甲苯胺蓝快速显示肥大细胞的制作方法探讨

目的为了使肥大细胞在标本制作过程中胞质内的颗粒不被水溶性液体所溶解,且使其标本上的颗粒颜色呈现异染性。方法选用20g以上的健康小白鼠为材料,用70%乙醇液为甲苯胺蓝染料的溶剂,一步完成标本的固定与染色操作。结果所显示的肥大细胞轮廓完整,与底衬背景对比鲜明,而且胞质内紫红色的嗜碱性颗粒粗大清晰、色彩鲜艳。结论在简化了操作程序的基础上,很好地保留了肥大细胞颗粒,又能使其颗粒染色呈现异染性。     

Microwaveable Toluidine Blue Stain for Surface Staining of Undecalcified Bone Sections

Abstract

Toluidine blue stain has been widely used for staining undecalcified bone sections, but previous techniques required removal of embedding plastic or surface etching with both alcohol and acid solutions. In this study, solutions of toluidine blue at different pHs were used to stain unetched and acidetched sections. A group of the test staining solutions were heated in an effort to enhance section staining. Acid-etching prior to staining was found to be necessary to achieve bone matrix staining, but methanol etching could be eliminated if staining solutions were heated to 55–60°C. Sections stained with the heated solutions buffered to pH 7.0 or 8.0 had optimum morphology. (The J Histotechnol 17:357, 1994)     

Staining behavior and applicability of spectrally pure Capriblue GN, Stella Blue, Oxonin and Punky Blue

The staining properties of 4 spectral pure derivates of Phenoxazin--Capriblue GN, Stella Blue, Oxonin, and Punky Blue--were investigated using human and animal tissues. Punky Blue and Stella Blue are newly synthesized derivates. A simplified staining technique was developed based on conventional fixing and mounting methods of histological materials. Punky Blue is a particularly contrasting metachromatic nucleus stain, and produces at pH = 4 and a staining period of 5 min a differentiated cell picture, the colours of which are comparable to conventional stains. The Oxonin-stain produces good contrasts after 2 min. Capriblue GN is only limited use as one-component tissue stain, due of its low metachromatic characteristics and Stella Blue, on its own, does not provide sufficient contract. The influence of electrolytes and hydrogen ions on the staining mechanisms will be discussed. An increase of hydrogen ions is used to control the selective staining of amphoteric biopolymers. The possible binding mechanisms of stains used are discussed in the light of their staining properties.     

Rapid Flagella Stain

Leifson stain was modified to produce rapid staining of bacterial flagella on untreated microscope slides. The procedure was reliable when tested against a variety of motile and nonmotile bacteria.     

Functions of Bacterial Flagella

Abstract

Many bacterial species are motile by means of flagella. The structure and implantation of flagella seems related to the specific environments the cells live in. In some cases, the bacteria even adapt their flagellation pattern in response to the environmental conditions they encounter. Swarming cell differentiation is a remarkable example of this phenomenon. Flagella seem to have more functions than providing motility alone. For many pathogenic species, studies have been performed on the contribution of flagella to the virulence, but the result is not clear in all cases. Flagella are generally accepted as being important virulence factors, and expression and repression of flagellation and virulence have in several cases been shown to be linked. Providing motility is always an important feature of flagella of pathogenic bacteria, but adhesive and other properties also have been attributed to these flagella. In nonpathogenic bacterial colonization, flagella are important locomotive and adhesive organelles as well. In several cases where competition between several bacterial species exists, motility by means of flagella is shown to provide a specific advantage for a bacterium. This review gives an overview of studies that have been performed on the significance of flagellation in a wide variety of processes where flagellated bacteria are involved.     

A Simplified Leifson Flagella Stain

Flagella of bacteria taken directly from 24- and 48-h blood agar plates can be stained by using a simple, reliable procedure.     

Flagella are virulence determinants of Burkholderia pseudomallei

Burkholderia pseudomallei, a facultatively intracellular pathogen, is a flagellated and motile gram-negative bacterium and is the causative agent of melioidosis in humans. Flagella are commonly recognized as important virulence determinants expressed by bacterial pathogens since the motility phenotype imparted by these organelles often correlates with the ability of an organism to cause disease. We used a virulent isolate of B. pseudomallei, KHW, to construct an isogenic deletion mutant with a mutation in the flagellin gene (fliC) by gene replacement transposon mutagenesis. The KHWDeltafliCKm mutant was aflagellate and nonmotile in semisolid agar. The isogenic KHWDeltafliCKm mutant was not impaired in terms of the ability to invade and replicate in cultured human lung cells compared with the wild type. It was also equally virulent in slow-killing assays involving Caenorhabditis elegans, but it was avirulent during intranasal infection of BALB/c mice. Very few bacteria, if any, were isolated from the lungs and spleens of KHWDeltafliCKm-infected mice. In contrast, the bacterial loads in the lungs and spleens were similar in mice infected with KHW and in mice infected with the complemented mutant, KHWDeltafliCKm/pUCP28TfliC. Unlike the Syrian hamster or diabetic rat models of infection, the B. pseudomallei flagellin was also a virulence factor during intraperitoneal infection of BALB/c mice. In this study, all animals infected with KHWDeltafliCKm remained healthy and did not succumb to disease regardless of the route of infection. The flagellum is therefore an important and necessary virulence determinant of B. pseudomallei during intranasal and intraperitoneal infection of mice.     

Night sleep in patients with vegetative state

Polysomnographic recording of night sleep was carried out in 15 patients with the diagnosis vegetative state (syn. unresponsive wakefulness syndrome). Sleep scoring was performed by three raters, and confirmed by means of a spectral power analysis of the electroencephalogram, electrooculogram and electromyogram. All patients but one exhibited at least some signs of sleep. In particular, sleep stage N1 was found in 13 patients, N2 in 14 patients, N3 in nine patients, and rapid eye movement sleep in 10 patients. Three patients exhibited all phenomena characteristic for normal sleep, including spindles and rapid eye movements. However, in all but one patient, sleep patterns were severely disturbed as compared with normative data. All patients had frequent and long periods of wakefulness during the night. In some apparent rapid eye movement sleep episodes, no eye movements were recorded. Sleep spindles were detected in five patients only, and their density was very low. We conclude that the majority of vegetative state patients retain some important circadian changes. Further studies are necessary to disentangle multiple factors potentially affecting sleep pattern of vegetative state patients.     

Flagella are a positive predictor for virulence inLegionella

Pathogenesis of Legionnaires» disease is strictly related to the ability of the legionellae to infect phagocytic cells, yet surface markers of virulence inLegionellaisolates are currently unknown. Rabbit antibodies raised against purified flagella ofLegionella pneumophilaserogroup 1 recognized a total of 24 of 30 laboratory-maintained isolates ofL. pneumophilaserogroups 1–15 and 16 of 24 otherLegionellaspecies tested by rapid immunoblot and indirect immunofluorescence assay. All isolates possessing flagella detectable with these anti-flagella antibodies, regardless of species, were capable of infectingHartmannella vermiformis. Isolates lacking immunologic cross-reactivity were shown to lack purifiable flagella. The majority of aflagellate isolates were not motile and failed to multiply intracellularly in co-culture withHartmannella vermiformis. Some isolates characterized as aflagellate when harvested from BCYE agar were able to multiply in amoebae, and flagella were subsequently detectable by immunologic methods. These data suggest that lack of immunologic recognition of flagella in laboratory-maintained isolates ofLegionellais due to their attenuation and a corresponding loss of expression of flagella. More importantly, the presence of flagella can serve as a positive predictive marker for strain virulence and is useful in determining the virulence status ofLegionellaisolates.     

Staining of cellular mitochondria with LDS-751.

We have found the dye LDS-751 to bind almost exclusively to mitochondria when incubated with viable, nucleated cells. Treatment of cells with the nuclear stain acridine orange and LDS-751 revealed little colocalization when the cells were examined by confocal microscopy. Staining with the dye rhodamine 123, which is known to bind polarized mitochondria, was virtually identical to the pattern observed with LDS-751. This staining pattern was observed to be consistent over a range of 0.02-20 microg/ml LDS-751 and was consistent between both fibroblasts and monocytes. Depolarization of mitochondria with the mitochondrial depolarizing agents phenyl arsine oxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) dramatically reduced both LDS-751 staining, and rhodamine 123 fluorescence. Taken together, these results suggest that LDS-751 is excluded from the nucleus and binds the polarized membranes of mitochondria. Given this, interpretation of LDS-751 fluorescence as being indicative of nuclear status, as is commonly done to discriminate between leukocytes and erythrocytes, is unwarranted and may lead to erroneous conclusions if mitochondria become depolarized upon processing.