A Trichrome Stain for Insect Material

Deparaffinized insect sections are brought down to water and overstained in a 0.1% solution of azocarmine G in 1% acetic acid. They are then destained in a saturated solution of orange G until the azocarmine G is removed from the endocuticle and the latter is colored pale yellow. After washing, the sections are transferred to a 5.0 % solution of phosphotungstic acid in water for 3 min. They are then rinsed in distilled water and stained in a 0.1% solution of methyl green in 1% acetic acid until the endocuticle is green. Differentiation is done in 2 changes of 95% alcohol. The sections are then dehydrated either in absolute alcohol or dioxane, cleared in a mixture of “camsal”, eucalyptol, dioxane, and paraldehyde (1:2:2:1), and mounted in Mohr and Wehrle's medium, a mountant of the Euparal type.     

A Trichrome Stain for Insect Material

Deparaffinized insect sections are brought down to water and overstained in a 0.1% solution of azocarmine G in 1% acetic acid. They are then destained in a saturated solution of orange G until the azocarmine G is removed from the endocuticle and the latter is colored pale yellow. After washing, the sections are transferred to a 5.0 % solution of phosphotungstic acid in water for 3 min. They are then rinsed in distilled water and stained in a 0.1% solution of methyl green in 1% acetic acid until the endocuticle is green. Differentiation is done in 2 changes of 95% alcohol. The sections are then dehydrated either in absolute alcohol or dioxane, cleared in a mixture of “camsal”, eucalyptol, dioxane, and paraldehyde (1:2:2:1), and mounted in Mohr and Wehrle's medium, a mountant of the Euparal type.     

Studies on Textile Dyes for Biological Staining: I. Pontacyl Blue Black SX, Pontacyl Violet 6R and Luxol Fast Yellow TN

After a series of commercial grade textile dyes were screened, three -were found to be excellent biological stains. Two of these, pontacyl blue black SX and pontacyl violet 6R, were nuclear stains, the best results being obtained after mordanting with chrome salts. The third dye, luxol fast yellow TN, imparted a brilliant yellow stain to the cytoplasm and collagen fibers when used in alcoholic solution. A technical procedure for the use of either of the nuclear stains combined with the yellow cytoplasmic stain is given.     

Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

Investigation of Thiazin Dyes as Biological Stains II. Influence of Buffered Solutions on Staining Properties

The effect of buffer solutions of varying reaction upon staining fixed sections with thionin, azures A, B, and C, and methylene blue has been studied. The buffer solutions were employed in one of three different ways: for pre-treatment of the sections, for post-treatment, or as solvents for the dyes. Regardless of the method of employing the buffer solutions it was found that the intensity of staining increased with increasing pH-values (a fact which is generally known to be true in the case of basic dyes). It is not certain whether this effect is due to varying the H-ion concentration or to altering the salt content of the solution, or to both. It was also noticed that there was one point where the staining intensify increased most rapidly. This point was either between pH 5 and pH 6 or between pH 6 and pH 7, its position varying with the method of fixation and of applying the buffer solutions. It was further observed that between pH 5 and pH 7 there were always more pronounced metachromatic effects than with either more acid or more alkaline buffer solutions.     

A Combination Verhoeffs Elastic and Masson's Trichrome Stain for Routine Histology

A method for the combined staining of elastic, muscle and connective tissue for routine use in histopathology is described. The elastica, stained black by Verhoeffs technique, is contrasted with the muscle and connective tissue stained red and green or blue respectively by a modification of Masson's trichrome. Cell nuclei stain blue-black with Weigert's iron hematoxylin. The procedure takes approximately two hours and is most suitable for the study of vascular pathology in surgical and autopsy sections.     

A Modified One-Step Trichrome Stain for Demonstration of Fine Connective Tissue Fibers

Gomori's one-step trichrome procedure was modified to improve coloration of fine connective tissue fibers. Paraffin sections from tissues fixed in alcohol, acetone, Zenkerformol, 10% formalin, Kaiserling's or Carnoy's fluid were mordanted 1 hr at 56 C in Bouin's solution, stained 1 min in a trichrome solution (chromotrope 2R-phosphomolybdic acidaniline blue WS) adjusted to pH 1.3 with HCl, rinsed in 1% aqueous acetic acid, dehydrated and covered. Collagen, reticulum fibers, basement membranes, ring fibers around splenic sinuses, intercalated discs in cardiac muscle and cartilage were colored blue. Nuclei, cytoplasm, fibrin, muscle fibers and elastic fibers were stained red. Pretreatment of sections with Bouin's solution enhanced the affinity of tissues for chromotrope 2R and was found essential for satisfactory coloration of material fixed in alcohol, acetone, formalin or Carnoy's fluid. Because this method does not require differentiation, it gave uniform results even in the hands of inexperienced laboratory trainees. No fading was observed in sections stored for more than 8 yr.     

Staining Problems with Eosin Y: A Note from the Biological Stain Commission

In 1980, eosin Y was the certified dye with which technologists encountered most problems. The specific problem most frequently brought to the attention of the Biological Stain Commission was that solutions of eosin Y formed a precipitate and failed to stain cytoplasm red when used as a counterstain to hematoxylin.     

Giemsa Stain for the Diagnosis of Bovine Babesiosis. I. Staining Properties of Commercial Samples and Their Component Dyes

SYNOPSIS. Studies on the composition of commercial Giemsa stain and its effect upon staining quality are reported. These studies were supplemented by observations on the preparation of the components of Giemsa stain and their staining properties in aqueous solution, in Nocht's solution, and in laboratory prepared Giemsa stains containing one azure component. Five groups of commercial batches were differentiated on the basis of their staining reactions on thick and thin films of bovine blood containing Babesia bigemina and B. argentina. Spectrophotometric and chromatographic analysis showed that four groups differed in the proportions of the thiazine components present, while the fifth-group did not appear to be Giemsa stain. Comparison of their staining effects with those obtained with each component in laboratory prepared stains indicated that the major effects of commercial batches on both blood cells and parasites were due to the thiazine component or components in highest proportions, with satisfactory staining of protozoa associated with those batches containing high proportions of methylene blue and azure B and low proportions of the remaining thiazine components.
The function of each component of Giemsa stain is defined and the need for the proper balancing of thiazine eosinates with free azure is shown. Close correlation was obtained between analysis by spectrophotometry and chromatography and direct staining tests when samples initially with low MX values were re-examined spectrophotometrically after removal of their methylene violet content. The existence of a leuco form of eosin is reported and its possible significance to the Romanowsky effect is discussed.     

The Mechanism of Staining Explained on A Chemical Basis II. General Presentation

At present the division of the factors influencing staining into those supporting a chemical mechanism or those supporting what is known as “adsorption” mechanism is questioned because the latter term cannot be definitely defined from general usage. In this paper the nature of adsorption is discussed. Data indicating definitely a chemical mechanism are not directly considered; but in many cases often cited by other writers as necessitating a non-chemical mechanism, the validity of such mechanism is questioned.     

A Selective Stain for Eosinophils Using Two Oxazine Dyes Applied Sequentially

A methanolic solution of the oxazine textile dye, C. I. basic blue 122, followed by an aqueous alkaline solution of the oxazine dye, C. I. basic blue 141, and a brief rinse in an acetate buffer at pH 3.45 produces intense black staining of eosinophil granules. This staining was selective for eosinophils while other types of peripheral blood leukocytes showed little if any staining under the same conditions. This staining procedure may be useful for detecting eosinophils in samples of blood, bone marrow, or urine when eosinophiluria results from interstitial nephritis.     

A Polychromatic Stain for Use in Parasitology

In sensory systems, insight into synaptic arrangements on cells of known physiological response properties has helped our understanding of the structural basis for these properties. To carry out these types of studies, however, synaptic types in the region of interest must be defined. Unfortunately, defining synaptic types in the brainstem has proved to be a challenging enterprise. Our study was done to classify synapses in the gustatory part of the nucleus solitarius using objective quantitative criteria and a cluster analysis procedure. Cluster analysis allows classification of a population of objects, such as synaptic terminals, into groups that exhibit similar characteristics. Six terminal types were identified using cluster analysis and subsequent analyses of variance and post hoc tests. Unlike classification schemes used for the cerebral cortex, where synaptic apposition density thickness and shape of vesicles is useful (Gray's Type I and II synapses), the concentration of vesicles in a terminal was a more useful measurement with which to classify terminals in the nucleus solitarius. To validate that vesicle density (vesicles/μm²) is a useful defining characteristic to classify terminals in the nucleus solitarius, terminals of a known type were used. GABAergic terminals were identified using postembedding immunohistochemical techniques, and their vesicle density was determined. GABAergic terminals fall into the range of two of the terminal types defined by the cluster analysis and, based on vesicle density, two types of GABAergic terminals were identified. We conclude that vesicle density is a helpful means to identify synapses in this brainstem nucleus.     

A Safe Spore Stain for Class Use

In looking for an efficient method of pre-treating bacterial spores for staining with carbol fuchsin, the author found that the ammonia in Lagerberg's formula and the chromic acid in Moeller's formula were both fairly effective in increasing permeability. Combining the two proved highly successful; and the following method has been used for four years by classes of beginners in the study of spore-formers isolated from soil by the students.

Cover the heat-fixed smear with five percent chromic acid and after thirty seconds add twice the amount of concentrated ammonia to the acid. After two minutes rinse and steam with carbol fuchsin for two or three minutes. Rinse, destain with one percent sulphuric acid for fifteen to thirty seconds, rinse again and flood slide with tap water. Add to this a few drops of Loeffler's methylene blue and allow to counter-stain for ten to thirty seconds. Rinse, blot, dry and examine. If instructions are followed the results are always good.     

A New Stain for Pollen Fertility Studies

The juice from the berries of Cocculus hirsutum was extracted and used for pollen fertility studies in various crops. Two stains were prepared: P. H. Ramanjini (PHR) stain and modified PHR stain. The modified PHR stain contains lactic acid and produces the best staining differentiation. The intensity of the staining was dependent on the thickness of the pollen cell walls, hence PHR stain is recommended for thick walled pollen grains and the modified PHR stain for pollen with relatively thin walls. The preparation of both the stains are very simple, quick and inexpensive.     

Milling Red SWB: A Textile Dye for Staining Epithelial Intercellular Bridges

Fourteen textile dyes were evaluated as histological stains for benign and malignant tissues. An acid dye, Milling red SWB (Acid Red 114, C.I. 23635) was found to possess great affinity for epithelial intercellular bridges. The intercellular bridges were demonstrated clearly when the Milling red SWB was used as a 1% solution in 1% potassium alum after hematoxylin and, also, when methylene blue ZX (Basic Blue 9, C.I. 52015) was used as a counter-stain, without hematoxylin. The intercellular bridges could not be demonstrated in pleomorphic cells which invaded the subepithelial connective tissue.     

Oxazine Dyes. Iv. Simultaneous Nuclear and Double-Contrast Cytoplasmic Staining from A Single Solution

A dye mixture, consisting of a celestine blue B dispersion (prepared according to Gray et al. 1956), orange G, and acid fuchsin in one solution, simultaneously stains nuclear elements and gives double contrast staining of cytoplasmic elements. Orange G, 0.16 gm, and acid fuchsin, 0.04 gm, dissolved in 100 ml of celestine blue B dispersion and adjusted to pH 0.8 gives, when applied for 1.5 min, results comparable or superior to other “triple contrast” stains on a wide variety of tissues. No differentiation other than that which occurs during dehydration is necessary.     

A New Technique for Staining Catecholic Residues in Biological Samples

This technique for localizing catecholic residues in biological samples is based on the condensation of Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with quinone residues obtained by the oxidation of catechols in the presence of ammonia. The product is a dark pink MBTH-quinone compound. This method is very sensitive and positive to catechol even at the 0.05 µg level and the final product is chemically stable.     

A One Step Esterase Stain for Use with a Hemacytometer

When harvesting cells for macrophage cell cultures, our laboratory sought a method which would simultaneously confirm cell identity and allow enumeration of these cells. Such a procedure would reduce the time needed to assess the readiness of the cells for culturing (measuring their viability, and indicating their identity as well as their number).