Modification of the bi-directional sliding movement of actin filaments along native thick filaments isolated from a clam

A reliable viability assay for Giardia is required for the development of disinfection process design criteria and pathogen monitoring by water treatment utilities. Surveys of single-staining nucleic acid dyes (stain dead parasites only), and double-staining vital dye kits from Molecular Probes (stain live and dead parasites) were conducted to assess the viability of untreated, heat-killed, and chemically inactivated Giardia muris cysts. Nucleic acid staining results were compared to those of in vitro excystation and animal infectivity. Nucleic acid stain, designated as SYTO-9, was considered the best among the single-staining dyes for its ability to stain dead cysts brightly and its relatively slow decay rate of visible light emission following DNA binding. SYTO-9 staining was correlated to animal infectivity. A Live/Dead BacLight was found to be the better of 2 double-staining viability kits tested. Logarithmic survival ratios based on SYTO-9 and Live/Dead BacLight were compared to excystation and infectivity results for G. muris cysts exposed to ozone or free chlorine. The results indicate that SYTO-9 and Live/Dead BacLight staining is stable following treatment of cysts with chemical disinfectants.     

The colorectal adenoma-carcinoma sequence: the limits between polypectomy and intestinal resection

A hemocytometer-based trypan blue dye exclusion cell quantitation and viability assay was compared with a similar assay using simultaneous fluorometric staining with fluorescein diacetate and propidium iodide. Viable and nonviable cell densities were measured, and culture viability was calculated both during the normal growth cycle of a murine hybridoma and in response to the application of millimolar concentrations of either tert-butyl hydroperoxide or ferrous iron. During the early phase of rapid hybridoma cell growth, assay-based differences in viable cell density were not significant. As the culture aged, the trypan blue dye exclusion assay significantly overestimated cell viability, thereby underestimating nonviable cell density and yielding an erroneous estimation of the overall viability of the culture. Because of its lack of ambiguity in the identification of stained, nonviable cells and its resulting increased accuracy in the estimation of culture viability, the fluorometric assay was considered a better choice for the evaluation of cell viability.     

Distribution and characterization of proteoglycans associated with exfoliation material

PURPOSE: To examine the distribution of proteoglycans in the exfoliation materials in order to investigate the nature of the materials. METHODS: The anterior parts of two eyes with exfoliation syndrome were examined by electron microscopy after staining with cupromeronic blue (cmb). Some specimens were treated with enzymes and/or nitrous acid prior to staining. The effects of the enzymes were evaluated statistically by counting the density of the cmb-positive filaments in the exfoliation materials, using a computer. One eye with exfoliation syndrome stained with alcian blue was observed with light microscopy. RESULTS: Exfoliation materials were observed along the epithelial cells of the iris and ciliary body, and in the trabecular meshwork and zonules. In tissue specimens treated with cmb, electron-dense filaments were seen associated with the exfoliation materials. Microfibrils in the trabecular meshwork and iris, and zonular fibrils themselves were free of any filament staining, while the exfoliation materials located closely to the fibrils contained the electron-dense filaments. In the tissue specimens treated with chondroitinase AC, chondroitinase B, chondroitinase ABC or nitrous acid before cmb staining, the amount of the filament associated with exfoliation materials decreased in comparison to the controls. Digestion with keratinase did not demonstrate any significant changes in staining. A combination treatment with chondroitinase ABC and nitrous acid eliminated almost all filaments associated with the exfoliation materials. In the eye stained with alcian blue, the zonules that did not stain for the dye demonstrated an accumulation of exfoliation materials that stained strongly for alcian blue. CONCLUSIONS: Exfoliation materials contain chondroitin sulfate, dermatan sulfate, heparan sulfate proteoglycans. Depositions of proteoglycans on the microfibrils may be closely associated with the formation of exfoliation materials.     

Use of a simple light absorbance assay to measure lymphocyte proliferation

The proliferative response of human lymphocytes to stimuli such as foreign histocompatibility antigens or mitogens is generally assessed by measuring the amount of tritiated thymidine which the cells incorporated in culture. In this paper, the possibility of assessing lymphocyte proliferation and viability by an empirical assay, using measurement of light absorbance on a ELISA reader in the yellow wave length (450 nm/air-550 nm/air), has been studied. The correlation of these measurements with a colormetric viability assay using MTS/PMS, with tritiated thymidine incorporation and with trypan blue exclusion viability counting, was determined. The results showed that the light absorbance assay correlated well with cell proliferation during 48-120 hours culture period and with cell viability after a 72 hour period. The MTS/PMS colormetric assay as well as trypan blue exclusion cell counting confirmed that the light absorbance assay was not merely caused by dead cells. This data confirm that the light absorbance assay is sufficiently sensitive to low levels of proliferation to allow detection of such responses at least as effectively as thymidine incorporation. The light absorbance assay procedure avoids the expense, time and hazards associated with scintillation counting, and is simple to perform without the necessity for reagents and preparative steps required by other assays.     

''We have to put up with it--don''t we?'' The experience of being the registered nurse on duty, managing a violent incident involving an elderly patient: a phenomenological study

Cyclospora cayetanensis, a newly emerging coccidian protozoa is world-wide in distribution. In the present study, different concentrations and staining techniques were used for identification of Cyclospora. Formol-ether sedimentation and Sheather's sugar flotation were used as concentration techniques and the different stains used were: the modified Ziehl-Neelsen, Giemsa, safranin-methylene blue, modifications of trichrome stain, calcoflour white and finally phenol-auramine. The safranin stain was the best, as it stained all the oocysts of Cyclospora uniformly, besides being rapid and easily applicable in the laboratories. Phenol-auramine stained the oocysts well, where both the wall and internal contents fluoresced brightly. With the calcoflour white stain, only the wall of oocysts took that fluorescent stain. The modified Ziehl-Neelsen stained some of the oocysts well, yet great variability in the staining pattern was noticed. Cyclospora oocysts were not efficiently stained with either trichrome modifications or Giemsa stains.     

Evaluation of preparation, staining and microscopic techniques for counting incremental lines in cementum of human teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.     

A packaging system for SV40 vectors without viral coding sequences

We studied the incidence of regurgitation in 100 patients undergoing elective gynecological laparoscopies under general anesthesia with intermittent positive pressure ventilation using a laryngeal mask airway (LMA). Patients ingested methylene blue capsules 10-15 min before induction of anesthesia. After induction and insertion of an LMA using the recommended insertion technique, a fiberoptic examination of the larynx was made for traces of dye and to site a pH probe in the bowl of the LMA for continuous monitoring. LMA insertion was successful in all patients within two attempts (95 at first attempt). Fiberoptic examination revealed the vocal cords or cords and posterior or anterior epiglottis in 96 and no trace of dye in 99 patients. One patient regurgitated dye immediately after induction, and the stain was seen on the LMA after removal. The remaining 99 LMAs were not stained. Thirty patients were randomly selected for fiberoptic examination of the laryngopharynx before neuromuscular block was antagonized. Methylene blue staining did not occur in any of these patients. In 91 patients with complete pH data, regurgitation (pH < 4.0) did not occur. The 95% confidence limit for a true probability of regurgitation in this study is 0.041 or a true rate of regurgitation of less than 4.1%. A larger study would be required to possibly demonstrate a lower incidence of regurgitation. This study confirms the clinical impression that the incidence of regurgitation during laparoscopies with a LMA is extremely low.     

Methylene blue dyeing of cellular nuclei during salpingoscopy, a new in-vivo method to evaluate vitality of tubal epithelium

The Fallopian tube can be damaged by different noxious substances that may change cellular ultrastructure and function. Alteration of the cell membrane allows the passage of certain aniline dyes, which can stain the nucleus. A total of 310 Fallopian tubes from 163 patients who underwent a surgical or diagnostic laparoscopy during fertility studies was analysed by salpingoscopy. Cellular nuclei were stained by injection of 20 ml of a 10% solution of methylene blue in saline solution (NaCl 10%) through the cervical cannula prior to salpingoscopy. Evaluation of nuclear staining with methylene blue, adhesions, vascular alterations, and the flattening of folds in relation to pregnancy outcome was undertaken. Quantification of salpingoscopic findings was carried out according to a score. Flattening of folds and vascular alterations showed no difference in the pregnant and non-pregnant groups. On the other hand, adhesions and nuclear dyeing were significantly greater in the non-pregnant group (adhesions 13.6 versus 26.8%, P < 0.004, and nuclear dyeing: 25 versus 41.7%, P < 0.009, pregnant versus non-pregnant). Methylene blue dye is a new tool to evaluate in vivo cyto-histological tubal damage, and is a useful and simple method to provide a prognosis of salpingean function.     

Blunt impact causes changes in bone and cartilage in a regularly exercised animal model

Confocal laser scanning microscopy combined with a vital stain has been used to study apoptosis in organogenesis-stage mouse embryos. In order to achieve optical sectioning through embryos, it was necessary to use low power objectives and to prepare the sample appropriately. Mouse embryos were harvested on gestation day 8 or 9 and stained with the vital lysosomal dye, LysoTracker Red. Following incubation in the stain, embryos were fixed in 2% paraformaldehyde overnight, dehydrated in a graded methanol series, and cleared in benzyl alcohol/benzyl benzoate. The resulting embryo is almost transparent and retains specific LysoTracker Red staining. The entire embryo can be optically sectioned and reconstructed in three dimensions to reveal areas of dye staining. To test this approach, the chemotherapeutic drug hydroxyurea was added to day 8 embryos in vitro to induce apoptosis. Our results demonstrated specific regions undergoing programmed cell death in normal development and increased apoptosis in embryos exposed to hydroxyurea. The observed patterns of LysoTracker Red staining correlate well with previous studies of cell death using other lysosomotropic dyes such as Nile blue sulfate, acridine orange, or neutral red. LysoTracker Red has the advantages of being aldehyde-fixable and highly fluorescent (bleaching was not observed even after multiple scans). This procedure allows for the optical imaging of whole day 9 (approximately 22 somites) embryos that were greater than 500 microns thick in the Z-axis.     

Effectiveness of SYTOX Green stain for bacterial viability assessment

The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations of Escherichia coli and Salmonella typhimurium. This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes. Its application to natural samples should be considered with caution.     

Macrophages as effector cells of protective immunity in murine schistosomiasis. III. Loss of susceptibility to macrophage-mediated killing during maturation of S. mansoni schistosomula from the skin to the lung stage

Newly transformed skin-stage and lung-stage schistosomula were compared in terms of their susceptibility to killing mediated by activated mouse macrophages in vitro. Although skin-stage schistosomula were readily killed by macrophages activated as a consequence of either BCG or Schistosoma mansoni infection and used either as cell monolayers or in suspension, lung-stage larvae appeared to be totally resistant to this effector mechanism and survived normally when reinjected into mice. Resistance of schistosomula to in vitro damage by macrophages was evident as early as 18 hr after host infection and was complete in worms recovered at 42 hr. The insusceptibility of lung-stage larvae is apparently not due to a defect in effector cell-target contact, because the induction of extensive macrophage adherence to the worms by the addition of anti-mouse red blood cell antisera to the cultures had no effect on parasite viability. These findings provide additional support for the concept that schistosomula during their development to the lung stage undergo a generalized change affecting their susceptibility to a variety of different immunologic effector mechanisms.     

Reversible myocardial dysfunction in a patient with alcoholic ketoacidosis: a role for hypophosphatemia

In order to easily assess growth and destruction of Toxoplasma gondii in vitro, this report describes two double staining assays that both visualize live and dead organisms: acridine orange--ethidium bromide (AO-EB) and bisbenzimide (Hoechst 33258)--propidium iodide (B-PI). EB and PI were chosen for dead organisms staining while AO and B stain viable organisms. Thus, both double staining assays seem more informative than Giemsa staining or indirect immunofluorescence. They offer methods to study internal structure of the parasite as well as information on host-parasite relationships. Moreover, detection in culture are sensitive, easier, and less time consuming than previous methods. So, they should to be useful in strains behaviour analysis.     

Cytomorphology of tyrosine-rich crystalloids in fine needle aspirates of salivary gland adenomas

OBJECTIVE: To evaluate the presence of tyrosine-rich crystalloids (TRC) in fine needle aspiration (FNA) specimens of pleomorphic adenomas of salivary gland. STUDY DESIGN: FNA specimens from 12 patients were reviewed, and the percentage of cases showing TRC was established. The staining properties of the TRC were evaluated as well as spontaneous fluorescence under ultraviolet (UV) light. RESULTS: Of the 12 pleomorphic adenomas, 4 showed TRC (30%) in the smears. Among the eight cytologically negative cases there were two that showed a few TRCs on histology. All positive cases were from African American patients. TRC stained weakly with Papanicolaou stain. TRC were deep blue with Diff-Quik. They fluoresced under UV light. CONCLUSION: TRC could be detected in FNA specimens. They were best seen under UV light. The Papanicolaou technique stained TRC very pale, making them difficult to see. Diff-Quik stained TRC dark blue, mimicking deposits of dye. The amount of TRC in histology paralleled the detection rate in cytology.     

The eye of the tiger sign in cortical-basal ganglionic degeneration

Expression of Galalpha(1-3)Gal on endothelium has been implicated in the rejection of porcine xenografts. The aim of this study was to determine whether expression of Galalpha(1-3)Gal on pig islets varies between pigs aged 5, 12 and 24 weeks, and to investigate whether it is expressed on islets isolated by collagenase digestion or islets maintained in tissue culture. Samples of pancreas were obtained from pigs aged 5, 12 and 24 weeks. Islets were isolated by manual collagenase digestion and density gradient separation. Samples were taken immediately after isolation or after maintenance in tissue culture. Pancreas and islet samples were processed, sectioned and stained with the lectin BS1-B4 (which binds to Galalpha(1-3)Gal residues), and anti-insulin antibody using a double staining technique. There was no significant difference in the staining patterns to sections of pancreas obtained from 5, 12 and 24 week old pigs. Vascular endothelium, connective tissue and the luminal surface of duct epithelial cells stained with BS1-B4 in all sections; endocrine and exocrine cells did not stain. Preliminary experiments showed that lectin staining to isolated islets was inconsistent between preparations, but expression did not appear to differ significantly between ages: lectin staining of some beta-cells was evident in the majority of freshly isolated preparations, but was not detectable on beta-cells following tissue culture. In conclusion, expression of Galalpha(1-3)Gal did not differ significantly in pancreata from 5, 12 and 24 week old pigs. Preliminary experiments showed that Galalpha(1-3)Gal was expressed by beta-cells immediately following isolation, but not after maintenance in culture.     

Kinetics of white blood cell staining by intravascular administration of rhodamine 6G

Rhodamine 6G is a vital dye accumulating in the mitochondria of cells. It is used in intravital fluorescence microscopy for contrast enhancement of white blood cells (WBC), enabling visualization of WBC in the microvasculature even at high center flow velocity. The aim of this study was to examine the kinetics of WBC staining after intravascular administration of rhodamine 6G in Lewis rats, Syrian golden hamsters and BALB/c mice. For this purpose, WBC were isolated from whole blood and the percentage of cells stained positively as well as their fluorescence intensity were measured by flow cytometry 5, 15, 30 and 60 min after dye administration. Injection of 0.06-0.2 mg/kg body weight of rhodamine 6G resulted in staining practically all granulocytes and monocytes over the entire observation period of 60 min. Fluorescence intensity of WBC was adequate to be detected in an experimental setup for intravital fluorescence microscopy in the hamster dorsal skinfold chamber. The degree of WBC staining was different in the species studied, yielding a higher percentage of stained lymphocytes in rats than in mice and hamsters. Staining of lymphocytes declined within 60 min after rhodamine application, the loss of fluorescent label being most pronounced in hamster cells. After 15-30 min, relative fluorescence intensity of stained lymphocytes had decreased considerably, indicating the need for reinjection of the dye or limiting microscopic analysis to approximately 15 min after rhodamine 6G administration. While the intravascular injection of rhodamine 6G results in adequate staining of granulocytes and monocytes, only a fraction of lymphoid cells are stained.     

Molecular analysis of P16(Ink4)/CDKN2 and P15(INK4B)/MTS2 genes in primary human testicular germ cell tumors

Seven blue nucleic acid dyes from Molecular Probes Inc. (SYTO-9, SYTO-11, SYTO-13, SYTO-16, SYTO-BC, SYBR-I and SYBR-II) were compared with the DAPI (4',6-diamidino-2-phenylindole) method for flow cytometric enumeration of live and fixed bacteria in aquatic systems. It was shown that SYBR-II and SYTO-9 are the most appropriate dyes for bacterial enumeration in nonsaline waters and can be applied to both live and dead bacteria. The fluorescence signal/noise ratio was improved when SYTO-9 was used to stain living bacteria in nonsaline waters. Inversely, SYBR-II is more appropriate than SYTO dyes for bacterial enumeration of unfixed and fixed seawater samples.     

Comparative study of classical, colorimetric and immunologic staining methods for the assessment of tumor cell viability

The trypan blue exclusion test, the MTT test and an immunostaining test for apoptosis were performed before and after incubation of SW620 human colonic carcinoma cells with different cytotoxic agents (CTAs) in order to assess tumor cell viability and CTA efficacy in vitro. A modified MTT test using light microscopy was also performed. A good correlation was found between the trypan blue assay and the MTT test, as determined by spectrophotometry. There was no 'overestimation' of cell viability as measured by the trypan blue test. The monitoring of formazan formation by light microscopy was feasible, but not very reliable since it did not show a good correlation with findings determined by spectrophotometry. The apoptosis test failed to show good correlation with other tests. Distilled water had no relevant cytotoxic effect, while chlorhexidine cetrimide (HAC 3.5%), chloramine 0.5% and polyvinylpyrrolidone iodine (PVP-I) > or = 0.05% damaged a large majority of cells. PVP-I at a concentration of > or = 5% was found to be the most effective CTA.     

A histochemical examination of the staining of kainate-induced neuronal degeneration by anionic dyes

Anionic dyes, notably acid fuchsine, strongly stain the nuclei and cytoplasm of neurons severely damaged by injury or disease. We provide detailed instructions for staining nervous tissue with toluidine blue and acid fuchsine for optimal demonstration of injured neurons. Degeneration was induced in the hippocampus of the mouse by systemic administration of kainic acid, and the resulting acidophilia was investigated using paraffin sections of the Carnoy- or Bouin-fixed brains. The affected cells were bright red with the toluidine blue-acid fuchsine sequence. Their nuclei were stainable also with alkaline Biebrich scarlet and with the 1,2-naphthoquinone-4-sulfonic acid-Ba(OH)2 method; all staining was blocked by benzil but was relatively refractory to deamination by HNO2. These properties indicated an arginine-rich protein. The nuclei were strongly acidophilic in the presence of a high concentration of DNA (strong Feulgen reaction), and acidophilia could not be induced in normal neuronal nuclei by chemical extraction of nucleic acids. The cytoplasmic acidophilia of degenerating hippocampal neurons was due to a protein rich in lysine (extinguished by alkalinity, easily prevented by deamination, and unaffected by benzil). Stainable RNA was absent from the perikarya of the affected cells, but normal neuronal cytoplasm did not become acidophilic after extraction of nucleic acids. We suggest that kainate-induced cell death is preceded by increased production of basic proteins, which become concentrated in the nucleus and perikaryon. Groups of small, darkly staining neurons were seen in the cerebral cortex in control and kainate-treated mice. These shrunken cells were purple with the toluidine blue-acid fuchsine stain, and were attributed to local injury incurred during removal of the unfixed brain.     

Road traffic accidents in Lusaka and blood alcohol

The moment of the conduction block was determined in the 10th sympathetic ganglion of the frog with the help of electrophysiological control of the total synaptic activity. After supravital staining with methylene blue the morphology of the synaptic pericellular apparatus was studied. It has been shown that most of synapses are in the intermediate phase (after Majorov) and have swollen, loosing their dye synaptic bouttons against the background of non-stained bodies of nerve cells. A supposition is made that due to the presence of single synapses with less pronounced changes under complete block of conducting it can not be excluded that the block may develop in the transition of the synapse from the phase of stained pericellular apparatus into an intermediate phase.     

The modified Steiner stain: a new use for an old stain? Staining cytomegalovirus-infected cells in gastrointestinal biopsies

The modified Steiner stain is a non-specific silver stain for identifying bacteria in formalin-fixed, paraffin-embedded tissues. The principle behind its use is that bacteria are first sensitized using uranyl nitrate solution, making them able to precipitate silver from a silver nitrate solution. It is used routinely for staining gastric biopsies to identify Helicobacter pylori. Upon staining a gastric biopsy from a patient with acquired immunodeficiency syndrome (AIDS) and cytomegalovirus gastritis, we recognized that this technique also stains the viral inclusions of cytomegalovirus-infected cells. We then proceeded to stain 43 consecutive cytomegalovirus-positive gastrointestinal biopsies from 33 immunocompromised patients based on positive cytomegalovirus immunohistochemistry (DAKO-cytomegalovirus monoclonal antibody, clones DDG9 and CCH2). We also stained eight cytomegalovirus-infected, non-gastrointestinal tissues, including lung, adrenal gland, ovary, skin and neural tissue, to ensure that the stain was staining the cytomegalovirus-infected cells and not argyrophilic or argentaffin neuroendocrine cells of the gastrointestinal tract. In 40 of the 43 cytomegalovirus-infected gastrointestinal biopsies, we saw positive staining with the modified Steiner stain (93% sensitivity). The cytomegalovirus-infected, non-gastrointestinal tissues all stained positively with the modified Steiner stain. Because the modified Steiner stain is frequently used to identify Helicobacter pylori in gastric biopsies, we propose that it be studied further for possible use either as a screen or as a confirmatory tool, or both, for cytomegalovirus inclusions in gastrointestinal biopsies.