylindole) has been shown in this investigation to be a convenient and reliable DNA-specific nuclear stain which will exhibit pale blue fluorescence under ultraviolet irradiation. It could be employed to stain the nucleus in living plant specimens by simply immersing them in highly diluted DAPI solution (ca. 0.5 ppm) for 2–24 hours. The specimen with stained nucleus is still capable of exhibiting cyclosis. The seedling may grow steadily in such solution. Hence, the whole procedure is well suited for the preliminary survey of occurrence of nuclear extrusion in various higher plants chosen at random. As most investigations on nuclear extrusion have been intensively conducted on two monocots (wheat and Allium), special attention is paid to dicots. Free hand sections consisting of several layers of cells in thickness are taken from the various parts of a plant (flower bracts, petals and stipule) to avoid artificial indlced by injury ,and stripping. The main findings are summarized in Table, and the microscopic record illustrated in figures. The preliminary survey has adequately demonstrated that the frequency of successful detection of nuclear extrusion in plant samples taken at random is rather high although its occurrence is rare. Patient search under microscope is usually required. In general it is present in embryonic as well as in senescent tissues and in tissues of transitory existence such as the leaf hairs, but it is absent in mature normal function-organs. The results are in conformity with those of prefious observations.     

Stabilized Romanowsky blood stain.

It has been shown that the degradation of thiazine dyes which normally occurs in methanolic solution, as in the case of Romanowsky blood stains, can be prevented by making the solution acidic. In a certain range of acidity, the stain precipitates in the form of monothiazine eosinate, but by making the solution sufficiently acidic, eosin is protonated and the precipitate cannot form. These observations have been used to develop a blood stain which is stable, even at elevated temperatures, for several months. For use the stain is neutralized by a specially formulated fixative solution.     

Stabilized Romanowsky Blood Stain

It has been down that the degradation of thiazine dyes which normally occurs in methanolic solution, as in the case of Romanowsky blood stains, can be prevented by nuking the solution acidic. In a certain range of oddity, the stain precipitates in the form of monothiazine cosinate, but by making the solution sufficiently acidic, eosin is protonated and the precipitate cannot form. These observations have been used to develop a blood stain which is stable, even at elevated temperatures, for sexed months. For use the stain is neutralized by a specially formulated fixative solution.     

Bismarck Brown As A Stain For Mucoproteins

Bismarck brown has been used for many years as a stain for mucin. But some types of mucoprotein are so water-labile that they cannot be demonstrated by the aqueous or weak alcoholic solutions usually employed. It has been found that Bismarck brown in slightly acidified, strong alcoholic, solution stains mucin. A simple method is given for using this solution for staining water-stable mucoproteins. Another method is included in which full precautions are given for avoidance of water at all stages subsequent to fixation. This method must be used for the more water-labile mucoproteins. By the use of these methods, it has been possible to demonstrate a wide range of mucoproteins including those of the mast cells of Hardie and that of the zona pellucida of the graafian follicle.     

The analysis of some commercial dyes and Romamowsky stains by high-performance liquid chromatography.

High-performance liquid chromatography has been used to quantitate batch variations in commericial samples of thiazine dyes, thiazine eosinates, and Romanowsky-type blood stains. It has been observed that all the dyes and eosinates examined, only methylene blue chloride and thionin were reasonably free of their methylated, demethylated, or oxidized homologs. Large variations in composition were observed between most of the samples of each type examined. In several instances the labeled compound was a minority species. In one instance a dye was apparently mislabeled. Large compositional variation was found between various batches of Wright and Giemsa stains, whereas significant differences between the thiazine composition of these two stain types were minor. Very little compositional variation was observed between the lots of LARC stain examined. The thiazine composition of Ames stain was similar for the three lots examined. Ames stain, however, was found to contain several components of unknown composition which have been linked to degradation products formed when stains are aged in methanolic solution.     

Delafield's Hematoxylin and Safranin for Staining Meristematic Tissues

The following technic is suggested for staining permanent preparations of meristematic tissues: Prepare and mount the sections by the usual paraffin method. From water, stain them 2-10 minutes in a solution made by adding 2-4 cc. of Delafield's hematoxylin to a Coplin jar full of tap water. As staining is progressive, the sections should be examined from time to time with a microscope. When the cell walls have become a deep purple, transfer the preparations, thru the usual series, to a mixture of xylol-absolute-alcohol in equal parts, and from this to a counterstain made by adding 4-6 cc. of a saturated solution of safranin in absolute alcohol to a Coplin jar full of xylol (75%) with absolute alcohol (25%). This stains the nuclei. Leave the sections in the counterstain at least 2 hours and then rinse them in xylol-absolute-alcohol (1:1) to remove excess safranin. Transfer them to pure xylol and then mount them in neutral balsam.     

Displacement

Displacement is a noncommital term for the reactions that occur when slides previously stained in phloxine or rose Bengal are immersed for varying lengths of time in a solution of another dye in ethyl Cellosolve. In most histotechnic tests Lendrum's (1947) phloxine-tartrazine is given as the stain for acidophilic inclusion bodies. However the lack of contrast between the phloxine and tartrazine has been a serious limitation. A number of dyes were tried as possible substitutes for the tartrazine. A rose Bengal-Bismark brown Y procedure was developed which stains similarly to Lendrum's phloxine-tartrazine and which doer have the needed contrast. After staining for 10 min in 1% aqueous rose Bengal and rinsing in isopropyl alcohol slides are placed for 20, 30, 40 and 50 min in 0.05% Bismark brown Y in ethyl Cellosolve. In various tissues and structures the rose Bengal is sequentially displaced by the Bismark brown Y. Thus collagen loses the red stain after 30 min while acidophilic structures like sperm heads and Paneth cell granules retain the red stain after 50 min in the displacement solution. The results are strikingly similar to staining with alkaline Biebrich scarlet.     

Displacement.

Displacement is a noncommital term for the reactions that occur when slides previously stained in phloxine or rose Bengal are immersed for varying lengths of time in a solution of another dye in ethyl Cellosolve. In most histotechnic texts Lendrum's (1947) phloxine-tartrazine is given as the stain for acidophilic inclusion bodies. However the lack between the phloxine and tartrazine has been a serious limitation. A number of dyes were tried as possible substitutes for the tartrazine. A rose Bengal-Bismark brown Y procedure was developed which stains similarly to Lendrum's phloxine-tartrazine and which does have the needed contrast. After staining for 10 min in 1% aqueous rose Bengal and rinsing in isopropyl alcohol slides are placed for 20, 30, 40 and 50 min in 0.05% Bismark brown Y in ethyl Cellosolve. In various tissues and structures the rose Bengal is sequentially displaced by the Bismark brown Y. Thus collagen loses the red stain after 30 min while acedophilic structures like sperm heads and Paneth cell granules retain the red stain after 50 min in the displacement solution. The results are strikingly similar to staining with alkaline Biebrich scarlet.