Use Of Vital Staining Research Articles

An Improved Evans Blue Staining Method for Consistent, Accurate Assessment of Plasmodiophora brassicae Resting Spore Viability.

Clubroot caused by Plasmodiophora brassicae is an important disease of brassica crops. The use of vital stains to determine the viability of P. brassicae resting spores can provide useful information regarding spore longevity, inoculum potential, or the efficacy of antimicrobial treatments. Evans blue is one example of a vital stain that has been reported to differentially stain viable and nonviable resting spores. Some previously published protocols using Evans blue to stain P. brassicae resting spores have not provided accurate or consistent results. In this study, we modified the Evans blue method by increasing the staining time to 8 h or more and evaluated P. brassicae resting spores after heat treatment at various combinations of temperature and time. Extending staining times significantly increased the numbers of stained resting spores up to 7 h, after which the numbers of stained spores did not change significantly (R2 = 96.88; P ≤ 0.001). The accuracy of the modified method to discriminate viable and nonviable spores was evaluated in repeated experiments and by comparing the staining data with those derived from inoculation assays and propidium monoazide quantitative PCR (qPCR). The results demonstrated that the modified Evans blue staining method improved the accuracy and consistency of measurement of P. brassicae resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores (R2 = 99.84; P ≤ 0.001) and confirmed in canola infection bioassays.

PM 7/28 (2) Synchytrium endobioticum

Specific scopeThis Standard describes a diagnostic protocol for Synchytrium endobioticum.This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.Specific approval and amendmentApproved in 2003‐09. Revision approved in 2017‐06.

An estimate of the percentage of non-predatory dead variability in coastal zooplankton of the southern Humboldt Current System

Non-predatory dead variability in zooplankton remains poorly quantified worldwide. Here, we make the first estimation of the percentage of dead organisms in coastal zooplankton communities in the Humboldt Current System (HCS) under in situ conditions. The study was conducted in four coastal sites of the southern HCS (between 36 and 37°S) over a period of one year. Percentages of dead organisms were based on the classification as live or dead of 158,220 holoplankton and 17,591 meroplankton individuals using neutral red staining technique. The percentage of dead organisms in total-zooplankton was between 4.3% in Coronel Bay (summer) and 76.9% in Llico (autumn). The percentage of dead total-holoplankton varied from 4.2% (Itata River Mouth; autumn) to 77.6% (Llico; autumn), while the percentage of dead total-meroplankton ranged from 1.5% to 56.8% in Coronel Bay and Coliumo Bay, respectively. The most abundant taxa analyzed were the copepods Acartia sp., Paracalanus sp., Calanoides sp., Cladocera, Polychaeta, and the eggs of anchoveta Engraulis ringens. Among these taxa, there was a high degree of interspecific variability in the estimation of the dead organisms. The Pearson correlation shows significant relationships between maximum temperature, and minimum salinity, with the percentage of dead individuals of Acartia sp. and Paracalanus sp. Environmental factors explaining those relationships were: the El Niño 2015–2016 event, and freshwater river runoff. The use of vital staining to estimate non-predatory death for total-zooplankton and selected sentinel species is a promising tool to establish baselines to evaluate natural perturbations (e.g. ENSO), and anthropogenic alterations in coastal pelagic ecosystems.

Study of Apoptosis by DNA Fragmentation and Phosphatidylserine Externalization, and of Meiotic Segregation in Ejaculated Sperm of Chromosomal Translocation Carrier Patients

Study of Apoptosis by DNA Fragmentation and Phosphatidylserine Externalization, and of Meiotic Segregation in Ejaculated Sperm of Chromosomal Translocation Carrier Patients

Use of Methylene Blue for Detection of Specialized Intestinal Metaplasia in GERD Patients Presenting for Screening Upper Endoscopy

The ability of randomly obtained biopsy specimens to identify intestinal metaplasia in the distal esophagus is low. Use of vital staining has been suggested, as stains are taken up by areas histologically identified as specialized intestinal metaplasia (SIM). This study evaluated the role of methylene blue (MB) staining for identification of SIM in GERD patients undergoing a screening endoscopy. Chromoendoscopy of the distal esophagus using 1% MB was performed on 52 GERD patients presenting for their first endoscopy. Biopsies were obtained from areas that were stained darkly, stained lightly, unstained, or macroscopically abnormal. In patients with no MB staining, four-quadrant biopsy of the distal esophagus was performed. Twenty-seven patients (52%) showed staining with MB, while 25 patients did not. Two hundred sixty-six biopsies were obtained. SIM was detected in 11 (21%) subjects (SIM+) but not in the remaining 41 (SIM-). One hundred sixty-five biopsies were unstained (25 SIM+, 140 SIM-) and 101 were stained (12 SIM+, 89 SIM-). The per-biopsy sensitivity and specificity of MB for detection of SIM were 32.4 and 85%, while the per-patient sensitivity and specificity were 63.3 and 51.2%. MB staining for detection of SIM in GERD patients without a macroscopic appearance suggestive of a columnar-lined esophagus is a poor screening tool for SIM.

Synchytrium endobioticum

<i>Synchytrium endobioticum</i>

‘Now you see it…now you don't.’ Carmoisine vital dye facilitates complete removal of cutaneous neoplasia by intraoperative visual enhancement

Patients presenting for excision of squamous cell carcinomata, including Bowen's disease, in sun-damaged skin often present with poorly defined, morphoeic or multifocal neoplasms, the extent of which can be difficult to identify intraoperatively. Use of vital staining has been commonly used to aid identification and appropriate excision of squamous lesions of the oral cavity and upper aerodigestive tract but has not been readily adopted for cutaneous lesions. We report a case of a morphoeic squamous cell neoplasm of the web space and fingers to illustrate the merits of vital staining cutaneous squamous neoplasms with the simple dye Carmoisine E122. This assists with intraoperative tumour identification and facilitates adequate oncological resection and appropriately planned reconstruction. By comparison with other methods such as Mohs, it is a simple, cheap, and rapid aid that may be used by surgeons of all grades to improve identification of the extent of the neoplasm, without special equipment.

OR 4 Use of vital stains in evaluation of bacterial viability in infected dentin

OR 4 Use of vital stains in evaluation of bacterial viability in infected dentin

The application of toluidine blue as a diagnostic adjunct in the detection of epithelial dysplasia

The detection of premalignant lesions of the oral mucosa allows for treatment that may be sufficiently early to prevent their progression to invasive carcinoma. Although a number of techniques for supplementing clinical examination have been described, on the whole they rely on laboratory-based investigations with variable false-negative rates. Toluidine blue vital staining, on the other hand, has been advocated as a simple, inexpensive, and sensitive chairside test. Most of the clinical series investigating its efficacy, however, have relied on clinically abnormal mucosa or tissue that stained. For the purpose of quantifying the true sensitivity of toluidine blue, the present study involved the examination of clinically normal mucosa as well as abnormal tissue and compared it with the presence or absence of staining. With this technique the false-negative rates of 42% and 58% obtained for carcinoma-in-situ and mild-to-moderate dysplasia, respectively, suggest restricting the use of vital staining to selective cases.

Contribution of interdigitation to the occlusal development of the dentition in Macaca fascicularis.

The contribution of interdigitation to the development of the dentition of juvenile Macaca fascicularis was studied on a series of dental casts and at the histological level by the use of vital staining. Fourteen laboratory-born monkeys were allocated to a control group (n = 7) or an experimental group (n = 7). They were followed from 31 to 152 weeks of age. In the animals of the experimental group, interdigitation was eliminated by gradually grinding the cusps of the molars and canines in both dental arches as soon as possible after emergence. Silicone impressions of the dental arches of each monkey were taken at regular intervals. Two experimental and two control animals received vital staining at regular intervals and were processed for histological evaluation at the end of the experimental period. Changes over time in the dimensions of the dentition were analysed. Locally, the maxillary dental arch in the experimental group broadened significantly faster than in the control group. No significant differences between the experimental and the control group were found for any of the mandibular parameters. The experimental intervention also led to less prevalence of anterior open bite in the experimental group than in the control group. It is concluded that interdigitation plays a role in the development of the maxillary dental arch and does not seem to affect mandibular dental arch development.

Juvenile freshwater mussel (bivalvia: unionidae) responses to acute toxicity testing with copper

AbstractThe sensitivities of newly metamorphosed juveniles of Villosa iris and Anodonta grandis to copper were determined using 24‐h static bioassays. Two methods of assessing postexposure response were compared: direct visual examination and the use of vital staining with neutral red. Both species responded to copper exposures, exhibiting valve closure at concentrations as low as 24 μg Cu per liter for Villosa iris and 17 μg Cu per liter for Anodonta grandis. EC50 values of 27 μg Cu per liter (Villosa iris) and 33 μg Cu per liter (Anodonta grandis) were calculated on the basis of the valve closure response. Use of vital staining with neutral red provided an EC50 of 29 μg Cu per liter for Villosa iris. The amber coloration of Anodonta grandis valves prevented determination of an EC50 using the vital staining. LC50 values of 83 μg Cu per liter (Villosa iris) and 44 μg Cu per liter (Anodonta grandis) were calculated from the results of vital staining. Neutral red was found to be effective in assessing postexposure mortality and sublethal responses after acute exposures to copper.

Evaluation of rabbit sperm acrosomal integrity and fertilizing ability by use of vital stains

Three staining procedures to detect sperm acrosome integrity were compared via electron microscopy. Stains were applied to epididymal, freshly ejaculated, in vivo capacitated, and sonicated sperm cells in addition to spermatozoa displaying sequentially removed plasma and outer and inner acrosomal membranes. Sequential membrane removal procedures resulted in removal of plasma membranes from 73% of all sperm cells, removal of plasma and outer acrosomal membranes from 74% of all sperm cells, and removal of plasma and outer and inner acrosomal membranes from 87% of all sperm cells as determined by electron microscopy. Live/dead staining results were not statistically different from subjective microscopic motility evaluations (P less than 0.005) for epididymal, sonicated, freshly ejaculated, and in vivo capacitated sperm samples. All three stains assessed were similarly capable of detecting the acrosome status of freshly ejaculated and of sonicated spermatozoa compared to data obtained by electron microscopy (P = 0.010). However, only the Bryan-Akruk stain afforded data that were closely correlated with data obtained via electron microscopy for all sperm types assessed; the latter included in vivo capacitated spermatozoa and sperm cells rendered free of plasma membranes. Results confirmed an earlier report by successfully effecting sequential removal of rabbit acrosomal membranes and documented use of the Bryan-Akruk acrosomal stain for evaluation of sperm cell populations for fertilizing ability. These findings should prove useful in further investigations of mechanisms involved in achievement of fertilizing ability by rabbit spermatozoa.

Ovarian development in mosquitoes: stages of growth and arrest, and follicular resorption

ABSTRACT. Christophers' stages of ovarian development are elaborated and redefined to provide a more useful descriptive tool for mosquito physiology. Vital staining is advocated to help identify the earliest stages of vitellogenic follicles. By use of vital staining it has been confirmed that in Aedes aegypti (L.) early vitellogenic ovaries contain negligible numbers of dormant follicles, and that fecundity is correlated with food intake by regulated resorption of developing follicles. The concept of developmental gates in mosquito oogenesis is discussed.

Distinction of macrophage subpopulations: measurement of functional cell parameters by flow cytometry.

Macrophages represent a functionally heterogeneous group of cells which belong to the mononuclear phagocyte system (1). Heterogeneity may exist between macrophages from different organs as well as among macrophages within one organ (2,3). Heterogeneity has been defined by differences of Ia-antigen expression (4,3); monoclonal antibodies (5,6) against cell surface determinants; receptors for the C3 complement component or the Fc part of IgG molecules; cell size (7,3); enzyme activities e.g. phosphatase, nucleotidase (8) peroxidase (9) and transglutaminase (10); wheat germ lectin binding (11); tumor cytoxicity (12); or phagocytosis (13) and adherence. Classification according to several parameters is necessary to identify small subpopulations of macrophages (1). Flow cytometry is a particularly useful method for this purpose, especially because functional parameters of living cells can be measured simultaneously at the single cell level in a fast and accurate way. Such parameters include cytoplasmic (14,15) or lysosomal (16) enzyme activities, transmembrane potential (17,18), intracellular pH (19) and phagocytosis. The use of vital stains also permits cell sorting. Sorted cells can be recultivated and further analyzed. Macrophages are often characterized as cells with high esterase activity (20,21,22), although there are some reports on low esterase activity in macrophages (23,24,25). It was the purpose of this study to characterize the low activity macrophages in more detail.

Cephalometric and clinical evaluation of Class I discrepancy cases treated by serial extraction

0 NE of the major topics of discussion among orthodontists has been treatment in the mixed-dentition stage, especially in cases with evidence of a tooth-materialto-basal-bone discrepancy. Before 1930 treatment in the deciduous- and mixed-dentition stages was popular because the orthodontist assumed that Wolff’s law, which states that “bone grows in response to functional demand,” applied to the maxilla and the mandible as well as to the long bones. The general consensus was that early intervention could affect the dental arches with respect to growth and development. Angle’s philosophy that gentle forces in orthodontic movement would stimulate bone growth was well accepted. Soon, however, research gave the orthodontist a greater appreciation of growth and development. Broadbent, with the aid of a. cephalometer, made some of the early contributions in 1931. Schour’s14 growth study made use of vital staining, and Brodie31 * concluded : “Only alveolar processes are affected by orthodontic tooth movement.” Practitioners became more skeptical about treatment in the mixed dentition, and most orthodontic treatment of malocclusions was carried out after complete eruption of the permanent teeth. It was thought that early treatment would result in damage to the teeth and supporting structures, with no assurance of success in treatment. Early intervention was recommended only in treatment of cross-bites, oral habits, drifting permanent teeth as a result of early loss of deciduous teeth, irregularities caused by supernumerary teeth, and ectopic eruption of first molars. A few years later, other men. in the field contended that mixed-dentition treatment would permit development of the face toward greater balance and

Immune cytolysis of mouse thymic lymphocytes

Summary 1.Immune cytolysis was carried out by mixing a suspension of mouse thymic lymphocytes containing 97 to 99% viable cells with complement and various dilutions of rabbit antiserum or isoantiserum. Cellular integrity was determined after incubation for 2 hr at 37° C, by use of vital staining. An end point of 50% cell deformation was employed. 2.As source of complement, hamster serum was almost nontoxic to mouse thymic lymphocytes. Two absorptions with mouse red cells and three absorptions with mouse liver removed the slight toxicity and any tendency for clumping. Guinea pig serum similarly absorbed was still slightly toxic. 3.The count of deformed cells in the assay tubes containing antiserum changed relatively little between 1 and 3 hr after the start of incubation. This could be attributed to a balance between the rate of increase in the number of stained cells and their rate of disappearance due to complete lysis. 4.Optimal conditions for assay of complement were determined. Complement titers determined with cells sensitized with varying concentrations of isoantiserum were relatively constant; in contrast, the complement requirement decreased with increasing concentrations of rabbit antiserum. This complement-sparing action of rabbit antiserum was only partially correlated with its complement-independent cytotoxicity. 5.Optimal conditions for assay of cytolysin were determined. Direct proportionality between cytolytic titer and cell concentration was obtained over a range from approximately 5 to 20 million cells/ml. There was progressive loss of proportionality at lower cell concentrations. 6.An unabsorbed rabbit antiserum to AKR thymic lymphocytes gave no evidence of specificity in its cytolysis of thymic lymphocytes taken from four different strains of inbred mice. The two isoantisera that were tested showed potent cytolysis of AKR thymic lymphocytes.

Immune Cytolysis of Mouse Thymic Lymphocytes

Summary Immune cytolysis was carried out by mixing a suspension of mouse thymic lymphocytes containing 97 to 99% viable cells with complement and various dilutions of rabbit antiserum or isoantiserum. Cellular integrity was determined after incubation for 2 hr at 37° C, by use of vital staining. An end point of 50% cell deformation was employed. As source of complement, hamster serum was almost nontoxic to mouse thymic lymphocytes. Two absorptions with mouse red cells and three absorptions with mouse liver removed the slight toxicity and any tendency for clumping. Guinea pig serum similarly absorbed was still slightly toxic. The count of deformed cells in the assay tubes containing antiserum changed relatively little between 1 and 3 hr after the start of incubation. This could be attributed to a balance between the rate of increase in the number of stained cells and their rate of disappearance due to complete lysis. Optimal conditions for assay of complement were determined. Complement titers determined with cells sensitized with varying concentrations of isoantiserum were relatively constant; in contrast, the complement requirement decreased with increasing concentrations of rabbit antiserum. This complement-sparing action of rabbit antiserum was only partially correlated with its complement-independent cytotoxicity. Optimal conditions for assay of cytolysin were determined. Direct proportionality between cytolytic titer and cell concentration was obtained over a range from approximately 5 to 20 million cells/ml. There was progressive loss of proportionality at lower cell concentrations. An unabsorbed rabbit antiserum to AKR thymic lymphocytes gave no evidence of specificity in its cytolysis of thymic lymphocytes taken from four different strains of inbred mice. The two isoantisera that were tested showed potent cytolysis of AKR thymic lymphocytes.

Identification of transplanted tissues in chick embryos by marking with phosphorus-32.

THE technique of transplantation in early chick blastoderms developed by Waddington1,2 has hitherto been handicapped by uncertainties in distinguishing host from graft tissues during analysis of the resulting embryo. In Amphibia this difficulty has been resolved by heteroplastic grafting between differently pigmented species3,4, or by the use of vital stains which could be fixed in the tissues5. Not a little of the superiority of amphibian embryos as experimental material is due to these techniques. Analogous methods have failed to give an adequate solution of the difficulty in chick embryology. Conveniently available species (as used by Waddington and Schmidt6) are not sufficiently distinct histologically, and vital staining is not apparently always well localized7 and its fixation for subsequent examination in paraffin sections, though possible8, is not reliable.

THE DEVELOPMENT OF THE VERTEBRATE EXCRETORY SYSTEM

SummaryI. The vertebrate excretory system is a holonephros. The entire organ arises from the one source, namely, from the intermediate cell‐mass which unites the somite with the lateral plate, and is originally segmental. The part of the intermediate cell‐mass adjacent to the somite develops into the nephrotome or nephric chamber, and the part connecting it with the body cavity becomes the peritoneal canal. The tubule is an outgrowth from the dorsal wall of the chamber and the duct arises from the union, in front and behind, of the distal extremities of the tubules and is therefore primitively segmental.2. The simplest relations appear to be found in myxinoids so far as can be ascertained from the somewhat incomplete knowledge we possess of this group. They are also primitive in Gymnophiona and detailed reference has therefore been made to this group.3. The original terms pronephros, mesonephros and metanephros are retained. The term pronephros should only be applied to the organ in larval Anamnia and to that of a few adult teleosts. In most vertebrates, behind the pronephros where this is present, or posterior to the zone of anterior rudimentary tubules, the intermediate cell‐mass is early separated from somite and lateral plate and forms the so‐called nephrogenic blastema from which the remainder of the excretory organ is derived. The later differentiation is one of size and complexity only. The gradual separation of the hinder portion into a well‐defined isolated metanephros, such as is present in the Amniota, is indicated in some elasmobranchs and in some urodeles, and is seen in various species of teleosts.4. Recent experimental work with Amphibia has located the area from which the organ develops in the gastrula. The methods of pronephrotomy and transplantation have substantiated the earlier observations as to the precise level at which the pronephros arises and that from which the duct takes its origin. The capacity of the duct to grow independently backwards towards the cloaca has been conclusively proved by the use of vital staining. By means of bisection, partial removal and transplantation an attempt has been made to ascertain the power of regeneration of the duct and also the interrelations between the latter and the developing mesonephric blastema. Interesting information has been acquired with regard to the role of induction in these interrelations.5. Attention is drawn to the distinction between the ciliated nephrostomial canal and the ciliated peritoneal canal; their initial derivation and later development in normal and abnormal conditions is discussed.6. The relationship between external and internal glomeruli is considered, and the passage from one to the other as met with at the anterior end of the mesonephros in many Amniota is described. It is emphasized that the function of such a condition is by no means clear and needs experimental investigation.7. It is concluded that the homogeneity of the pro‐, meso‐ and metanephros has now been convincingly established from many points of view. The nephrogenic material arises from a single source and exhibits identical properties throughout, and there is striking uniformity in the structure and function of the various parts of the system.

NODULAR SUBEPIDERMAL FIBROSIS (DERMATOFIBROMA VERSUS HISTIOCYTOMA)

THIS article is based on clinical and pathologic studies of 95 patients who had 1 or more lesions of nodular subepidermal fibrosis. Unna,¹in 1896, first described the condition as "fibroma simplex." It has become widely known as "dermatofibroma lenticulare"²or as "nodular subepidermal fibrosis."³For many years a fibroblastic, and possibly traumatic, origin of the condition was accepted. However, in articles published from 1931 to 1933, Woringer and his associates⁴questioned the fibroblastic origin of all these lesions and described what Woringer believed formed a separate entity under the title "histiocytoma." He and his associates found phagocytic reticuloendothelial cells. Their observations were supported by the studies of Senear and Caro⁵with the use of vital stains. More recently, in 1943, Gross and Wolbach⁶described a series of cases similar to those described by Woringer and his associates but advanced the belief that the