A Two Step Stain for Normal and Leukemic Monocytes Using Two Different Dyes Applied in Sequence

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

Modifcation of the French Azure C Tissue Stain

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

Basic Blue 148: A Rapid Stain for T Helper Cells

After brief exposure to an aqueous solution of the oxazine textile dye C. I. basic blue 148 following fixation in 37% formalin, 95% ethanol and glacial acetic acid, T helper cell nuclei and cytoplasm in specimens of peripheral blood displayed a deep red-violet color. No other cell in normal blood or bone marrow specimens showed intense staining of this type. The total staining time is 1 min. Basic blue 148 stain is a promising technique for hematology and immunology laboratories as a rapid screening test for T helper cells in blood specimens using a microscopic slide and ordinary incandescent illumination.     

Basic Blue 54: a New Colorant for Monocytes

C.I. basic blue 54, a sulfur containing azo textile dye, stained the nucleus and cytoplasm of normal and leukemic monocytes bright red-violet. Essential for the staining reaction was a brief final rinse in a pH 3.6 acetic acid-sodium acetate buffer. Coloration of the type found in monocytes was not observed in other types of mature and immature leukocytes.     

Rapid Identification of T Helper and T Cytotoxic/Suppressor Lymphocytes with an Oxazine Dye

Peripheral blood lymphocytes displayed a plurality of sues and colors when exposed first to a methanolic solution of C.I. basic blue 141, then to an aqueous alkaline solution of the Same dye and Msed in a neutral HEPES buffer containing trace amounts of various salts. As confirmed with purified lymphocyte subpopula-tions obtained with a cell sorter, T helper cells (CD4) were small and their nuclei and cytoplasm stained deep blue. T cytotoxic/suppressor cells (CD 8) were larger than T helper cells, their nuclei stained pale green or blue green and their cytoplasm contained a cluster of magenta colored granules. From start to finish, the stain takes 15 min to perform. Used in the manner described, basic blue 141 holds promise as a rapid means of identifying and differentiating CD4 and CD8 cells under the ordinary light microscope without using monoclonal antibodies or fluorescence.     

Basic Blue 75: a New Stain for Erythroblasts

C.I. basic blue 75 in an aqueous alkaline solution stains the nuclei of mature anc immature erythroblasts bright red. Simultane ously, the stain colors the cytoplasm of erythro blasts blue in immature cells and purple in ma ture cells. Colors of the type described were noi found in other normal and abnormal hemato poietic cells.     

Alcian Blue Pyridine Variant–a Superior Alternative to Alcian Blue 8GX: Staining Performance and Stability

We compared the staining performance, dye content, solubility, and visual absorption maximum of two batches of alcian blue pyridine variant and of five batches of alcian blue 8GX (C.I. 74240). Whenever possible, we also compared results to those obtained with the same dye batches produced at an earlier date to provide information concerning dye stability. Both alcian blue pyridine variant batches were of high dye content, stable, of satisfactory solubility, and performed well in both the routine Mowry mucin stain and in the critical electrolyte concentration (CEC) stain. Of the five alcian blue 8GX samples, some were also of appropriate dye content, were sufficiently stable, and gave good staining in the two procedures. Two batches, however, were unstable, and three batches were unsatisfactory in staining performance and solubility in the CEC stain. Consequently alcian blue pyridine variant is a superior substitute for alcian blue 8GX.     

Alcian Blue Pyridine Variant-a Superior Alternative to Alcian Blue 8GX: Staining Performance and Stability

We compared the staining performance, dye content, solubility, and visual absorption maximum of two batches of alcian blue pyridine variant and of five batches of alcian blue 8GX (C.I. 74240). Whenever possible, we also compared results to those obtained with the same dye batches produced at an earlier date to provide information concerning dye stability. Both alcian blue pyridine variant batches were of high dye content, stable, of satisfactory solubility, and performed well in both the routine Mowry mucin stain and in the critical electrolyte concentration (CEC) stain. Of the five alcian blue 8GX samples, some were also of appropriate dye content, were sufficiently stable, and gave good staining in the two procedures. Two batches, however, were unstable, and three batches were unsatisfactory in staining performance and solubility in the CEC stain. Consequently alcian blue pyridine variant is a superior substitute for alcian blue 8GX.     

New stains for blood and bone marrow cells.

Traditionally, blood and bone marrow cells have been identified based on their characteristic shapes and colors when stained with one of several panoptic stains including Wright's or Giemsa's. As questions arose regarding the origin of normal and leukemic cells, cytochemical stains were developed. These stains help identify cells on the basis of a distinctive metabolite or enzyme. As part of an ongoing tradition in which textile dyes are used for biological staining, several new stains have been applied to hematologic staining. These include C.I. basic blue 41, basic blue 141, basic blue 93, and an asymmetrical polymethine dye. As additional cell-selective stains are developed, we can anticipate further improvements in our ability to identify normal and malignant hematopoietic cells.     

New Stains for Blood and Bone Marrow Cells

Traditionally, blood and bone marrow cells have been identified based on their characteristic shapes and colors when stained with one of several panoptic stains including Wright's or Giemsa's. As questions arose regarding the origin of normal and leukemic cells, cytochemical stains were developed. These stains help identify cells on the basis of a distinctive metabolite or enzyme. As part of an ongoing tradition in which textile dyes are used for biological staining, several new stains have been applied to hematologic staining. These include C.I. basic blue 41, basic blue 141, basic blue 93, and an assymetrical polymethine dye. As additional cell-selective stains are developed, we can anticipate further improvements in our ability to identify normal and malignant hematopoietic cells.     

A Hematoxylin and Eosin-Like Stain for Glycol Methacrylate Embedded Tissue Sections

A staining procedure is described for use with glycol methacrylate embedded tissue sections which does not stain the plastic embedment or remove the sections from the glass slides. The basic dye is celestine blue B. It is prepared by treating 1 g of the dye with 0.5 ml concentrated sulfuric acid. It is then dissolved with the following solution. Add 14 ml glycerine to 100 ml 2.5 percent ferric ammonium sulfate and warm the solution to 50 C. Finally adjust the pH to 0.8 to 0.9 The acid staining solution consists of 0.075 percent ponceau de xylidine and 0.025 percent acid fuchsin in 10 percent acetic acid. Slides containing the dried plastic sections are immersed in the celestine blue solution for five minutes and in the ponceau-fuchsin solution for ten minutes with an intervening water rinse. After a final wash, the sections are air dried and coverslipped. This staining procedure colors the tissues nearly the same as hematoxylin and eosin procedures.     

Luxol Fast Blue Arn: A New Solvent Azo Dye with Improved Staining Qualities for Myelin and Phospholipids

Luxol fast blue ARN (Du Pont, C.I. solvent blue 37) is a diarylguanidine salt of a sulfonated azo dye. This dye was compared with other Luxol blue and Luxol black dyes. Luxol fast blue ARN has improved staining qualities for phospholipids and myelin, and can advantageously be substituted for Luxol fast blue MBS (MBSN). Appropriate staining times for a 0.1% dye solution in 95% ethanol (containing 0.02% acetic add) at 35°-40° C range from 2-3 hr. After staining, the sections should be rinsed in 95% ethanol, rinsed in distilled water, and differentiated for 2 sec in 0.005% Li₂CO₃, rinsed in 70% ethanol, washed in water, and counterstained as required. Phospholipids and myelin selectively stain deep blue. A fixative containing CaCl₂, 1%; cetyltrimethylammonium bromide, 0.5%; and formaldehyde, 10%, in water gave excellent results with brain. However, 10% formalin can be used. The staining of the phospholipids is probably due to the formation of dye-phospholipid complexes.     

Standardization of Staining in Giycosaminoglycan Histochemistry: Alcian Blue, its Analogues, and Diamine Methods

Glycosaminoglycans are identified in tissue sections by various histochemical techniques including staining with alcian blue and its analogues, such as cuprolinic blue and cupromeronic blue, or with high and low iron diamine methods. The variation in staining results is particularly confusing in the case of alcian blue, where not only are several different brands of alcian blue available but also several different staining protocols are used. If the results obtained by these techniques are compared, they often do not match. We have developed a dot blot technique for quality control of glycosaminoglycan histochemistry to standardize the staining protocols. This staining technique enables his-tochemists to test particular batches of alcian blue or its analogues for selective glycosaminoglycan staining, thus improving control of histochemical results. The results obtained using the dot blot assay indicate that it is necessary to test each batch of dye individually to obtain valid results in glycosaminoglycan histochemistry.     

Vital Staining with Two Dis-Azo Textile Dyes

Subcutaneous injections of 0.25% saline solutions of two dis-azo textile dyes, calcodur pink 2BL, C. I. 353, also known as benzo fast pink 2BL and amidine fast rose 2BL, and a blue dye, dianil blue G, C. I. 508, were made on alternate days on albino rats for one week. The blue dye is closely similar to Niagara blue 4B, C. I. 520, and dianil blue R, C. I. 465. Staining reactions were much like those of other vital blue disazo dyes. Although the pink dye exhibited a similar staining pattern, there were notable differences. The tissues of most glands were stained pink or red. Nuclei of the tubular epithelial cells of the kidney contained red granules as did the cytoplasm of the Kupfer cells. Most unusual was the bright red staining of the elastica interna of medium and large sized arteries.     

Gram staining and lectin binding properties of Myxosporea and Sporozoea

The staining method developed by Christian Gram was introduced as a simple and highly selective tool for demonstrating myxosporean and coccidian sporogonic stages. When using standard blood staining procedures for those enigmatic parasites it is sometimes difficult to distinguish them from fish host tissue. They clearly exhibit a partial Gram-positive reaction in histological sections, but staining is variable in air dried fish organ imprints. To visualize the Gram-negative background of different host tissue components in histological sections, the conventional safranin counterstain of the Gram protocol may be modified as follows: after application of 2% crystal violet (basic violet 3) and Lugol's solution, sections are stained with 0.1% nuclear fast red-5% aluminum sulfate and 0.35% aniline blue (acid blue 22) dissolved in saturated aqueous picric acid. Replacement of the Gram-specific dye crystal violet with 2% malachite green gave similar results in organ imprints containing myxospores or coccidia, but only in sections containing myxosporea. Staining for 1 min with an aqueous solution of 0.5% malachite green and followed 1 min washing was sufficient for rapidly demonstrating the parasite spores in organ imprints of both myxosores and oocysts. With regard to the role of acid mucopolysaccharides and other carbohydrates in the Gram reaction of spores, alcian blue 8GX staining was compared to the binding of FITC-labeled WGA, GS I and GS II. Each lectin was applied at 20 μl/ml PBS, HEPES for 1 hr. Whereas WGA yielded a nonspecific pattern like the alcian blue staining, GS II resulted in a pattern similar to the Gram staining results. This binding was weak in untreated specimens, but was significantly enhanced when digested first within trypsin overnight in a humid chamber at 37 °C. The binding of GS II to both myxosporidian and coccidian spores suggests that they are both composed of polymers containing N-acetyl-D-glucosamine residues. Furthermore, the results suggest that this hexosamine plays a key role in the Gram reaction.     

A new stain for identification of avian leukocytes

Differential staining of avian leukocytes was achieved within 6 min following brief fixation in a methanolic solution of C.I. acid red 360 followed by immersion in a mixture containing C.I. basic blue 41, C.I. basic blue 141, and C.I. acid red 52. Heterophils contained black angular and punctate granules. Eosinophils contained bright purple granules. Lymphocytes displayed red nuclei and blue cytoplasm. Monocytes contained red-brown nuclei and lavender cytoplasm. Basophils showed red-orange granules. Thrombocytes stained deep purple. Compared to traditional panoptic stains like Wright's or Giemsa's, the new staining method provides brighter colors, more precise details of cellular structures, and shorter staining time. Significantly, it facilitates identification of avian leukocyte species based on differences in color as well as differences in size and shape.     

The effect of selected basic dyes on the blood cells,in particular,the basophils,of the Pacific oyster,Crassostrea gigas

It was determined by means of light and electron microscopy that the granular blood cells of Pacific oysters could absorb basic dyes from dye-coated plastic beads injected into the mantles of oysters; in doing so, the granular blood cells became altered morphologically and behaviorally. In addition, it was observed that oysters could absorb the basic dye, toluidine blue O, in low concentration from sea water. It was shown that the absorption of toluidine blue O caused a shift in the blood cell levels of oysters, and it was suggested that this shift was occasioned by the absorption of toluidine blue O by granulocytes.     

A new stain for identification of avian leukocytes

Differential staining of avian leukocytes was achieved within 6 min following brief fixation in a methanolic solution of C.I. acid red 360 followed by immersion in a mixture containing C.I. basic blue 41, C.I. basic blue 141, and C.I. acid red 52. Heterophils contained black angular and punctate granules. Eosinophils contained bright purple granules. Lymphocytes displayed red nuclei and blue cytoplasm. Monocytes contained red-brown nuclei and lavender cytoplasm. Basophils showed red-orange granules. Thrombocytes stained deep purple. Compared to traditional panoptic stains like Wright's or Giemsa's, the new staining method provides brighter colors, more precise details of cellular structures, and shorter staining time. Significantly, it facilitates identification of avian leukocyte species based on differences in color as well as differences in size and shape.     

History of Staining the Staining of Blood and Parasitic Protozoa

By the term “blood stain” one ordinarily means a compound dye formed from the chemical union of an acid and a basic dye, and usually a compound of the eosin-methylene-blue group. It is well known today that the sodium salt of a color acid (e. g. eosin) and the chloride of a dye base (e. g. methylene blue) may be converted by simple metathesis into sodium chloride plus the compound dye (e. g. methylene blue eosinate), the latter being insoluble in water unless an excess is present of either the acid or the basic dye. In modern blood stains a compound dye of this type is dissolved in methyl alcohol and mixed with water on the slide at the moment of staining.     

A Tribasic Stain for Thin Sections of Plastic-Embedded, OsO4-Fixed Tissues

Thin (0.5-1 μ) sections of plastic-embedded, OsO₄-fixed tissues were attached to glass slides by heating to 70 C for 1 min. A saturated solution combining toluidine blue and malachite green was prepared in ethanol (8% of each dye) or water (4% of each dye). Methacrylate or epoxy sections were stained in the ethanol solution for 2-5 min. The water solution was more effective for some epoxy sections (10-80 min). Epoxy sections could be mordanted by 2% KMnO₄, in acetone (1 min) before use of the aqueous dye, reducing staining time to 5-10 min and improving contrast. Aqueous basic fuchsin (4%) was used as the counter-stain in all cases; staining time varied from 1-30 min depending upon the embedding medium and desired effects, methacrylate sections requiring the least time. In the completed stain, nuclei were blue to violet; erythrocytes and mitochondria, green; collagen and elastic tissue, magenta; and much and cartilage, bright cherry red. Sections were coated with an acrylic resin spray and examined or photographed with an oil-immersion lens.