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Understanding Romanowsky staining. I: The Romanowsky-Giemsa effect in blood smears
Abstract
Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of 'toxic' granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).
... The major problem of unsuccessful visualization of cells is insufficient staining of the cytoplasm. The hematoxylin-eosin staining method resulted in the fewest lost smears; however, it was difficult to distinguish between nuclear chromatin and basophilic cytoplasm and between granules and cytoplasm compared with that following Romanowsky staining [30]. A large percentage of bat blood smears were unsuccessfully stained using the Romanowsky-Giemsa method, even though many cell compartments were well distinguishable. ...
... However, these problems were not permanent and varied greatly. Our results are in consistent with the previous study conducted by Horobin and Walter [30] who stated that the main problems regarding the use of the Romanowsky-Giemsa stain and influences of practical variables such as dye structure, solvent composition, and pH, and of staining time all remain enigmatic." Another problem with the morphological determination of WBCs is that there is no standard stain for the staining of most types of bat leukocytes. ...
Background and Aim: A drawback of studies on bat blood smears in the field is the lack of time for fixation because blood sampling using a non-lethal method often provides less time for fixation in smear preparations due to the small volume of blood collected. Usually, there is insufficient blood for another smear preparation, so it is necessary to use blood smears as rationally as possible, especially for rare bats. Many stains are used for staining peripheral blood smears, and they have advantages and disadvantages. This study aimed to examine commonly used stains for blood smears to select the best stain for staining peripheral blood smears in bats.
Materials and Methods: In this study, 48 blood smears of Rhinolophus spp. bats were examined using several blood staining methods. Four methods that showed the best results were used in further experiments: Romanowsky-Giemsa, Pappenheim, hematoxylin-eosin, and eosin methylene blue.
Results: Comparative analysis of different methods for staining bat blood smears revealed that the most convenient method for analyzing blood cells is Pappenheim method.
Conclusion: Staining blood smears using Pappenheim method yield the least number of unsuccessful blood smear stains and are quite effective for the morphological analysis of blood cells.
... 3 If the left lobe is tied off, we use a volume of 0.8 ml to lavage the right lung. 4 A typical recovery is 75-90 % of the fluid volume. 5 Injured mice often yield a BAL that is visibly bloody. 6 If the left lobe is tied off, at this point it can be removed by cutting below the suture. ...
... In addition, the composition of the leukocytes is evaluated by morphology coupled with differential staining, a process that employs more than one chemical dye to help the investigator differentiate the cell types from one another. The staining technique was developed by Dmitri Romanowsky and further modified by others (Wright, Giemsa, Leishman) [5,6,[17][18][19] to combine methanol-based fixation and staining in a single step, using the basic dye methylene blue to detect nucleic acid and the acidic dye eosin to detect protein. Commercial kits (variously known as Diff-Quik, Diff-Quick, or Kwik-Diff™) are readily available that split the fixation and staining into three separate steps. ...
The mouse has been a popular organism for studying mechanisms of acute lung injury (ALI), given the small size, rapid life cycle, and genetic malleability of this animal. The major features of ALI in mice include disruption of the alveolar capillary barrier, histopathological changes in lung architecture, localized inflammation, and physiological dysfunction. In this chapter, we describe the basic techniques that we use in our laboratory to qualitatively and quantitatively assess ALI in mice, focusing particularly on permeability and inflammation. Detailed methodology for performing bronchoalveolar lavage and analyzing components of the lavage is outlined, along with guidelines for processing and evaluation of lung tissue. Mastery of these essential tools will allow any investigator to reliably and reproducibly measure the experimental outcome in multiple mouse models of acute lung injury.
... 26,27 Stains prepared from the initial method (MGG, Wright's, Leishman's and Giemsa's stains, Diff-Quik â ) in which azure B and eosin Y are mixed in an aqueous solution are called 'Romanowsky stains'. 5,[29][30][31][32] Standardisation: the search for the perfect Romanowsky's stain A recurring problem of Romanowsky's stains is their 'notorious capriciousness' 30 and their batch-to-batch variations. It is the reason why some authors consider that combining stains from two different manufacturers is the best way to ensure adequate staining of cells. ...
... Lopez Cardozo 19 and Spriggs and Boddington 45 both estimate that the correct pH should be 6.8. HEPES buffer was recommended by the ICSH in 1984 and by Horobin and Walter in 1987, 29 but not by the EQALM in 2004 that simply recommends phosphate buffer 0.067 M according to S€ orensen, pH 6.8. 1 ...
Objective:
Since the guidelines of the International Committee for Standardisation in Haematology (ICSH) in 1984 and those of the European Committee for External Quality Assessment Programmes in Laboratory Medicine (EQALM) in 2004, no leading organisation has published technical recommendations for the preparation of air-dried cytological specimens using May-Grünwald-Giemsa (MGG) staining.
Data sources:
Literature data were retrieved using reference books, baseline-published studies, articles extracted from PubMed/Medline and Google Scholar, and online-available industry datasheets.
Rationale:
The present review addresses all pre-analytical issues concerning the use of Romanowsky's stains (including MGG) in haematology and non-gynaecological cytopathology. It aims at serving as actualised, best practice recommendations for the proper handling of air-dried cytological specimens. It, therefore, appears complementary to the staining criteria of the non-gynaecological diagnostic cytology handbook edited by the United Kingdom National External Quality Assessment Service (UK-NEQAS) in February 2015.
... At the stage of staining when chromatin becomes purple, basophilic cytoplasms still are blue, but if staining times are prolonged suffi ciently, these cytoplasms also become purple. With extremely extended times, many other substrates become purple as well (Horobin and Walter 1987). The causes of such rate effects are varied and are considered as appropriate for each of the staining applications, e.g., blood smears, G-banding, resin section staining etc., discussed below. ...
... If staining times are lengthened or eosin binding is enhanced by lowering the pH of the dye bath (Lillie 1977), the purple staining is restored. It has been established, however, by comparing preparations fi xed in alcohol followed by formaldehyde to those fi xed in formaldehyde followed by alcohol that the effect is due to the chemical changes (Horobin and Walter 1987). ...
... At the stage of staining when chromatin becomes purple, basophilic cytoplasms still are blue, but if staining times are prolonged suffi ciently, these cytoplasms also become purple. With extremely extended times, many other substrates become purple as well (Horobin and Walter 1987). The causes of such rate effects are varied and are considered as appropriate for each of the staining applications, e.g., blood smears, G-banding, resin section staining etc., discussed below. ...
... If staining times are lengthened or eosin binding is enhanced by lowering the pH of the dye bath (Lillie 1977), the purple staining is restored. It has been established, however, by comparing preparations fi xed in alcohol followed by formaldehyde to those fi xed in formaldehyde followed by alcohol that the effect is due to the chemical changes (Horobin and Walter 1987). ...
Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of toxic granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).
... The production of a third color, purple, in addition to the blue and pink staining seen with similar dyes, was unique to these solutions. The ability of Romanowsky-type stains to produce three distinct colors is termed the " Romanowsky effect " and appears to arise from the ability of azure B, one of the oxidative products of methylene blue, and eosin to combine in solution to form a molecular complex responsible for the Romanowsky effect (Wittekind 1983, Woronzoff-Dashkoff 2002, Horobin and Walter 1987. Giemsa, while not required to produce the desired Romanowsky effect, may be added to intensify nuclear features and neutrophil granulation. ...
... To minimize stain precipitate from forming in the working Wright ' s-buffer solution, the solution should be changed after two batches of slides have been processed or after 1 h, whichever comes fi rst. Precipitate also may result from inadequate washing of the slides, inadequate fi ltration of the stain, a buffer concentration that is too high, or a methanol concentration that is too low (Horobin andWalter 1987, Rodak et al. 2007). If precipitate is present on the stained slides, this may be removed by dipping the slides in absolute methanol in a Coplin jar followed by a quick rinse with deionized water. ...
Abstract Wright-Giemsa staining is a common procedure that is performed routinely in hematology laboratories. Consistency in intra-laboratory staining quality is essential for accurate morphological interpretation of blood smears. Although the Wright-Giemsa stain can be challenging to perform, the methods illustrated here have provided consistent, high quality stains in the Special Hematology Laboratory at the University of Minnesota for over half a century. We outline methods for collecting blood specimens, preparing the slides and performing a Wright-Giemsa stain using our combination of reagents. Various techniques that have been passed down in our laboratory for troubleshooting suboptimally stained specimens are shared as well.
... At the stage of staining when chromatin becomes purple, basophilic cytoplasms still are blue, but if staining times are prolonged suffi ciently, these cytoplasms also become purple. With extremely extended times, many other substrates become purple as well (Horobin and Walter 1987). The causes of such rate effects are varied and are considered as appropriate for each of the staining applications, e.g., blood smears, G-banding, resin section staining etc., discussed below. ...
... If staining times are lengthened or eosin binding is enhanced by lowering the pH of the dye bath (Lillie 1977), the purple staining is restored. It has been established, however, by comparing preparations fi xed in alcohol followed by formaldehyde to those fi xed in formaldehyde followed by alcohol that the effect is due to the chemical changes (Horobin and Walter 1987). ...
An introduction to the nomenclature and concept of "Romanowsky stains" is followed by a brief account of the dyes involved and especially the crucial role of azure B and of the impurity of most commercial dye lots. Technical features of standardized and traditional Romanowsky stains are outlined, e.g., number and ratio of the acidic and basic dyes used, solvent effects, staining times, and fixation effects. The peculiar advantages of Romanowsky staining are noted, namely, the polychromasia achieved in a technically simple manner with the potential for stain intensification of "the color purple." Accounts are provided of a variety of physicochemically relevant topics, namely, acidic and basic dyeing, peculiarities of acidic and basic dye mixtures, consequences of differential staining rates of different cell and tissue components and of different dyes, the chemical significance of "the color purple," the substrate selectivity for purple color formation and its intensification in situ due to a template effect, effects of resin embedding and prior fixation. Based on these physicochemical phenomena, mechanisms for the various Romanowsky staining applications are outlined including for blood, marrow and cytological smears; G-bands of chromosomes; microorganisms and other single-cell entities; and paraffin and resin tissue sections. The common factors involved in these specific mechanisms are pulled together to generate a "universal" generic mechanism for these stains. Certain generic problems of Romanowsky stains are discussed including the instability of solutions of acidic dye-basic dye mixtures, the inherent heterogeneity of polychrome methylene blue, and the resulting problems of standardization. Finally, a rational trouble-shooting scheme is appended.
... In order to ensure reliability of the automated results, differential white cell count was performed by optical microscopy after preparation of blood smears and staining, according to the Romanowsky methodology [11]. The differential count was carried out in a blinded fashion by the same analyst for all samples. ...
Background
Dipyrone is a non-narcotic analgesic/antipyretic widely used in some countries but prohibited in others due to suspected risk of agranulocytosis. The primary goal of this study was to evaluate hematological alterations in healthy adult volunteers after treatment with dipyrone.
Methods
The study enrolled 30 healthy volunteers of both genders, aged 19–37 years. They received tablets containing 500 mg of dipyrone sodium to be used four times daily for 7 consecutive days. Before the first administration, arterial pressure was measured and blood was collected in order to evaluate hematological baseline parameters. On the 8th day after the beginning of treatment, the volunteers had their blood pressure assessed once more and underwent a second blood draw. Total and specific leukocyte counts, creatinine, urea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), erythrocytes, and platelets were quantitatively determined.
Results
No statistically significant difference was observed among total or specific leukocyte counts. Number of platelets, erythrocytes, hemoglobin, and hematocrit decreased after treatment. Diastolic pressure, mean arterial pressure (MAP), and urea concentration declined, while creatinine, AST, and ALT showed no significant alterations. It is noteworthy that, even for parameters that showed statistically significant changes, the highest and lowest values remained within the normal ranges.
Conclusions
Although dipyrone has historically been associated with agranulocytosis, leukocyte counts remained practically unchanged after oral administration of dipyrone. On the other hand, the present study adds evidence that dipyrone is able to produce statistically relevant decrease in number of platelets, erythrocytes, hemoglobin, and hematocrit in healthy adults, even after short-term treatment.
... After fixation, the slide is stained to distinguish the cells from each other. Diff-Quik, commercial Romanowsky stain variant was used to rapidly stain and differentiate a variety of smears, commonly blood and non-gynecological smears, including those of fine needle aspirates [3][4][5]. Briefly, Dipped peripheral blood smears slide into fixative reagent (Triarylmethane dye and methanol), then dipped slide into stain solution 1 (Eosin G in phosphate buffer) followed by stain solution 2 (Thiazine dye in phosphate buffer) and allow excess ...
... Diff-Quik, a commercial Romanowsky stain variant, was used to stain rapidly and differentiate a variety of smears, commonly blood and nongynecological smears, including those of fine-needle aspirates. [59][60][61] Briefly, dipped peripheral blood smears were slid into fixative reagent (triarylmethane dye and methanol), then slides dipped into stain solution 1 (eosin G in phosphate buffer), followed by stain solution 2 (thiazine dye in phosphate buffer), and excess was allowed to drain after each dip. Slides were rinsed in distilled water (pH 7.2) and allowed to dry in air, then visualized under microscopy (Eclipse E600; Nikon, Tokyo, Japan). ...
Manganese (Mn) is an important mineral element required in trace amounts for development of the human body, while over- or chronic-exposure can cause serious organ toxicity. The current study was designed to evaluate the protective role of quercetin (Qct) against Mn-induced toxicity in the liver, kidney, lung, and hematological parameters in acute and subchronic rat models. Male Sprague Dawley rats were divided into control, Mn (100 mg/kg for acute model and 15 mg/kg for subchronic model), and Mn + Qct (25 and 50 mg/kg) groups in both acute and subchronic models. Our result revealed that Mn + Qct groups effectively reduced Mn-induced ALT, AST, and creatinine levels. However, Mn + Qct groups had effectively reversed Mn-induced alteration of complete blood count, including red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, and white blood cells. Meanwhile, the Mn + Qct groups had significantly decreased neutrophil and eosinophil and increased lymphocyte levels relative to the Mn group. Additionally, Mn + Qct groups showed a beneficial effect against Mn-induced macrophages and neutrophils. Our result demonstrated that Mn + Qct groups exhibited protective effects on Mn-induced alteration of GRP78, CHOP, and caspase-3 activities. Furthermore, histopathological observation showed that Mn + Qct groups effectively counteracted Mn-induced morphological change in the liver, kidney, and lung. Moreover, immunohistochemically Mn + Qct groups had significantly attenuated Mn-induced 8-oxo-2′-deoxyguanosine immunoreactivity. Our study suggests that Qct could be a substantially promising organ-protective agent against toxic Mn effects and perhaps against other toxic metal chemicals or drugs.
... Combined ultrasound and photoacoustic imaging with 1 μm resolution was performed on a blood smear stained with Wright-Giemsa. This colorimetric Romanowksy dye stains the different types of organelles found in leukocytes different colors, enabling visual identification of the cell types as well as different pathological conditions that may be present 12 . Common leukocytes including a neutrophil, lymphocyte and a monocyte were imaged with ultrasound and photoacoustics at 532 nm and 600 nm and a 1000 MHz ultrasound transducer. ...
An acoustic/photoacoustic microscope was used to create micrometer resolution images of stained cells from a blood smear. Pulse echo ultrasound images were made using a 1000 MHz transducer with 1 μm resolution. Photoacoustic images were made using a fiber coupled 532 nm laser, where energy losses through stimulated Raman scattering enabled output wavelengths from 532 nm to 620 nm. The laser was focused onto the sample using a 20x objective, and the laser spot co-aligned with the 1000 MHz transducer opposite the laser. The blood smear was stained with Wright-Giemsa, a common metachromatic dye that differentially stains the cellular components for visual identification. A neutrophil, lymphocyte and a monocyte were imaged using acoustic and photoacoustic microscopy at two different wavelengths, 532 nm and 600 nm. Unique features in each imaging modality enabled identification of the different cell types. This imaging method provides a new way of imaging stained leukocytes, with applications towards identifying and differentiating cell types, and detecting disease at the single cell level.
... However leukocytes are transparent in the visible spectrum, and difficult to identify without the use of a colorimetric stain. Metachromatic Romanowksy type dyes such as the Wright stain differentially stain cellular organelles and are often used for visual identification [38]. However, even when stained it can be difficult to identify abnormalities present in the blood cells. ...
High resolution ultrasound and photoacoustic images of stained neutrophils, lymphocytes and monocytes from a blood smear were acquired using a combined acoustic/photoacoustic microscope. Photoacoustic images were created using a pulsed 532 nm laser that was coupled to a single mode fiber to produce output wavelengths from 532 nm to 620 nm via stimulated Raman scattering. The excitation wavelength was selected using optical filters and focused onto the sample using a 20x objective. A 1000 MHz transducer was co-aligned with the laser spot and used for ultrasound and photoacoustic images, enabling micrometer resolution with both modalities. The different cell types could be easily identified due to variations in contrast within the acoustic and photoacoustic images. This technique provides a new way of probing leukocyte structure with potential applications towards detecting cellular abnormalities and diseased cells at the single cell level.
... A common example is the Wright stain, which is applied to a blood smear to aid in visual identification of the different leukocytes present in a blood sample [109]. The Wright stain is typically composed of methylene blue, azure B and eosin Y, which are selectively absorbed by the different structures within each leukocyte type to give red, pink, blue and purple hues [110]. The range of colors, and hence, optical absorption properties, are ideal for photoacoustic imaging using multiple optical wavelengths. ...
Photoacoustic imaging has experienced exponential growth over the past decade, with many applications in biomedicine. One application ideally suited to the analysis of single cells is photoacoustic microscopy (PAM). Using PAM, detailed submicrometer resolution images of single cells can be produced, with contrast dependent primarily on the optical absorption properties of the cell. A multiwavelength approach for targeting specific endogenous or exogenous chromophores can enhance cellular detail and resolve single organelles with contrast not possible with traditional optical microscopy. A quantitative analysis of the photoacoustic signals acquired from single cells can provide insight into their anatomical, biomechanical, and functional properties. This information can be used to identify specific cells, or to enhance the understanding of biological processes at the single cell level. This comprehensive review on PAM covers recent advances in high-resolution PAM, signal processing methods, and potential clinical applications targeting single cells in vitro and in vivo.
... The dimers are attracted to the negatively charged phosphate groups of DNA and adhere to the macromolecule as a result of hydrophobic interactions with the purine and pyrimidine rings of the DNA bases. Attraction of eosin anions by un-neutralized positive charges of bound azure B dimers changes the color of the stained DNA from blue to purple(16). Eosin is also attracted to proteins with excess protonated amino and guanidino groups (mainly side chains of lysine and arginine) over ionized carboxy groups (mainly glutamic and aspartic acids); these proteins are hemoglobin in erythrocytes and the major basic protein of eosinophil granules(17). ...
Kiernan, J. A. 2010. On chemical reactions and staining mechanisms. Connection (Dako Inc.
Scientific Magazine). Vol. 14, pp. 127-136. [What is the difference between argentaffin and
argyrophilic reactiuons? (p. 127-128). How does pH influence the staining mechanism of
Schiff's reagent in biological tissue samples? (p. 129-131). What is Giemsa’s stain and how
does it color blood cells, bacteria and chromosomes? (p. 132-136).]
... ) http://www.cytojournal.com/content/9/1/*** the cation dye (methylene blue derivative Azure B) and anion dye (Eosin Y) are in the same solution. Second, the purple (metachromatic) stain of the nuclei is believed to be due to the formation of a stain–stain attraction of the methylene blue derivative Azure B and Eosin Y complex to the cell chromati. [19,20] Some destaining was evident during the last step of rehydration (70% ethanol). This may support the theory that water is slightly basic and thus extracts the acid (anion) dye. It might also be that the cells swell and the dyes aggregate more in aqueous solution than in pure alcohol. ...
Protocols for immunocytochemical staining (ICC) and in situ hybridization (ISH) of air-dried Diff-Quick or May-Grünwald Giemsa (MGG)-stained smears have been difficult to establish. An increasing need to be able to use prestained slides for ICC and ISH in specific cases led to this study, aiming at finding a robust protocol for both methods.
The material consisted of MGG- and Diff-Quick-stained smears. After diagnosis, one to two diagnostic smears were stored in the department. Any additional smear(s) containing diagnostic material were used for this study. The majority were fine needle aspirates (FNAC) from the breast, comprising materials from fibroadenomas, fibrocystic disease, and carcinomas. A few were metastatic lesions (carcinomas and malignant melanomas). There were 64 prestained smears. Ten smears were Diff-Quick stained, and 54 were MGG stained. The antibodies used for testing ICC were Ki-67, ER, and PgR, CK MNF116 (pancytokeratin) and E-cadherin. HER-2 Dual SISH was used to test ISH. Citrate, TRS, and TE buffers at pH6 and pH9 were tested, as well as, different heating times, microwave powers and antibody concentrations. The ICC was done on the Dako Autostainer (Dako(®), Glostrup, Denmark), and HER-2 Dual SISH was done on the Ventana XT-machine (Ventana / Roche(®) , Strasbourg, France).
Optimal results were obtained with the TE buffer at pH 9, for both ICC and ISH. Antibody concentrations generally had to be higher than in the immunohistochemistry (IHC). The optimal microwave heat treatment included an initial high power boiling followed by low power boiling. No post fixation was necessary for ICC, whereas, 20 minutes post fixation in formalin (4%) was necessary for ISH.
Microwave heat treatment, with initial boiling at high power followed by boiling at low power and TE buffer at pH 9 were the key steps in the procedure. Antibody concentrations has to be adapted for each ICC marker. Post fixation in formalin is necessary for ISH.
... There are a large number of variables to consider during a cell staining procedure with Giemsa in order to get the most optimal results. Among these are: the structure of the dyes in the Giemsa stain, the pH of the buffer, the concentration of the dye and incubation time of the staining (Horobin and Walter, 1987). Also, fresh ''working solution'' has to be prepared every time a staining procedure is performed and that the result of such solution has to be checked in a preliminary test. ...
Leishmaniasis is a group of endemic diseases produced by infection with Leishmania parasites and affects tropical and subtropical regions of the world. Due to the severe problems related to the treatment of this condition (resistance and toxicity), further studies are needed to evaluate new antileishmanial compounds. The activity of antileishmanial prototypes should be analyzed in models that allow a better interpretation of the findings with respect to natural infection. In this sense, the use of an infection model with macrophages and dendritic cells is a better than using promastigotes alone, in order to establish the potential leishmanicidal activity of a prototype compound. For infection analysis, staining with polychromatic dyes such as Giemsa plus microscopic examination is the gold standard. However, it is common to find problems associated with color uniformity, expertise of the observer, sensitivity and specificity of the technique. For this reason, it's necessary to develop tools and protocols to overcome such limitations. This study assessed the utility of the SYBR® Safe fluorescent dye, considering its affinity for nucleic acids as a useful property for staining the nucleus and kinetoplast of Leishmania parasites within an infected cell. Infection (and subsequent treatment) assays were performed in dendritic cells and macrophages infected with Leishmania panamensis parasites to compare SYBR® Safe and Giemsa stain for the same assay. Correlation coefficients were found to be above 0.9 for both techniques; however, unlike Giemsa, SYBR® Safe staining was easier and provided a clearer observation of internalized parasites. These results support the use of SYBR® Safe as a promising tool for evaluating potential antileishmanials given its advantages over the traditional technique.
... Cytoplasmic RNA is typically stained blue; however, nuclei, in which DNA is concentrated, appear purple. The Giemsa effect can be achieved using two pure dyes, eosin Y and azure B; the purple color results from the formation of complexes of eosin Y and DNA-bound azure B (51)(52)(53). The stain colors Plasmodium nuclei reddish purple, and cytoplasm bluer; it is not, however, readily possible to use measurements of the absorption of the component dyes to derive quantitative measures of DNA or RNA content. ...
In many resource-poor areas, the HIV/AIDS epidemic coexists with epidemics of two much older diseases, malaria and tuberculosis, and the three diseases together kill approximately six million people per year. Although HIV/AIDS is treatable, but not curable, many if not most cases of malaria and tuberculosis (TB) can be cured if diagnosed correctly and promptly. The diagnosis of both malaria and TB is cell-based, typically made by microscopy of stained smears of blood (malaria) and sputum (TB). Cytometry has been shown to be effective for diagnosis of both conditions; however, conventional cytometers have been too complex and costly to be widely applied. It is likely that a newly developed small, simple, robust, inexpensive, energy-efficient low-resolution fluorescence image cytometer, employing a light-emitting diode for excitation and a megapixel digital camera chip for detection, could be used in resource-poor areas for malaria and TB diagnosis and for rapid (24-48 h) determination of antimicrobial susceptibility of Mycobacterium tuberculosis.
Objectives
To evaluate on animal models the health effects of the combined or separate exposure to main chemical and physical hazards of plasma-based material processing technology environment.
Materials and methods
Male Wistar rats were exposed to actual levels of hazardous factors in plasma technology occupational environment: i.e., ozone and nitrogen oxides (O3 and NOx) in respective concentrations of 0.5 mg/m3 and 1.0 mg/m3 and high-frequency (1000–1600 Hz) of 112 dB intensity noise for 3 h/day, 5 days/week for 12 weeks, with a recovery period of 1 month.
Results
Exposure to noise or its combination with chemical factors (ozone, nitrogen oxides) causes non-specific CNS changes testifying for significant excitation dominance, especially in the case of joint exposure. Histological examination of rats’ brain in experimental revealed a pronounced increase in blood filling of small vessels on the tenth day of the experiment, with subsequent intensification of vascular alterations and eventually to cerebral edema. The exposure to noise significantly reduced total thymus, bone marrow and spleen cell numbers and these was also more pronounced under the joint impact of noise and toxic gases. Thymus, but not bone marrow or spleen, mitotic activity was as well reduced under the same modes of exposure. Cytological investigation of film preparations of subcutaneous connective tissue revealed that joint exposure led to microcirculatory disorders, increased number of dark mast cells and reduced degranulation processes indicative of increased autoregulatory processes effective at microvasculature level.
Conclusions
High-frequency and intensity noise is main stressor factor that has negative impact on CNS and immune system, morphology and functioning of hematopoietic organs (spleen, bone marrow, thymus) and connective tissue. Its negative impact is significantly potentiated by concurrent exposure to ozone and nitrogen oxide, while exposure only to these toxic gases has no significant effect on the above targets.
I give an historical account and analysis of the scientific priority of the discovery of the polychrome staining of microscopic biological preparations provided by mixtures of eosin plus methylene blue and its derivatives, especially azure B. I maintain that both the formal priority for the discovery of the polychrome staining phenomenon and credit for initiating the development of a technique of polychrome staining properly belong to D. L. Romanowsky. His scientific work demonstrated the possibility of using a simple technique to stain hematological preparations selectively to give good contrast, high resolution and the ability to identify malaria parasites. Romanowsky's approach constituted the starting point for the development of a family of polychrome stains for microscopic investigation of hematological preparations by a number of his contemporaries.
Alveolar epithelial injury is a feature of ARDS. In this chapter, we will address strategies to analyze alveolar epithelial cell function in lung injury and repair processes. These include an overview of cell isolation procedures for murine and human alveolar epithelial type II cells and examples for phenotypic characterization of ATII cells in the context of lung injury. Moreover, we will discuss functional in vitro assays to evaluate differentiation and regeneration potential of alveolar epithelial cells.
Flow cytometry is a technology that simultaneously measures and analyzes multiple physical characteristics of single particles, usually cells, using an optical-to-electronic coupling system. A wide range of applications are discussed for this tool, ranging from traditional immunology to monitoring of cell transfection and/or screening of protein libraries. Moreover, new exciting insights into ongoing developments are introduced, including automation, laboratory integration, and software advances.
May-Grünwald-Giemsa (MGG) stain is a Romanowsky-type, polychromatic stain as those of Giemsa, Leishman and Wright. Apart being the reference method of haematology, it has become a routine stain of diagnostic cytopathology for the study of air-dried preparations (lymph node imprints, centrifuged body fluids and fine needle aspirations). In the context of their actions of promoting the principles of quality assurance in cytopathology, the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP) and the French Society of Clinical Cytology (SFCC) conducted a proficiency test on MGG stain in 2013. Results from the test, together with the review of literature data allow pre-analytical and analytical steps of MGG stain to be updated. Recommendations include rapid air-drying of cell preparations/imprints, fixation using either methanol or May-Grünwald alone for 3-10minutes, two-step staining: 50% May-Grünwald in buffer pH 6.8 v/v for 3-5minutes, followed by 10% buffered Giemsa solution for 10-30minutes, and running water for 1-3minutes. Quality evaluation must be performed on red blood cells (RBCs) and leukocytes, not on tumour cells. Under correct pH conditions, RBCs must appear pink-orange (acidophilic) or buff-coloured, neither green nor blue. Leukocyte cytoplasm must be almost transparent, with clearly delineated granules. However, staining may vary somewhat and testing is recommended for automated methods (slide stainers) which remain the standard for reproducibility. Though MGG stain remains the reference stain, Diff-Quik(®) stain can be used for the rapid evaluation of cell samples.
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Mixtures of polychrome methylene blue-eosin Y (i.e., Giemsa stain) are widely used in biological staining. They induce a striking purple coloration of chromatin DNA (the Romanowsky-Giemsa effect), which contrasts with the blue-stained RNA-containing cytoplasm and nucleoli. After specific prestaining treatments that induce chromatin disorganization (giving banded or harlequin chromosomes), Giemsa staining produces a differential coloration, with C- and G-bands appearing in purple whereas remaining chromosome regions are blue. Unsubstituted (TT) and bromo-substituted (BT) DNAs also appear purple and blue, respectively. The same occurs in the case of BT and BB chromatids.
In addition to discussing the use of Giemsa stain as a suitable method to reveal specific features of chromosome structure, some molecular processes and models are also described to explain Giemsa staining mechanisms of chromatin.
Degranulation of intestinal mast cells in rainbow trout was studied ex vivo by means of texture analysis and related to the maximal intestinal contraction elicited by degranulation itself. Two strips from the same intestinal segment from ten trout were sampled, processed for light microscopy and stained with Giemsa solution. One of the two strips was exposed to an incremental dose of compound 48/80 in an isolated organ bath before processing. Gray-level RGB channel equivalent and 8-bit gray-level images of five granular cytoplasm areas of mast cells for each section were analyzed for texture features and to evaluate discrimination possibility between treatment groups by means of linear discriminant analysis according to feature selection methods and RGB stacks. Differential mean values (after–before compound 48/80) of the green (r
2 = 0.84, p < 0.01) and blue (r
2 = 0.83, p < 0.01) RGB channels and 8-bit grayscale (r
2 = 0.76, p < 0.05) image correlated significantly with the respective value of maximal intestinal contraction. A possible acidic (anionic) nature for the putative pro-contractile basophil agonist can be inferred.
A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18 100 cm–1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations.Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18 100 cm–1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions.The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly orientated dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence. Subsequently, the absorption spectra of the highly orientated dye complexes were measured with plane polarized light. We found that the transition moments, m
AB, of the bound AB cations in DNA-AB and DNA-AB-EY are orientated almost perpendicular to k, i.e. m
ABk. On the contrary, the transition moments, m
EY, of the bound EY anions in DNA-AB-EY are polarized parallel to k, i.e. m
EY k. The transition moments m
AB and m
EY lay in the direction of the long axes of the AB and EY chromophores. For that reason, in both DNA-AB and DNA-AB-EY the long molecular axes of the AB cations are orientated approximately perpendicular to the DNA chains, while the long molecular axes of the EY chromophores are polarized in the direction of the DNA chains. Therefore, in DNA-AB-EY the long axes of AB and EY are perpendicular to each other, m
ABm
EY. This molecular arrangement fully agrees with our quantitative measurements and with the theory of the absorption of plane polarized light by orientated dye complexes, which has been developed and discussed in detail.
Romanowsky staining of suspension-fixed lymphocytes and fibroblasts, deposited as monolayers on slides, involves an initial basic dyeing process followed by formation of a hydrophobic Azur B/Eosin Y complex at the more permeable and so faster staining cellular sites. This mechanism is shared with blood and marrow smears. However certain morphological features peculiar to suspension-fixed, cell culture-derived preparations also influence the staining pattern via rate control: namely the irregular and bulky profiles of fibroblasts, compared to the smoother and thinner lymphocytes; and the occasional superficial occlusion of cells by culture medium.
We studied the antithrombotic activity of 2-acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester (NCX 4016), a novel nitric oxide (NO)-releasing aspirin derivative, in vivo in different animal models of platelet-dependent and independent pulmonary thromboembolism and compared it with that of aspirin. NCX 4016 protected mice from death induced by the intravenous (i.v.) injection of collagen plus epinephrine, of 9,11-dideoxy-11α,9α-epoxymethano-prostaglandin F2α (U46619) and of thrombin while aspirin was only active against collagen plus epinephrine. The drop in platelet count and number of lung emboli were reduced by NCX 4016 more effectively than aspirin. NCX 4016 protected mice also from mechanical pulmonary embolism (i.v. injection of hardened rat red blood cells) while aspirin was ineffective. In rabbits, NCX 4016 significantly reduced the accumulation of [111In]oxine-labeled platelets in the pulmonary vasculature induced by collagen and by thrombin while aspirin produced reductions which were significant only versus collagen. In conclusion, NCX 4016 exerts a more pronounced antithrombotic activity than aspirin in vivo in two different animal species, largely due to a deeper inhibitory effect on platelets. NCX 4016 may represent a better antithrombotic agent than aspirin.
Romanowsky stains are used routinely by veterinary clinical pathology laboratories for cytologic and blood film evaluations. Automated stainers are available for both aqueous and methanolic Romanowsky stains. Mast cell granules and canine distemper virus inclusions are known to stain differently by these 2 methods, but we have noticed differences in the staining characteristics of other granulated cells.
The aim of this study was to investigate and document the variable appearance of basophils and large granular lymphocytes in blood films stained using aqueous and methanolic Romanowsky methods.
Cytologic preparations from 1 canine mast cell tumor and blood films from 8 dogs, 1 cat, 1 rabbit, and 1 ostrich were stained using an automated aqueous stain (Aerospray 7120, with and without a predip fixative) and an automated methanolic stain (Hematek). Staining quality and intensity of the cytoplasmic granules in mast cells, basophils, and large granular lymphocytes was evaluated subjectively.
Cytoplasmic granules of mast cells, basophils, and large granular lymphocytes stained poorly or not at all with the automated aqueous stain but stained prominently and were readily identified with the automated methanolic stain. Use of the predip fixative with the Aerospray method improved the visibility of basophil granules but not mast cell granules, and had a variable affect on the visibility of granules in large granular lymphocytes.
Clinical pathologists should be aware of the staining methodology used on the slides they evaluate to avoid incorrect interpretation of granulated cell populations.
Standardized specimens with reproducible staining properties were fabricated from extracts of biological objects (bovine liver, nucleoprotamine and defatted muscle). The standard specimens were stained with two formulations of the Romanowsky-Giemsa stain (RG), using the same azure B and eosin Y. One formulation used methanol and Sorensen's buffer and the other DMSO and Hepes buffer as solvents. The standard specimens were stained either in the composite stain or in the individual dyes dissolved in the same solvents and at the same concentration as the composite stain. Solution spectroscopy demonstrated different spectra for the two formulations with some wavelength regions varying by more than an order of magnitude. The RG spectra were also very different from those of the individual dyes dissolved at the RG concentration in the respective solvents. The stained standard specimens were analyzed by microspectrophotometry and were found to have spectra similar to those of cell smears. Furthermore, the standard specimens were shown to be a repeatable substrate for stain uptake. The transmitted light intensity from random fields of the same standardized specimen varied +/- 5%. When specimens were stained at the same time, the specimen-to-specimen variation depended on preparation conditions and the measurement wavelength, but was as good as +/- 5% for some conditions. The quantitative stain performance of both formulations was studied and compared. The standardized specimens provide a tool for the quantitative study of staining processes and specimen preparation procedures and for stain calibration.
The present paper gives a review of the actual state of standardization of biological dyes and stains. In a first part general information is given on practical problems encountered by the routine user of dyes with special emphasis on dye contamination. Some theoretical aspects of standardization are discussed. The second part of the paper gives more detailed information on commercial batches of hematoxylin-eosin-, Giemsa- and Papanicolaou-stains and on their standardization. Special problems arising with the application of image analysis techniques are briefly mentioned. User-oriented specifications for the standardization of dyes, stains and staining procedures are given. Fluorescent dyes and dyes used in chromogenic reagents such as the Feulgen-Schiff reaction are not included in this review.
A new and technically simple Romanowsky-Giemsa (RG) stain is proposed as a standardized technique for use in histology. An RG stock solution (pure azure B 7.5 g/l, eosin Y as eosinic acid 1.2 g/l in dimethylsulfoxide) is diluted to form the working solution with HEPES-buffer, pH 6. Staining time is 30-90 min after formol-calcium solution (or 2-4 hr after formaldehyde-organic acid mixtures). The resulting overstained sections are to be differentiated. A tannic acid-acetic acid combination in an isopropanol-water mixture was found to give optimum results within 100 sec. Subsequent dehydration is in isopropanol only. The staining pattern obtained is polychrome. The distribution of colors in detail is influenced by the modes of pre- and posttreatment. Of practical interest is the development of green and greenish blue colors on collagen fibrils which contrast strongly against the pink of sarcoplasm. For this and other reasons, this RG stain version seems suitable to replace the trichrome Gomori-type trichrome stains under appropriate processing conditions.
Bone marrow smears were made and fixed in methanol or formaldehyde. Marrow sections of various thicknesses were also prepared from formaldehyde fixed marrows embedded in paraffin or plastic (glycol methacrylate). The different smears and sections were then stained by a Romanowsky--Giemsa procedure. Some specimens were stained using a standard microwave-stimulated method previously used diagnostically. The effects of technical variations were studied, including degree of microwave irradiation and the staining time. Comparisons of the resulting staining outcomes showed that microwave stimulated Romanowsky--Giemsa staining of plastic sections is a rate controlled process. Unusual aspects of the staining pattern of plastic sections (namely the purple basophilic cytoplasms and nucleoli, and blue chromatin) are due to microwave stimulation and formaldehyde fixation respectively.
Romanowsky staining of suspension-fixed lymphocytes and fibroblasts, deposited as monolayers on slides, involves an initial basic dyeing process followed by formation of a hydrophobic Azur B/Eosin Y complex at the more permeable and so faster staining cellular sites. This mechanism is shared with blood and marrow smears. However certain morphological features peculiar to suspension-fixed, cell culture-derived preparations also influence the staining pattern via rate control: namely the irregular and bulky profiles of fibroblasts, compared to the smoother and thinner lymphocytes; and the occasional superficial occlusion of cells by culture medium.
The objective of this study was to review and compare rapid protocols for fixation and staining of cytologic smears. We used fresh surgical specimens from dogs and horses to evaluate and modify, if necessary, previously described rapid staining protocols. Slides were wet-fixed, rehydrated or air-dried. Rapid Papanicolaou, hematoxylin and eosin (H&E), and Romanowsky stains were applied, including modification of Diff-Quick stain. The modified rapid staining protocols were simple to use and gave results within 5 minutes that were comparable to those obtained with traditional methods. Advantages of rehydrated vs wet-fixed smears included consistent preparations, a clean background, and equally good or superior nuclear detail.
For ultrastructural localization of acid mucosubstances in rabbit granulocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde, or osmium tetroxide, sectioned at 40 micro, and stained with the Rinehart and Abul-Haj solution of dialyzed iron (DI). Heterophils revealed DI staining on the outer surface of the plasma membrane, in the Golgi complex involved in primary granulogenesis, and in primary granules. The intragranular distribution of DI-stained material varied at different stages in the maturation of primary granules. Immature granules of heterophils fixed by any of the three methods contained a peripheral concentric band of DI-positive material; however, fully mature primary granules possessed a core of DI-reactive material in heterophils fixed with osmium tetroxide, but they contained little or no staining in heterophils fixed with formalin or glutaraldehyde. Secondary granules of rabbit heterophils failed to stain with DI. Tertiary granules, observed only in late heterophils, contained distinct DI-positive particles. Basophil granules exhibited intensely DI-stained material distributed in an orderly pattern throughout the granule. In eosinophils, DI staining was localized in the Golgi complex and in the rims of a few immature cytoplasmic granules.
For ultrastructural localization of acid mucosubstances in rabbit granulocytes, bone mar- row and buffy coat specimens were fixed with formalin, glutaraldehyde, or osmium tetrox- ide, sectioned at 40 p, and stained with the Rinehart and Abul-Haj solution of dialyzed iron(DI).Heterophils revealed DI staining on the outer surface of the plasma membrane, in the Golgi complex involved in primary granulogenesis, and in primary granules . The intragranular distribution of DI-stained material varied at different stages in the matura- tion of primary granules . Immature granules of heterophils fixed by any of the three meth- ods contained a peripheral concentric band of DI-positive material ; however, fully mature primary granules possessed a core of DI-reactive material in heterophils fixed with osmium tetroxide, but they contained little or no staining in heterophils fixed with formalin or glutaraldehyde . Secondary granules of rabbit heterophils failed to stain with DI . Tertiary granules, observed only in late heterophils, contained distinct DI-positive particles . Baso- phil granules exhibited intensely DI-stained material distributed in an orderly pattern throughout the granule . In eosinophils, DI staining was localized in the Golgi complex and in the rims of a few immature cytoplasmic granules .
"Toxic" neutrophils from humans with severe bacterial infections, identified by the presence of Döhle bodies, "toxic" granules, and vacuoles were shown to differ from normal neutrophils both in ultrastructure and in lysosome activity.
Döhle bodies were identified as lamellar aggregates of rough endoplasmic reticulum. Toxic granules corresponded to the azurophilic granules usually identified by Romanowsky stains only in neutrophil precursors. By electron microscopy such granules were large, electron-dense, and peroxidase positive; they could usually be distinguished from the smaller, less dense, "specific" granules also present in control neutrophils, but in the latter they became visible by light microscopy only after prolonged staining or following fixation with glutaraldehyde. These observations suggest that toxic granules represent an abnormal staining reaction of the large dense granules in the toxic cells, and not phagocytized material, newly formed abnormal granules or autophagic bodies.
Alkaline phosphatase activity was significantly greater in toxic neutrophils than in normal ones; 80% of the activity of both was located in the lysosome fraction. Beta glucuronidase was normal. Total acid phosphatase was normal, but the percentage located in the nonlysosome fraction of toxic neutrophils was increased, suggesting that lysosomes were "labilized."
Formation of neutral red vacuoles in supravitally stained preparations, an index of lysosome activity, occurred more rapidly in toxic neutrophils. This reaction paralleled degranulation and the formation of clear vacuoles in unstained wet mounts and could be blocked by colchicine, a lysosome stabilizer, or enhanced by procedures which activate lysosomes. "Autophagic" vacuoles were observed by electron microscopy in some toxic neutrophils.
These observations are discussed in relation to the concept that the "toxic" neutrophils in severe bacterial infection reflect cellular immaturity and/or stimulation or degeneration.
Acid mucopolysaccharides have been extracted from whole rabbit polymorphonuclear leucocytes and from the cytoplasmic granules of these cells.
The leucocyte acid mucopolysaccharides can be separated into two fractions by the solubility of their CPC complexes in solutions of differing salt concentration. One of these fractions appears to be identical with hyaluronic acid; the other appears to be an atypical chondroitin sulfate.
On both a dry weight and total protein basis the polymorphonuclear leucocyte granule contains approximately 2.6 times as much acid mucopolysaccharide as does the whole cell. Hyaluronic acid is concentrated in the granules in particular; its function is unknown.
These results do not indicate that all lysosomes contain abundant acid mucopolysaccharides, for no detectable carbohydrate of this class could be extracted from lysosome-rich alveolar macrophages.
Unlabelled:
Human malaria parasites, including the most lethal Plasmodium falciparum, are increasingly resistant to existing antimalarial drugs. One remarkable opportunity to selectively target P. falciparum stems from the unique AT-richness of its genome (80% A/T, relative to 60% in human DNA). To rationally explore this opportunity, we used drugs (adozelesin and bizelesin) which distinctly target AT-rich minisatellites and an in silico approach for genome-wide analysis previously experimentally validated in human cells [Woynarowski JM, Trevino AV, Rodriguez KA, Hardies SC, Benham CJ. AT-rich islands in genomic DNA as a novel target for AT-specific DNA-reactive antitumor drugs. J Biol Chem 2001;276:40555-66]. Both drugs demonstrate a potent, rapid and irreversible inhibition of the cultured P. falciparum (50% inhibition at 110 and 10+/-2.3 pM, respectively). This antiparasital activity reflects most likely drug binding to specific super-AT-rich regions. Relative to the human genome, the P. falciparum genome shows 3.9- and 7-fold higher frequency of binding sites for adozelesin and bizelesin, respectively. The distribution of these sites is non-random with the most prominent clusters found in large unique minisatellites [median size 3.5 kbp of nearly pure A/T, with multiple converging repeats but no shared consensus other than (A/T)(n)]. Each of the fourteen P. falciparum chromosomes contains only one such "super-AT island" located within approximately 3-7.5 kbp of gene-free and nucleosome-free loci. Important functions of super-AT islands are suggested by their exceptional predicted potential to serve as matrix attachment regions (MARs) and a precise co-localization with the putative centromeres.
Conclusion:
Super-AT islands, identified as unique domains in the P. falciparum genome with presumably crucial functions, offer therapeutically exploitable opportunity for new antimalarial strategies.
The present study introduces the principle of atomic force microscopy (AFM) and reviews our results of human metaphase chromosomes obtained by AFM. AFM imaging of the chromosomes revealed that the chromatid arm was not uniform in structure but had ridges and grooves along its length, which was most prominent in the late metaphase. The arrangement of these ridges and grooves was roughly symmetrical with the counterpart of the paired sister chromatids. AFM imaging of banded chromosomes also showed that the ridges and grooves were related to the G/Q-positive and G/Q-negative bands, respectively. At high magnification, the chromatid was seen to be produced by the compaction of highly twisted chromatin fiber loops, and its compaction tended to be stronger in the ridged regions of the chromosomes than in the grooved regions. Our AFM studies also showed the presence of catenation of chromatin fibers between the ridged portions of the chromatid in the late metaphase. Thus, AFM is useful for obtaining the three-dimensional surface topography not only in ambient conditions but also in physiological liquid conditions, and is expected to be an attractive tool for investigating the structure of chromosomes.
The value of the Giemsa stain on smears and imprints has long been acknowledged, but few pathologists have been consistently successful with histologic sections because of the peculiar problems of the dyes and the mysterious vagaries of the methodology. A Giemsa stain giving reproducible differential staining of tissue sections is particularly desirable for the examination of hematopoietic tissues. A modified Giemsa stain which provides such reproducibility is described. It differs from other methods primarily in the use of a stock solution which contains known amounts of azure A–eosin and azure B–eosin. In addition, acetic acid is used instead of phosphate buffers to obtain an acidic working solution. The results suggest that the Giemsa stain may represent the most useful means of evaluating lymphoid tissue in section. It is of particular value in identifying the immunoblast (basophilic stem cell), as well as providing differentiation of granulocytic elements.
Despite extensive quantitative work on the thermodynamics of textile dyeing (Vickerstaff, 1954), probably only Weiss (1954), who estimated the heat of reaction of elastin with orcein in vitro, has attempted to apply similar methods to a histological problem. In the present article a semi-quantitative investigation into the effect of temperature on the staining of sections by the basic dye azure A (M.W. 292) is reported, and certain aspects of the thermo- dynamics of staining are discussed. Despite the fact, discussed in the text, that some of the results reported in the present article can be accepted only with reservations, it will be shown that the thermodynamical approach to staining promises to throw light on several aspects of the staining process.
Column and paper chromatography of four thiazin dyes revealed both inorganic and organic impurities. In thionin, azure A, azure B and methylene blue, sodium and other metal cations were found as inorganic impurities. The analysis for organic impurities revealed that the dyes were mixtures; specifically each dye contained one or more of the other dyes as impurities. Inorganic impurities were detected by ashing the dyes in the presence of H2SO4 and chromatographing the sulfate salts on paper. They were removed by filtration through ion exchange resins. Organic impurities were detected by paper chromatography and removed by column chromatography on Woelm's neutral alumina.
This paper describes a comparison of the performance of two standardized Romanowsky blood stains, namely those of Marshall et al. and Wittekind et al., both containing azure B and eosin alone. Stain performance is assessed objectively by the use of three complementary techniques, all based on the visible absorbance spectra of stained cellular substrates. The first of these techniques is a simple comparison of the shapes and heights of the absorbance spectra. The second technique uses the CIE Colorimetric System, and thus permits the quantitation of colour in a manner that agrees with human observation. CIE co-ordinates (chromaticity points, luminance) are calculated directly from absorbance spectra. The third technique is that of spectral subtraction, which yields a set of factors which describe the quantities of component dyes which are bound by the object. This technique, unlike the other two, requires a priori knowledge of the dyes used in the stains, and their spectra when bound to cellular substrates.
Although the differences between the two methods are subtle, and hard for the subjective observer to define, the objective methods described here do show statistically significant differences. Wittekind's stain produces less intense staining, except for lymphocyte and monocyte cytoplasms. To the human eye, the differential coloration of these two substrates is more pronounced, but the difference between all nuclei and cytoplasms is less marked. The major difference in the uptake of dye components is in the small quantities of eosin dimer that are bound in this technique.
This paper describes a microspectrophotometric study of blood smears stained by a simple, standardized Romanowsky technique, using only the dyes azure B and eosin. Absorbance spectra are presented for twenty-two classes of cellular object, and for the two dyes in solution, together with tabulations of spectral maxima, and suitable wavelengths for use in automated image processing. The colours of objects stained with azure B/eosin are discussed in terms of absorbance spectra. By a spectral subtraction technique, it is shown that the differential colouration of various cell structures may be explained satisfactorily in terms of the varying proportions of only four dye components. These are the monomers and dimers of azure B and eosin. Polymerization was found to occur both in solution and on binding to biopolymers. A similar analysis of a conventional Romanowsky stain would present much greater difficulties, due to the greater number of dye components, which, however, contribute little to the colours observed.
A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18 100 cm–1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations.Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18 100 cm–1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions.The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly orientated dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence. Subsequently, the absorption spectra of the highly orientated dye complexes were measured with plane polarized light. We found that the transition moments, m
AB, of the bound AB cations in DNA-AB and DNA-AB-EY are orientated almost perpendicular to k, i.e. m
ABk. On the contrary, the transition moments, m
EY, of the bound EY anions in DNA-AB-EY are polarized parallel to k, i.e. m
EY k. The transition moments m
AB and m
EY lay in the direction of the long axes of the AB and EY chromophores. For that reason, in both DNA-AB and DNA-AB-EY the long molecular axes of the AB cations are orientated approximately perpendicular to the DNA chains, while the long molecular axes of the EY chromophores are polarized in the direction of the DNA chains. Therefore, in DNA-AB-EY the long axes of AB and EY are perpendicular to each other, m
ABm
EY. This molecular arrangement fully agrees with our quantitative measurements and with the theory of the absorption of plane polarized light by orientated dye complexes, which has been developed and discussed in detail.
Of the batches of dye examined only one (Azure B, Merck number YE 132) can be regarded as being of acceptable quality. This material retails at 6.60/g for a 250 g lot, and may be considered to be reasonably priced. The quality of all Serva¢s < img src="/content/QNHU33T34552Q11P/xxlarge8216.gif" alt="lsquo" align="BASELINE" border="0pure < img src="/content/QNHU33T34552Q11P/xxlarge8217.gif" alt="rsquo" align="BASELINE" border="0 dyes is alarmingly poor and leaves much to be desired. In view of the fact that these dyes retail at6.60/g for a 250 g lot, and may be considered to be reasonably priced. The quality of all Serva's pure dyes is alarmingly poor and leaves much to be desired. In view of the fact that these dyes retail at 700.00/g, the purchaser should expect to obtain analytically pure material. It is hoped that this note may persuade users of commercially available pure Azure dyes to check the purity of any samples they might possess: manufacturers' claims should not be accepted at face value.
Eine Methode zur Anfrbung von Blut- und Knochenmarkausstrichen wird beschrieben, dadurch gekennzeichnet, da nur mit zwei Farbstoffen, reinem Azur B und Eosin, gefrbt wird. Die Ergebnisse sind denen gleichwertig, die mit der May-Grnwald-Giemsa-Frbung (MGG) erreicht werden. Die Azur B-Eosin-Frbung lt sich jedoch standardisieren, sie ist auch einfacher durchzufhren. Beziehungen zwischen Azur B-Eosin- und May-Grnwald-Giemsa-Frbung werden kurz besprochen.A new method is described for staining blood and bone marrow smears. It is characterized by the presence of only two dyes, purified azure B and eosin in methanol, as stock solutions. Staining results are equivalent to those obtained by using the traditional dye mixtures according toMay andGrnwald, Giemsa, Leishman orWright. Different from these azure B-eosin staining can be standardized and is easier to be handled. Correlations between the azure-B-eosin and May-Grnwald-Giemsa (MGG) staining methods are briefly discussed.
This is not a "how to" book but a "how the methods work". And might help you solve a problem with this methodology in some new way.
The use of prometaphase chromosomes prepared for high-resolution imaging is essential for accurate cytogenetic investigations. The process of Giemsa chromosome banding (G-banding), however, is often time consuming and difficult to standardize owing to differing ambient conditions and inter-operator variability. Consequently, many laboratories currently are introducing automatic metaphase finder and analysis systems to achieve the goals of higher throughput of samples and more consistent chromosome quality. In this context, we investigated the use of automation to improve the G-banding process. We investigated the use of the Shandon Thermo Varistain Gemini automatic stainer to replicate the manual process of G-banding. We compared the current manual method and the automated procedure and found that automation provided equivalent quality, and increased consistency while decreasing the time required and reducing the cost per preparation.
A thorough understanding of the mechanisms of R-, C- and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral absorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. - Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not necessary component for banding. - The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their absorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.
A panel of 17 eminent haematologists has assessed the performance of 5 Romanowsky stains prepared from pure component dyes, comparing the suitability and acceptability of these stains for the preparation of routine blood and bone-marrow films. It was found that the results obtained using the stain described by Marshall et al (1975) were comparable to those obtained using a modification of the stain described by Wittekind et al (1976). The performance of the 3 other stains was less acceptable. Variations in stain formulation have been correlated with stain performance.
This paper reviews the nature of Romanowsky staining and the relationship between Romanowsky dyes and the Romanowsky-Giemsa effect (RGE). On blood and bone marrow smears the RGE is characterized by a purple colouration of nuclei and neutrophil granules. The nuclear purple contrasts strongly with the blue cytoplasmic staining of cells rich in RNA. Requirement for the occurrence of RGE are: I A cationic dye: The best dye is azure B and, though azure A gives the nuclear purple colour, the cytoplasmic blue is inferior. No other cationic dye such as methylene blue is suitable. 2 An anionic dye: Most commonly eosin Y is used, but it can be replaced by the erythrosins. Full halogenation of the fluorescein (four atoms of bromine or iodine) is not necessary. Phloxine and rose bengal are unsuitable. 3 An appropriate substrate: These are proteins with acidic side groups or proteins bound to a polyanion. For the interaction with the dyes substrates must provide a suitable three-dimensional network which is why the RGE is not obtained in solutions. A tentative theory of RGE is advanced and briefly discussed.
A quantitative study of azure B-eosin-stained blood cells is reported. The effects of variation in stain formulation and staining technique on the binding of azure B and eosin by acidophilic, basophilic and neutrophilic substrates were measured by scanning microdensitometry. The variables considered were stain concentration (the azure B-eosin concentration ratio was constant), azure B concentration (at constant eosin concentration), eosin concentration (at constant azure B concentration), staining time, buffer pH, metal salt contamination, dye contamination, buffer concentration and fixation time. The last two were the only variables which failed to produce changes in dye uptake. Tentative explanations of the observed effects are advanced.
Commercial samples of Erythrosin B (CI 45430), Erythrosin Y (CI 45425), Fluorescein (CI 45350), Phloxine (CI 45410) and Rose Bengal (CI 45440) have been analysed by thin-layer chromatography. The Erythrosins were found to be mixtures consisting in the main of 4′-iodofluorescein, 4′,5′-di-iodofluorescein, 2′,4′,5′-triiodofluorescein and 2′,4′,5′,7′-tetraiodofluorescein, in some instances together with 2′,4′,5′-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2′,4′,5′,7′-tetraiodo-4,5,6,7-tetrachlorofluorescein. Samples of Fluorescein were mixtures of the nominal dye usually with traces of several unidentified, fluorescent components. Those of Phloxine consisted mainly of mixtures of 4′-bromo-4,5,6,7-tetrachlorofluorescein, 4′,5′-dibromo-4,5,6,7-tetrachlorofluorescein, 2′,4′,5′-tribromo-4,5,6,7-tetrachlorofluorescein and 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachlorofluorescein, often with 4,5,6,7-tetrachlorofluorescein Samples of Rose Bengal were mixtures of 4′-iodo-4,5,6,7-tetrachlorofluorescein, 4′,5′-di-iodo-4,5,6,7-tetrachlorofluorescein, 2′,4′,5′-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2′,4′,5′,7′-tetraiodo-4,5,6,7-tetrachlorofluorescein together with some unidentified components.
Most of the commercial dye samples gave an insoluble residue when extracted with methanol. This residue was usually inorganic carbonate or halide. Some possible practical consequences of the various impurities are discussed.
Glycol methacrylate (GMA) sections of animal tissues were stained with a group of twenty-seven reagents of very varied chemical characteristics. The artefactual background staining of the resin was found to be dependent on the hydrophilic/lipophilic character of the staining reagent, as estimated from the logarithm of its octanol-water partition coefficient (log P). Intense background staining occurred with lipophilic stains, whose log P greater than 2. In keeping with this, use of GMA semi-permeable membranes for enzyme histochemistry failed to give staining when using a lipophilic substrate, probably because the substrate was trapped in the membrane. An analysis of other routine histochemical stains--in terms of the probable occurrence of high resin background staining and low tissue sensitivity--is made. A numerical guide is provided to help avoid artefacts resulting from hydrophobic and size effects. Note: small, hydrophilic reagents (log P less than 0; molecular weight less than 550 Da) are least likely to show either type of artefact. Conversely, reagents which are lipophilic, or/and of intermediate size (log P greater than 2; 550 less than ionic weight less than 1000 Da), give strong background staining.
A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18,100 cm-1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations. Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18,100 cm-1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB approximately 1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions. The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly oriented dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence.(ABSTRACT TRUNCATED AT 400 WORDS)
Penetration of hydrophilic acid and basic dyes into sections cut from glycol methacrylate (GMA)-embedded tissues was studied; as were the effects on such staining of superficial coatings of thin layers of GMA. Dye size was a major factor in controlling penetration of resin and staining of tissues. 'Large' dyes (greater than 1000 Da) entered GMA very slowly, and only stained those tissue components poorly infiltrated by resin. 'Small' dyes (less than 550 Da) penetrated GMA readily, and stained tissue components whether or not they were resin-infiltrated. Dyes of intermediate size penetrated the resin, but the staining of resin-infiltrated tissue elements was slow. Background staining of resin also varied with dye size. Large dyes gave no staining of GMA. Small dyes did, but were readily removed by water washing. Dye of intermediate size penetrated resin slowly, and once inside were lost slowly. This gave background staining which required use of the plasticizing solvent ethanol for its removal. Increases in resin cross-linking also reduced staining rates. As a consequence, it is possible to predict the probable suitability, or otherwise, of various staining reagents proposed for use with GMA sections; and also the probable influences of histoprocessing on stain penetration. In particular it is suggested that penetration of colloidal metals and macromolecular reagents (e.g. labelled antibodies and lectins) will be limited to resin-free structures, and to the surface of resin sections. The use of superficial GMA coatings as convenient semipermeable membranes for enzyme histochemistry is also noted.
Bone marrow smears were made and fixed in methanol or formaldehyde. Marrow sections of various thicknesses were also prepared from formaldehyde fixed marrows embedded in paraffin or plastic (glycol methacrylate). The different smears and sections were then stained by a Romanowsky--Giemsa procedure. Some specimens were stained using a standard microwave-stimulated method previously used diagnostically. The effects of technical variations were studied, including degree of microwave irradiation and the staining time. Comparisons of the resulting staining outcomes showed that microwave stimulated Romanowsky--Giemsa staining of plastic sections is a rate controlled process. Unusual aspects of the staining pattern of plastic sections (namely the purple basophilic cytoplasms and nucleoli, and blue chromatin) are due to microwave stimulation and formaldehyde fixation respectively.
Romanowsky staining of suspension-fixed lymphocytes and fibroblasts, deposited as monolayers on slides, involves an initial basic dyeing process followed by formation of a hydrophobic Azur B/Eosin Y complex at the more permeable and so faster staining cellular sites. This mechanism is shared with blood and marrow smears. However certain morphological features peculiar to suspension-fixed, cell culture-derived preparations also influence the staining pattern via rate control: namely the irregular and bulky profiles of fibroblasts, compared to the smoother and thinner lymphocytes; and the occasional superficial occlusion of cells by culture medium.
The suitability of reverse-phase thin layer chromatography using a commercial adsorbant and aqueous methanol as an analytical tool for biological stains was investigated. The wide range of applicability of this technique is shown by the fact that of 120 dyes used as biological stains, 84 of diverse chemical character were successfully chromatographed by varying only the water content of the eluent. Unsuccessful chromatography was due either to immobility or streaking. Dyes exhibiting this behavior can be identified prior to chromatography by inspection of their structural formulae. Rf values were found to be significantly correlated with the calculated partition coefficients. This relationship provides information for the identification of dye components revealed by chromatography and a discussion of its use in the chemical characterization of various dyestuffs is presented.
The value of the Giemsa stain on smears and imprints has long been acknowledged, but few pathologists have been consistently successful with histologic sections because of the peculiar problems of the dyes and the mysterious vagaries of the methodology. A Giemsa stain giving reproducible differential staining of tissue sections is particularly desirable for the examination of hematopoietic tissues. A modified Giemsa stain which provides such reproducibility is described. It differs from other methods primarily in the use of a stock solution which contains known amounts of azure A eosin and azure B eosin. In addition, acetic acid is used instead of phosphate buffers to obtain an acidic working solution. The results suggest that the Giemsa stain may represent the most useful means of evaluating lymphoid tissue in section. It is of particular value in identifying the immunoblast (basophilic stem cell), as well as providing differentiation of granulocytic elements.
It is shown that the dyes used to produce banding patterns on chromosomes, quinacrine and Giemsa, are bound to DNA, and not to non-histone protein, the other chromosomal component remaining after acetic acid fixation. Studies on fixed nuclei and on extracted DNA in gelatine films show that the amount of dye bound is not affected by whether the DNA is native or denatured, and is not directly related to the amount of DNA present. Quinacrine is bound to the DNA ionically. With Giemsa, a new magenta compound is formed in situ, consisting of two molecules of methylene blue and one of eosin; this compound is attached to the chromosome by hydrogen bonds. Both quinacrine and the magenta compound formed from Giemsa appear to be attached to DNA molecules at two separate points, and the available evidence suggests that the amount of dye bound is related to the concentration of the DNA. It is suggested that the dye molecules bridge longitudinally separated sites brought into close proximity by folding of the DNA, and that the spatial arrangement of sites in the chromosome is influenced by non-histone proteins. It is concluded that chromosome banding is thus a consequence of the reduction of dye binding in those regions where the DNA chains become sufficiently dispersed to prevent bridging by the dye molecules. Possible indirect effects of base composition and repetition on dye binding at certain chromosomal sites are discussed.
Cytoplasmic granules of polymorphonuclear leukocytes (PMN) from blood resolve during velocity centrifugation into well separated bands I, II, and III (sedimentation increasing in that order). Band I comprises empty vesicles and 85% of the biochemically measured alkaline phosphatase of the whole PMN. Specific granules, which are peroxidase negative and contain 50% of the PMN lysozyme and 77% of the lactoferrin, constitute band II. Their average diameter is 0.13 μm. The azurophil granules, with average diameters of 0.3 μm constitute band III and contain 88% of the immunochemically measured myeloperoxidase, 84% of the neutral protease, and 46% of the lysozyme. Both specific granules and azurophils are bounded by membranes. Band III can be further resolved into 2 narrowly separable bands: IIIs (slow), which contains more myeloperoxidase, neutral protease, and β glucuronidase; and IIIf (fast), which contains lysozyme. Histochemically, both IIIs and IIIf granules contain peroxidase. Measured immunochemically, lactoferrin and myeloperoxidase, respectively, appear to afford unambiguous markers for specific and azurophil granules of PMN. Although various blood cells may contaminate suspensions of PMN, only specific granules of eosinophils created problems.
Batch variations in commercial dyes employed for Romanowsky-type staining have been studied by thin-layer chromatography.
The colored components of numemus batches of the thiazine dyes azure A, azure B, azure C, methylene blue, methylene violet hthsen, thionine and tduidine blue, and of the xanthene dyes emin B and eosin Y, have been compared. It has been found that of these dyes only methylene blue and thionine are commercially available reasonably free of colored contaminants. The azures, methylene violet Bernthsen and toluidine blue are complex mixtures of thiazine dyes of the thionine and thionoline series. Samples of eosin B are mixtures of the nominal dye with win Y, fluorescein and tribromofluorescein. Thoee of eosin Y are mixtures of this dye with fluorescein and tribromofluorescein.
The mechanisms whereby Giemsa is bound to chromosomes to produce G-banding patterns have been studied. The magenta colour produced in chromosomes by Giemsa staining appears to be due to the same 2:1 thiazine-eosin compound that precipitates from Giemsa solutions. This precipitate is formed in chromosomes in regions in which DNA phosphates occur at the correct distance apart to bind two thiazine molecules, which are subsequently able to bind to the same eosin molecule. Banding appears to be produced as a result of this precipitation occurring preferentially in hydrophobic regions of chromosomes.
The staining of chicken erythrocyte nuclei with Alcian blue followed by a treatment with ethanol-NaOH, showed to be highly resistant to acid extraction (5 N HCl) due to the alkaline conversion of Alcian blue into the insoluble pigment Monastral fast blue. It is assumed that the intercalative binding of the planar Monastral fast blue molecule to DNA accounts for its competition effect toward the intercalating fluorochromes acridine organe, ethidium bromine and daunomycin.
Many of the difficulties of staining plastic embedded tissues for light and electron microscopy derive from physical exclusion of hydrophilic staining reagents by hydrophobic embedding media. Structures which stain most intensely with hydrophilic reagents usually contain less hydrophobic plastic than do non-staining structures. Such incomplete infiltration is apparently caused by exclusion of viscous, hydrophobic monomers by physically dense and/or well hydrated tissue elements. In keeping with this, generalized staining of tissues embedded in hydrophobic media does occur when hydrophobic reagents are used. Staining of plastic-free structures with single hydrophilic reagents or with sequences of such reagents, is, however, largely rate-controlled. The surprising similarity of hydrophilic and hydrophobic plastic embedding media is discussed. Limits of this simple model are explored, with a consideration of the roles of fixative and of monomer-tissue reactions.
The chances of Romanowsky-Giemsa (RG) staining becorning a reliable and useful histochemical procedure are reviewed, based on the now proven fact that RG staining requires two dyes only, namely, cationic Azure B and anionic Eosin Y. These two dyes differe from otherwise similar dye combinations in that they give, on distinct biological substrates, one additional colour, purple, which cannot be obtained by the use of either dye alone. The purple colour characterizes the Romanowsky-Giemsa effect (RGE), which is the essential feature of RG staining.
Consideration is given to the physico-chemical and morphological implications of RGE. Of primary importance is the nature of the biological substrates where RGE occurs, and also of those where it has never been observed. The way substrates react to RG stains largely depends on the kind of pretreatment they have received; for instance, alcoholic fixation preserves RGE but formaldehyde may inhibit it. Physico-chemical factors are considered which, by altering either the biological substrates or the composition of the staining solutions, may modify the RG staining pattern.
This review also serves as an introduction for a series of experimental papers that will follow and which are intended to consolidate the basis of RG staining, a method which holds much promise as a useful histochemical tool.
Accurate and reproducible interference microscopy of unstained sections requires (a) the use of a shearing system, preferably with a half-shade eye-piece; (b) complete removal of embedding wax from the section; (c) complete replacement of one mounting medium by any successive medium; (d) accurate measurement of the refractive index (RI) of the mounting media at the temperature and wave-length used for microscopy. The optical path through a specimen mounted in water is consistently lower than expected from measurements in non-polar fluids—this is attributed to swelling of the tissues in water. Cellulose, glycogen and some mucins have a relatively low RI, and secretory granules and cytoplasmic chromidial substance a relatively high one. The highest RI observed was in calcified cartilage matrix. The “effective thickness” of tissue components, especially certain mucins, is often considerably less than the geometrical thickness of the section. This is attributed to the presence in the tissues of substantial spaces containing mounting medium.
The relation of the RI and the effective thickness of tissue components in sections to their permeability to histological dyes is discussed. It is concluded that both the RI and the effective thickness may be of some value in predicting the ease with which dye particles of a given size can penetrate the tissue.
Histochemistry: an explanatory outline of histochemistry and biophysical staining Gustav Fischer, Stuttgart ; Butterworths, London International Committee for Standardisation in haematology (1984) ICSH Reference Method for staining of blood and marrow films by azure B
- Rw Horobin
Horobin RW (1982) Histochemistry: an explanatory outline of
histochemistry and biophysical staining. Gustav Fischer, Stuttgart ; Butterworths, London
International Committee for Standardisation in haematology
(1984) ICSH Reference Method for staining of blood and marrow films by azure B and eosin Y (Romanowsky-Giemsa stain)