Detection of mineralized structures in very early stages of development of Marine Teleostei using a modified Alcian blue-Alizarin red double staining technique for bone and cartilage

We have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100-200 pm in length can be clearly visualized. The method is suitable for both ontogenic studies during early stages of skeletal development in most marine fishes (e.g., Sparus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 1000/0 ethanol for up to two years.

A novel method to count the red blood cells in thin blood films

This paper describes a novel idea to identify the total number of red blood cells (RBCs) as well as their location in a Giemsa stained thin blood film image. This work is being undertaken as a part of developing an automated malaria parasite detection system by scanning a photograph of thin blood film in order to evaluate the parasitemia of the blood. Not only will this method eliminates the segmentation procedures that are normally used to segment the cells in the microscopic image, but also avoids any image pre-processing to deal with non uniform illumination prior to cell detection. The method utilizes basic knowledge on cell structure and brightness of the components due to Giemsa staining of the sample and detects and locates the RBCs in the image.

Topical delivery of a naproxen-dithranol co-drug: in vitro skin penetration, permeation, and staining

Purpose This work probed the topical delivery and skin-staining properties of a novel co-drug, naproxyl-dithranol (Nap-DTH), which comprises anti-inflammatory (naproxen) and anti-proliferative (dithranol) moieties. Method Freshly excised, full-thickness porcine ear skin was dosed with saturated solutions of the compounds. After 24 h, the skin was recovered and used to prepare comparative depth profiles by the tape-stripping technique and to examine the extent of skin staining. Results Depth profiles showed that Nap-DTH led to a 5-fold increase in drug retention in the skin compared to dithranol. The application of Nap-DTH also demonstrated improved stability, resulting in lower levels of dithranol degradation products in the skin. Furthermore, significantly less naproxen from hydrolysed Nap-DTH permeated into the receptor phase compared to naproxen when applied alone (0.08 ± 0.03 nmol cm-² and 180 ± 60 nmol cm-², respectively). Moreover, the reduced staining of the skin was very apparent for Nap-DTH compared to dithranol. Conclusions Topical delivery of Nap-DTH not only improves the delivery of naproxen and dithranol, but also reduces unwanted effects of the parent moieties, in particular the skin staining, which is a major issue concerning the use of dithranol.

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6%) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5%) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.

Diagnosis of clinical bovine mastitis by fine needle aspiration followed by staining and scanning electron microscopy in a Prototheca zopfii outbreak

Biopsy by fine needle aspiration together with microbiological examination and scanning electron microscopy were evaluated in diagnosis of clinical bovine mastitis in a Prototheca zopfii outbreak. Fine needle aspiration was performed in 21 mammary quarters from ten Holstein cows presenting clinical mastitis caused by P. zopfii. The algae were previously identified in the microbiological examination of milk collected from these cows. Material aspirated from these 21 mammary glands was submitted to cytological staining (Gram, Giemsa and/or Shor staining). Fine needle aspiration enabled cytological identification of the algae in these 21 mammary glands, from which P. zopfii was isolated in the milk. Simultaneously, five mammary fragments collected by fine needle aspiration from these 21 mammary glands presenting clinical mastitis were also submitted to microbiological examination. P. zopfii was also isolated from these five fragments. Scanning electron microscopy technique also identified three of these five P. zopfii strains isolated from mammary fragments collected by cytological aspiration. These results suggest that fine needle aspiration may be an alternative method for the diagnosis of clinical mastitis.

Staining methods applied to glycol methacrylate embedded tissue sections

The use of glycol methacrylate (GMA) avoids some technical artifacts, which are usually observed in paraffin-embedded sections, providing good morphological resolution. on the other hand, weak staining have been mentioned during the use of different methods in plastic sections. In the present study, changes in the histological staining procedures have been assayed during the use of staining and histochemical methods in different GMA-embedded tissues.Samples of tongue, submandibular and sublingual glands, cartilage, portions of respiratory tract and nervous ganglion were fixed in 4% formaldehyde and embedded in glycol methacrylate. The sections of tongue and nervous ganglion were stained by H&E. Picrosirius, Toluidine Blue and Sudan Black B methods were applied, respectively, for identification of collagen fibers in submandibular gland, sulfated glycosaminoglycans in cartilage (metachromasia) and myelin lipids in nervous ganglion. Periodic Acid-Schiff (PAS) method was used for detection of glycoconjugates in submandibular gland and cartilage while AB/PAS combined methods were applied for detection of mucins in the respiratory tract. In addition, a combination of Alcian Blue (AB) and Picrosirius methods was also assayed in the sublingual gland sections.The GMA-embedded tissue sections showed an optimal morphological integrity and were favorable to the staining methods employed in the present study. In the sections of tongue and nervous ganglion, a good contrast of basophilic and acidophilic structures was obtained by H&E. An intense eosinophilia was observed either in the striated muscle fibers or in the myelin sheaths in which the lipids were preserved and revealed by Sudan Black B. In the cartilage matrix, a strong metachromasia was revealed by Toluidine Blue in the negatively-charged glycosaminoglycans. In the chondrocytes, glycogen granules were intensely positive to PAS method. Extracellular glycoproteins were also PAS positive in the basal membrane and in the region occupied by the lamina externa and reticular fibers surrounding each smooth muscle cells of the blood vessels. In the epithelial cells of the respiratory tract, acid and neutral mucins were histochemically detected by AB and PAS methods, respectively. Moreover, granules containing acid and neutral mucins were revealed in purple by AB and PAS concomitantly. In the sublingual gland sections, a distinct affinity of acid mucins by AB (in turquoise-blue) and collagen fibers by Picrosirius (in red) was obtained when these methods were combined. Although some routine dyes used in paraffin sections have showed a weak stain in historesin sections, our results showed that different dyes could be applied in GMA sections if modified staining procedures were assayed. Therefore, appropriate staining contrast and, thus, detection of one or different substances in a same section can be acquired in association to the good morphological resolution provided by GMA. (C) 2003 Elsevier Ltd. All rights reserved.

Evaluation of Staining Methods for Cytologic Diagnosis of Oral Lesions

ObjectiveTo compare the efficacy of Papanicolaou, hematoxylin-eosin (H-E), Leishman and periodic acid-Schiff (PAS) staining for Cytologic diagnosis of oral lesions.Study DesignPatients from the Discipline of Stomatology, Sao Jose dos Campos Dental School, from the wards of Hospital Heliopolis and from the dentistry outpatient clinic of the University Hospital, University of São Paulo Medical School, with the following diseases, were selected: erythematous candidiasis (n = 9), pseudomembranous candidiasis (n = 10), squamous cell carcinoma In = 19), herpes simplex (n = 8), paracoccidioidomycosis In = 8) and pemphigus vulgaris (n = 1).ResultsThe different staining methods were compared regarding the quality of definition of cytoplasmic and nuclear morphologic characteristics and the identification of bacteria, fungi, inflammatory cells and secretions. Papanicolaou and H-E staining were considered better methods. In cases of fungal infections, PAS staining is useful and should be applied as a complementary method.ConclusionWithin the limitations of this study, it can be concluded that the cytologic diagnosis of oral lesions along with different staining methods is a useful tool for oral diagnosis. (Acta Cytol 2008;52:697-701)

Myxosporidiosis in intensively-reared Piaractus mesopotamicus: Histopathological diagnosis by means of Ziehl-Neelsen staining

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Simple method for culture of peripheral blood lymphocytes of Testudinidae.

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

The Staining Effect of the Hydrochloric Acid-Chromate Trioxide Solution on the Minerals of the Chalcocite-Stibnite-Galena Ternary System.

The object of this work has been to devise a method by which the different phases in the chalcocite-stibnite-galena ternary system may be identified. As the mineralogists have no precise methods for the identification of these phases, a hydrochloric acid-chromate trioxide staining solution was employed.

The 'triple contrast' method in experimental wound ballistics and backspatter analysis.

In practical forensic casework, backspatter recovered from shooters' hands can be an indicator of self-inflicted gunshot wounds to the head. In such cases, backspatter retrieved from inside the barrel indicates that the weapon found at the death scene was involved in causing the injury to the head. However, systematic research on the aspects conditioning presence, amount and specific patterns of backspatter is lacking so far. Herein, a new concept of backspatter investigation is presented, comprising staining technique, weapon and target medium: the 'triple contrast method' was developed, tested and is introduced for experimental backspatter analysis. First, mixtures of various proportions of acrylic paint for optical detection, barium sulphate for radiocontrast imaging in computed tomography and fresh human blood for PCR-based DNA profiling were generated (triple mixture) and tested for DNA quantification and short tandem repeat (STR) typing success. All tested mixtures yielded sufficient DNA that produced full STR profiles suitable for forensic identification. Then, for backspatter analysis, sealed foil bags containing the triple mixture were attached to plastic bottles filled with 10 % ballistic gelatine and covered by a 2-3-mm layer of silicone. To simulate backspatter, close contact shots were fired at these models. Endoscopy of the barrel inside revealed coloured backspatter containing typable DNA and radiographic imaging showed a contrasted bullet path in the gelatine. Cross sections of the gelatine core exhibited cracks and fissures stained by the acrylic paint facilitating wound ballistic analysis.

Rapid method to express and purify human membrane protein using the Xenopus oocyte system for functional and low-resolution structural analysis.

Progress toward elucidating the 3D structures of eukaryotic membrane proteins has been hampered by the lack of appropriate expression systems. Recent work using the Xenopus oocyte as a novel expression system for structural analysis demonstrates the capability of providing not only the significant amount of protein yields required for structural work but also the expression of eukaryotic membrane proteins in a more native and functional conformation. There is a long history using the oocyte expression system as an efficient tool for membrane transporter and channel expression in direct functional analysis, but improvements in robotic injection systems and protein yield optimization allow the rapid scalability of expressed proteins to be purified and characterized in physiologically relevant structural states. Traditional overexpression systems (yeast, bacteria, and insect cells) by comparison require chaotropic conditions over several steps for extraction, solubilization, and purification. By contrast, overexpressing within the oocyte system for subsequent negative-staining transmission electron microscopy studies provides a single system that can functionally assess and purify eukaryotic membrane proteins in fewer steps maintaining the physiological properties of the membrane protein.

Pseudo-ternary phase diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by the lipid-film hydration method

Pseudo-ternary phase diagrams of the polar lipids Quil A, cholesterol (Chol) and phosphatidylcholine (PC) in aqueous mixtures prepared by the lipid film hydration method (where dried lipid film of phospholipids and cholesterol are hydrated by an aqueous solution of Quil A) were investigated in terms of the types of particulate structures formed therein. Negative staining transmission electron microscopy and polarized light microscopy were used to characterize the colloidal and coarse dispersed particles present in the systems. Pseudo-ternary phase diagrams were established for lipid mixtures hydrated in water and in Tris buffer (pH 7.4). The effect of equilibration time was also studied with respect to systems hydrated in water where the samples were stored for 2 months at 4degreesC. Depending on the mass ratio of Quil A, Chol and PC in the systems, various colloidal particles including ISCOM matrices, liposomes, ring-like micelles and worm-like micelles were observed. Other colloidal particles were also observed as minor structures in the presence of these predominant colloids including helices, layered structures and lamellae (hexagonal pattern of ring-like micelles). In terms of the conditions which appeared to promote the formation of ISCOM matrices, the area of the phase diagrams associated with systems containing these structures increased in the order: hydrated in water/short equilibration period < hydrated in buffer/short equilibration period < hydrated in water/prolonged equilibration period. ISCOM matrices appeared to form over time from samples, which initially contained a high concentration of ring-like micelles suggesting that these colloidal structures may be precursors to ISCOM matrix formation. Helices were also frequently found in samples containing ISCOM matrices as a minor colloidal structure. Equilibration time and presence of buffer salts also promoted the formation of liposomes in systems not containing Quil A. These parameters however, did not appear to significantly affect the occurrence and predominance of other structures present in the pseudo-binary systems containing Quil A. Pseudo-ternary phase diagrams of PC, Chol and Quil A are important to identify combinations which will produce different colloidal structures, particularly ISCOM matrices, by the method of lipid film hydration. Colloidal structures comprising these three components are readily prepared by hydration of dried lipid films and may have application in vaccine delivery where the functionality of ISCOMs has clearly been demonstrated. (C) 2003 Elsevier B.V. All rights reserved.

Using intervention time series analyses to assess the effects of imperfectly identifiable natural events: A general method and example

BACKGROUND: Intervention time series analysis (ITSA) is an important method for analysing the effect of sudden events on time series data. ITSA methods are quasi-experimental in nature and the validity of modelling with these methods depends upon assumptions about the timing of the intervention and the response of the process to it. METHOD: This paper describes how to apply ITSA to analyse the impact of unplanned events on time series when the timing of the event is not accurately known, and so the problems of ITSA methods are magnified by uncertainty in the point of onset of the unplanned intervention. RESULTS: The methods are illustrated using the example of the Australian Heroin Shortage of 2001, which provided an opportunity to study the health and social consequences of an abrupt change in heroin availability in an environment of widespread harm reduction measures. CONCLUSION: Application of these methods enables valuable insights about the consequences of unplanned and poorly identified interventions while minimising the risk of spurious results.

Comparative assessment of seller’s staining test (SST) and direct fluorescent antibody test for rapid and accurate laboratory diagnosis of rabies.

Background: Rabies causes 55, 000 annual human deaths globally and about 10,000 people are exposed annually in Nigeria. Diagnosis of animal rabies in most African countries has been by direct microscopic examination. In Nigeria, the Seller’s stain test (SST) was employed until 2009. Before then, both SST and dFAT were used concurrently until the dFAT became the only standard method. Objective: This study was designed to assess the sensitivity and specificity of the SST in relation to the ‘gold standard’ dFAT in diagnosis of rabies in Nigeria. Methods: A total of 88 animal specimens submitted to the Rabies National Reference Laboratory, Nigeria were routinely tested for rabies by SST and dFAT. Results: Overall, 65.9% of the specimens were positive for rabies by SST, while 81.8% were positive by dFAT. The sensitivity of SST in relation to the gold standard dFAT was 81.0% (95% CIs; 69.7% - 88.6%), while the specificity was 100% (95% CIs; 76% - 100%). Conclusion: The relatively low sensitivity of the SST observed in this study calls for its replacement with the dFAT for accurate diagnosis of rabies and timely decisions on administration of PEP to prevent untimely deaths of exposed humans.