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.     

Influence of staining on fast automated cell segmentation, feature extraction and cell image analysis

FAZYTAN, a system for fast automated cell segmentation, cell image analysis and extraction of nuclear features, was used to analyze cervical cell images variously stained by the conventional Papanicolaou stain, the new Papanicolaou stain and hematoxylin and thionin only; the last two dyes are used as the nuclear stains in the two versions of the Papanicolaou stain. Other dyes were also tried in cell classification experiments. All cell images in the variously stained samples could be described by the same nuclear features as had been adapted for the discrimination of conventional-Papanicolaou-stained cells. Variances were lower for thionin-stained cells as compared with hematoxylin-stained cells. By application of spectrophotometry, it was confirmed that the spectra of the cytoplasmic counterstains are superimposed on those of the nuclear stains. It appears that a variety of dyes are suitable as cytologic stains for cell classification by the FAZYTAN system, provided that they achieve sufficiently strong nuclear-cytoplasmic contrast by precisely delineating the chromatin texture.     

Stain Technic for Nissl's Granules Following Alcohol-Formol Fixation

1. Nerve tissue is fixed 2-4 hrs in a 5% solution of strong formalin in commerical 95% alcohol.

2. If dehydration is perfect, either chloroform or xylol may be used as a clearing agent.

3. A slow method of paraffin infiltration is advisable.

4. Sections should be cut 10-12 microns in thickness.

5. Coplin staining jars should be annealed by placing them on a rack in a pan of cold water, bringing the water to the boiling point, and allowing the jars to stand in boiling water for twenty minutes.

6. One per cent aqueous solutions of either methylene blue or Grübler's Neutral Roth are used as specific stains for Nissl's granules.

7. These stains are heated to boiling in a beaker, the slides are placed in the Coplin jars which are partially submerged in boiling water, and the hot stain poured into the jars. The flame beneath the water bath is turned down and the slides left for 20 minutes.

8. The excess of primary stain is washed off in 25% and 50% alcohol and the slides passed rapidly thru the alcohol series to absolute alcohol, and finally to xylol.

9. When counterstaining is desired, nigrosin in 1% aqueous solution, methyl orange, saturated solution in 50% alcohol, or, a 0.5% solution of eosin in 50% alcohol are recommended. These stains are used cold, and the slides are merely dipped in them after the excess of primary stain has been washed out in 25% and 50% alcohol.

10. If a cold primary stain is desired, a saturated solution of thionin in distilled water, acidified with 1% carbolic acid, will prove specific for the Nissl substance. Sections should be stained 5–10 minutes in thionin, then passed rapidly thru to absolute alcohol, and xylol. The same counterstains may be used as in the hot method.

11. Sections prepared by the hot method show little tendency to fade after ten years use.

12. Excepting neutral red, all the stains used in this technic are carried by the National Aniline and Chemical Company and are satisfactory. Coleman and Bell neutral red may be substituted for Grübler's Neutral Roth with good results.     

Standardization of the Papanicolaou stain. I. A comparison of five nuclear stains.

The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy.     

Newer applications of the histological stain prepared from Pterocarpus santalinus

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn. has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.     

Newer Applications of the Histological Stain Prepared from Pterocarpus Santalinus

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn, has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.     

Standardized Thionin-Eosin Y: A Quick Stain for Cytology

Two stains long used in exfoliative cytology, the hematoxylin-eosin Y and Papanicolaou stains, have not been standardized even today. Some dozens of hematoxylin and eosin and Papanicolaou staining recipes have been recommended in the literature. Consequently, the staining pattern of hematoxylin and eosin, and Papanicolaou stained cytological material varies from laboratory to laboratory. To a certain degree this is due to batch-to-batch variations of commercial samples of the natural dye hematoxylin (C.I. 75290). The present paper describes a simple, standardized and reproducible procedure using thionin bromide to replace hematoxylin in the hematoxylin and eosin stain.     

The Oxidation Products of Methylene Blue

The mechanism of the oxidation of methylene blue varies with the conditions. The formation of trimethyl thionin (azure B) and of asymmetrical dimethyl thionolin (azure A) is followed under alkaline conditions by that of dimethyl thionin (methylene violet) and under acid conditions by that of monomethyl thionin (named by authors azure C).

Simple and practical methods are given for the preparation of azure A and azure C. The latter product, which has not been obtained from methylene blue hitherto, has valuable staining properties as a nuclear and bacterial stain in tissue and may also be employed satisfactorily as a substitute for azure A in the MacNeal tetrachrome formula as a blood stain or substitute for the Giemsa stain.

Azure B has no particular merit in staining.

Azure C proves to be a very valuable stain. A procedure is given for its use with eosin Y and orange II as counterstains, by which it is possible to demonstrate bacteria in tissue and at the same time the cytological elements of the tissue.     

A comparative study of quantitative stains for DNA in image cytometry.

In this study we examined the reproducibility of several stains used to measure nuclear DNA by image cytometry. The specimens were touch preparations of liver and testis from mouse and liver, intestine and brain from rat, fixed in either neutral formalin or Carnoy's solution. The tested stains included four Feulgen methods (pararosaniline, azure-A, thionin and acriflavine), the gallocyanine-chromalum stain and two fluorescent stains (acridine orange and propidium iodide). Absorbance measurements employed a video image analysis system; fluorescence measurements were from a scanning microspectrophotometer. The acriflavine-Feulgen stain was analyzed for both absorbance and fluorescence. All seven stains were quantitative for DNA and gave reproducible results. The absorbance measurements had a lower coefficient of variation (CV) than the fluorescence values. In a nested analysis of variance of the pararosaniline Feulgen stains, cell-to-cell variability accounted for 67% of the total variance; slide-to-slide, 9%; and batch-to-batch, 24%. These values did not change significantly when the staining was performed in an automatic staining machine. For DNA analysis using image cytometry, we conclude that the Feulgen staining technique is the most useful. In particular, acriflavine-Feulgen-stained cells fixed in Carnoy's fluid give the least variation between measurement values and the most accurate ratios between the separate ploidy groups. For fluorescence cytometry we recommend Carnoy's fixation and the acriflavine-Feulgen stain because of its narrow CV as compared to acridine orange and propidium iodide.     

The new and reproducible Papanicolaou stain. Morphologic and spectrophotometric observations on the influence of stain composition on staining results

The morphologic and spectroscopic characteristics of a new and reproducible modification of the Papanicolaou stain are briefly described. The main features of this modification are (1) replacement of the natural dye hematoxylin by the synthetic and chemically well defined dye thionin, (2) introduction of an alcoholic counterstain consisting of eosin gamma and fast green FCF only and (3) employment of alcoholic solutions only. The absorption characteristics of hematoxylin and thionin bound to chromatin are influenced by the cytoplasmic counterstaining, especially by the two green dyes, the absorption peaks of which are close to those of the nuclear stains. The implications of these results for visual and automated cytologic diagnosis are briefly discussed.     

Aldehyde-Thionin: A Stain Having Similar Properties to Aldehyde-Fuchsin

A mixture containing thionin, 0.5 gm; paraldehyde, 7.5 ml; concentrated HCl, 1 ml; and 70% ethanol, 91.5 ml, when allowed to ripen for several days, produces a stain which, when applied to sections of tissue fixed in a Zenker-based fixative, resembles in its effects the aldehyde-fuchsin stain of Gomori, but presents certain advantages.     

Cinematography of Brain Structures Prepared by Perfusion Staining

Brains of rats were stained by perfusion, frozen, and sectioned. Of more than 10 stains tried only thionin and a silver stain (Weil-Davenport) gave good contrast upon penetrating the brain by way of its vascular system. Each section was discarded but the exposed surface of the block was photographed as single frames on 16 mm motion picture film. Proper projection at 16 frames/sec was obtained by cutting 10-14 μ sections, with 3 exposures after each section. Thus the viewer could readily visualize neural organization in 3 dimensions. The method is valuable for teaching neuroanatomy and for determining the extent of lesions in neurological investigations.     

Short Nissl Staining for Incubated Cryostat Sections of the Brain

Nissl stain often binds poorly to cryostat sections which have been incubated in solutions of radiolabeled ligands. Such incubation is used in receptor autoradiography of the brain when using the in vitro method. We have developed a rapid (16 min) modification of Nissl staining for sections that bind stain poroly, e.g., incubated sections. The method stains well sections which cannot be stained with other rapid Nissl staining methods.     

Short Nissl Staining for Incubated Cryostat Sections of the Brain

Nissl stain often binds poorly to cryostat sections which have been incubated in solutions of radiolabeled ligands. Such incubation is used in receptor autoradiography of the brain when using the in vitro method. We have developed a rapid (16 min) modification of Nissl staining for sections that bind stain poroly, e.g., incubated sections. The method stains well sections which cannot be stained with other rapid Nissl staining methods.     

Pinacyanol Erythrosinate as A Stain for Mast Cells

An alcoholic solution of the compound dye, pina-cyanol erythrosinate when diluted to the optimum dissociation point is a differential tissue stain which, in addition, selectively stains and differentiates mast cells. It can be made up and used like any other compound dye (e.g., Bowie's stain, neutral gentian, etc. or like a blood stain). It can be used after any of the common fixatives and has the advantage of selectively staining all types of mast cells in their various functional phases, even in those species (notably rabbit and man) in which they may be difficult to demonstrate with other mast cell stains after aqueous fixatives.     

Pinacyanol Erythrosinate as A Stain for Mast Cells

An alcoholic solution of the compound dye, pina-cyanol erythrosinate when diluted to the optimum dissociation point is a differential tissue stain which, in addition, selectively stains and differentiates mast cells. It can be made up and used like any other compound dye (e.g., Bowie's stain, neutral gentian, etc. or like a blood stain). It can be used after any of the common fixatives and has the advantage of selectively staining all types of mast cells in their various functional phases, even in those species (notably rabbit and man) in which they may be difficult to demonstrate with other mast cell stains after aqueous fixatives.     

Staining of the elastic fibers in insect connective tissue after tannic acid/glutaraldehyde fixation.

Locust neural lamella and Calpodes connective tissue fixed in glutaraldehyde have a fibrous component which stains after reaction with DAB and osmication and after staining sections with PTA. The fibers also stain when fixed in glutaraldehyde with tannic acid followed by osmication and section staining with lead citrate.     

A rapid connective tissue stain for glycol methacrylate embedded tissue

No reliable connective tissue stains for GMA sections were available until recently. However, the use of toluidine blue in combination with basic fuchsin appeared to be a rapid and reliable connective tissue stain for glycol methacrylate (GMA) embedded tissue.     

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.     

Fluorescent stains for quantification of DNA by confocal laser scanning microscopy in 3-D

Confocal microscopy requires the use of fluorophores to visualize structures of interest within a specimen. To perform reliable measurements of the intensity of fluorescence, the stain should be specific, penetrate well into tissue sections, and bind stoichiometrically. Furthermore, emission must be linear with respect to DNA content and brightness, and fluorescence should be stable. Confocal microscopy is used to determine DNA ploidy and to analyze texture of nuclei, which is accomplished in three dimensions, because nuclei can be measured within the original tissue context. For this purpose the sample must be stained with a DNA binding fluorophore with the properties described above. Stains with different properties have been developed for different applications. We review here the advantages and disadvantages of these different stains for analyzing DNA ploidy and nuclear texture using three-dimensional microscopy. We conclude that SYBR green I and TO-PRO-3 are the most suitable stains for this purpose at present.