Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.     

Cytologic detection of Wuchereria bancrofti microfilariae in urine collected during a routine workup for hematuria

A 23-year-old Guyanese man experienced intermittent, total, painless, gross hematuria for a month for which he sought medical attention at the Kings County Hospital Center in Brooklyn. Hematuria was accompanied by weakness but not by frequency of urination or burning on urination. Catheterized urine at the time of cystoscopy and each of two subsequent voided specimens examined cytologically contained sheathed microfilariae. Distinguishing features of the microfilariae were well demonstrated with the Papanicolaou stain. The well-stained nuclei, which did not extend into the clear zone, and the distinct, pale-stained sheath led to the positive identification of the microfilariae as Wuchereria bancrofti. The Papanicolaou stain may well be the stain of choice for the identification of microfilariae in the blood. The excellent detail obtained with this routine cytologic stain is as good as that with Giemsa, which does not stain the sheath.     

正常人和肝病患者血中“致密体”及其与纳米细菌的关系

目的研究正常和肝病患者血液中圆球体样微生物的生物学特性,初步确定纳米细菌与该种可滤过物的关系。方法将正常人和肝病患者培养阴性的血培养物进行细菌L型、穿菌和厌氧菌培养。同时取沉淀用透射电镜观察。L型培养液用0．45μm和0．22μm滤器过滤,接种RPMI 1640培养基,细胞培养条件下培养45d,用鼠抗纳米细菌单克隆抗体8D10免疫组化和钙盐染色进行纳米细菌鉴定。血浆纳米细菌培养阳性者12000×g离心后,进行普通和L型细菌培养。结果36／39患者和60／60健康对照血培养液中呈现类似于L型的巨型体、圆球体、原生小体的不明微生物。电镜观察圆球体内为电子致密样物质,周围未见细胞壁结构。电镜和光镜下可见其粘附在红细胞上或存在于红细胞内。滤过后培养物钙盐染色有5／39阳性,但纳米细菌免疫组化染色均阴性。纳米细菌阳性的培养物转种,未见一般细菌、细菌L型和上述血中“致密体”。结论血培养中致密体样微生物与纳米细菌无关,可能在维持机体正常免疫功能上具有一定意义。     

Quantification of small amounts of hemoglobin in polyacrylamide gels with benzidine.

Benzidine-hydrogen peroxide is a sensitive blood stain. Since benzidine is carcinogenic, less hazardous substitutes have been sought which retain the specificity and sensitivity of benzidine. We have tested two benzidine derivatives, 3,3′-dimethoxybenzidine and 3,3′, 5,5′-tetramethylbenzidine, reported to be satisfactory substitutes, but have found both to be unsatisfactory for quantifying small amounts of hemoglobin (Hb) on polyacrylamide gels. However, our data show that benzidine stains gels with an intensity proportional to the amount of Hb present when the staining time and temperature are controlled. The stain is fast, sensitive, and specific for Hb, gives very little background stain, and is stable if the gels are thoroughly rinsed and stored in distilled-deionized water. The relative amounts of Hb in different bands or different gels may be quantified later by densitometry. Procedures are suggested for using benzidine while giving adequate attention to governing regulations and personnel safety.     

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.     

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.     

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.     

A Formalin-Wright Staining Technique for Avian Blood Cells

A rather concentrated alcoholic staining solution, an aqueous formalin-containing diluent, and a mixture of ethyl ether and absolute methyl alcohol are required. Formulas: A. Wright's stain (Harleco, Cert. No. LWr-52 was used), 3.3 gm; methyl alcohol, 500 ml. B. Formaldehyde solution 40% USP (Fisher's used), 0.25 ml; distilled water, 500 ml with its pH adjusted to 6.8 by addition of either 0.25% Na₂CO₂ or 0.25% HCl, as needed. C. A I:I mixture of ethyl ether and absolute methyl alcohol. Procedure: Prepare thin smears of normal or pathological avian blood, air dry, place the slides on a drying rack, cover with solution A, and let stand for about 8 min. Dilute the stain by dropping on a volume of B estimated to be equal to the volume of the partially evaporated stain, and let stand for 2-5 min, or until the surface is well covered by a metallic sheen. Wash with distilled water adjusted to pH 6.8 with the 0.25% Na₂CO₂ solution or 0.25% HCl. Dry the preparations quickly by blotting with filter paper. Differentiate and adjust the color intensities by dipping 6-10 times into C. Check the results microscopically and differentiate further if the colors are not properly balanced. Dry, uncovered preparations may be examined under oil; or, a cover glass can be applied with balsam or a synthetic resin for permanent mount. Results are similar to those described in textbooks, but have been more consistent than those obtained with other techniques for blood cells of chicken, pheasants, American and Indian partridge, quail, pigeon, turkey, goose, canary, and the Himalayan snow partridge.     

A New Hematological Stain: I. Constituents and Methods of Use

The stain proposed by the author is quickly and simply prepared by mixing equal parts of the following permanent stock solutions:

The mixed stain is usable, for at least eight months, and is applicable to practically all hematological purposes: blood smears, fixed sections, frozen sections, and touch preparations. In the technics utilized to produce its action on preparations treated in different ways, the only variants are the methods for treating the cells, while the stain itself remains totally unchanged for all purposes. For blood smears fixed with methyl alcohol, the technic consists merely of pouring the stain on the slide, leaving it for 5 to 7 minutes, and then washing it off. On sections, a further process of differentiation with acid acetone is rapidly carried out. The various types of granules, including megakaryocyte and platelet granules, are clearly demonstrated. For frozen sections, the technic is extremely rapid, yet yields excellent differentiation.     

Concentrating and Staining Bone Marrow; An On-the-Slide Technique

A 0.5-1 ml sample of bone marrow is aspirated into a syringe containing 3 drops of 15% K₂-EDTA and an additional 1-2 drops of the EDTA solution previously placed on a slide, is then drawn into the syringe. All of the contents are ejected onto this slide, which is carefully tilted 2 or 3 times to an angle of 5-10°, and the edge brought to the center of another slide. The slide with the aspirate is then slowly tilted to 80-90°. Most of the blood and part of the marrow will drain off, leaving spicules of marrow and some blood on the original slide. A small drop of this concentrated marrow is dragged off with the edge of a third slide and deposited about 2 cm from the edge of a fourth slide on which the smear is to be made. The smear is made by bringing a clean (smearing) slide to the slide with the deposited marrow with flat surfaces parallel and the edges at a 90° angle. With gentle pressure, the smearing slide is pushed toward the empty end of the slide upon which the smear is made. This separates the marrow from the circulating blood. Before staining the smear is air dried and heated in an oven at 120-125 C for 2 min; or alternately for satisfactory but less uniform results the smear is heated over a microburner for 10 sec; then the smear is covered with 1 part of undiluted Wright's stain for 30—45 sec which is then diluted with 2 parts of a solution of 0.1-0.2 gm of Na₂S₂O₃ in 1 liter of distilled water and stained for 10-13 min with this diluted stain. Smears made in this manner have 3 concentric zones; the central zone contains the myeloid tissue; the middle, erythropoetic tissue; the outer, a mixture of blood and marrow.     

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.     

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.     

Use of urinary gram stain for detection of urinary tract infection in childhood

In this study, urinary culture, urinary Gram stain, and four tests within the urinalysis, leukocyte esterase, nitrite, microscopyfor bacteria, and microscopyforpyuria, were examined in 100 children with symptoms suggesting urinary tract infection. Our purpose was to determine the validity of the urinary Gram stain compared with a combination of pyuria plus Gram stain and overall urinalysis (positiveness of nitrite, leukocyte esterase, microscopy for bacteria, or microscopy for white blood cell). Of 100 children, aged two days to 15 years, 70 (70 percent) had a positive urinary culture: 40 girls (57 percent) and 30 boys (43 percent). Escherichia coli was the most common isolated agent. The sensitivity and specificity of the urinary Gram stain were 80 percent and 83 percent, and that of the combination of pyuria plus Gram stain 42 percent and 90 percent, and that of the overall urinalysis 74 percent and 3.5 percent respectively. Our findings revealed that neither method of urine screen should substitute for a urine culture in the symptomatic patients in childhood.     

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.     

Simplified method for the discrimination of acute myelogenous and non-myelogenous leukemias

Rapid discrimination of acute myelogenous and non-myelogenous leukemias is of great importance when chemotherapy is urgently needed in severely ill patients. For decades the most reliable cytochemical method for this classification is the demonstration of myeloperoxidase in blast cells [1-4, 6, 7]. We combined the simplified myeloperoxidase stain as described by Kaplow with a brief stain similar to Pappenheim's procedure or with commercially available Hemacolor rapid blood smear: this proved to be a simple staining method that permits good morphological judgment of the cells as well as reliable demonstration of peroxidase activity. This procedure takes less than 10 min using Hemacolor and can easily be done with prepared solutions without technical assistance.     

A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.     

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.     

A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.

We have developed a highly sensitive stain for visualizing proteins in polyacrylamide gels. Our modification of the procedure for de Olmos' neural, cupric-silver stain is 100 times more sensitive than the conventional Coomassie blue stain (e.g., detection of 0.38 vs 38 ng/mm² of serum albumin), and is comparable to the sensitivity attained with an autoradiogram of ¹⁴C-methylated proteins following a 5-day exposure. This silver stain will be especially useful for analysis of patterns of proteins from tissue where attainment of the high specific activity of isotope labeling which is necessary to detect minor protein components is expensive, technically difficult or, as in humans, prohibited. In preliminary results with material such as unconcentrated cerebrospinal fluid, the silver stain revealed a complex pattern of proteins not visible with Coomassie blue.     

Cholesterol-free phospholipid domains may be the membrane feature selected by N epsilon-dansyl-L-lysine and merocyanine 540

We have used N epsilon-dansyl-L-lysine as a fluorescent membrane probe, to study cells taken from tissues concerned with immune function. There is a striking similarity between the staining selectivity of this compound and that reported by others for merocyanine 540. Both compounds stain leukemic, human, peripheral leukocytes, an erythroleukemia line, and some mouse bone marrow cells, suggesting common selectivity for a membrane feature of hemopoietic cells. Both compounds fail to stain red blood cells, normal human leukocytes, mouse spleen and thymus cells. We have recently reported that dansyl-lysine apparently selects for cholesterol-free phospholipid domains in liposomes and now report similar selectivity for merocyanine 540 staining of liposomes.