Rapid Staining Research Articles

A Case of Perforated Sigmoid Diverticulitis in Which Gram Staining of Ascitic Fluid Was Useful for Diagnosis

An 85-year-old woman was admitted to our hospital for steroid therapy for relapsing nephrotic syndrome. During hospitalization, she complained of sudden epigastric pain at night. Although there were signs of peritoneal irritation, CT showed a large amount of ascitic fluid, but no free intraperitoneal gas. Gram staining of ascitic fluid obtained by abdominal paracentesis showed Gram-negative rods, which raised a strong suspicion of gastrointestinal perforation and peritonitis. Therefore, emergency surgery was performed. Exploration of the colon showed multiple sigmoid diverticula, one of which was perforated. The patient underwent an emergency Hartmann's procedure. Imaging studies failed to reveal any evidence of gastrointestinal perforation, presenting a diagnostic challenge. However, a physician performed rapid Gram staining of ascitic fluid at night when laboratory technicians were absent, had a strong suspicion of gastrointestinal perforation, and performed emergency surgery. Gram staining is superior in rapidity, and ascitic fluid Gram staining can aid in diagnosis, suggesting that it should be actively performed. We report this case, with a review of the literature on the significance of rapid diagnosis by Gram staining.

Negative Stain EM of Myosn-S1 Bound to Actin and Thin Filaments in Mgatp after Rapid Mixing

We have used rapid mixing negative stain electron microscopy (EM) to observe myosin heads (S1) bound to F-actin and thin filaments in the presence of ATP. Myosin V S1 was mixed with excess ATP, incubated for 2 sec to allow binding and hydrolysis, and then mixed with either F-actin or cardiac thin filaments. This mixture was applied to an EM grid and negatively stained with uranyl acetate ∼20 ms later. Short times on the grid allow protein concentrations as high as 10 uM to be used, which is critical to observe weakly binding complexes, such as S1-ADP-Pi with actin filaments. This approach has advantages compared to hand mixed solutions. It has improved time resolution (0.3 s vs 3 s between mixing and staining), the mixing is ∼1000x faster compared to manual manipulation (∼ 2 ms vs 2 s) and mixing is more uniform. The entire automated procedure used an apparatus with computer controlled stepper motors previously built for time-resolved cryo-EM (White et al, J Struct Biol:144, 246-52, 2003), which was modified for negative staining. Slow and non-uniform mixing produced by hand mixing can suggest cooperative binding. After automated mixing, S1-ADP-Pi with cardiac thin filaments at low calcium concentrations, S1 bound randomly and only rarely were bare or fully decorated filaments observed. Without ATP, thin filaments were mostly either bare or fully decorated, suggestive of cooperativity. These results may explain the apparently contradictory observations that binding of S1 to thin filaments appeared to be cooperative in micrographs, whereas kinetic and structural measurements indicate that individual rigor heads activate 12-14 actin subunits.

Exfoliative cytology of buccal squames: A quantitative cytomorphometric analysis of patients with diabetes.

Background:Diabetes is a third leading cause of mortality and morbidity in the world. Diabetes is one of the most common endocrine metabolic disorders and its prevalence has been increasing worldwide. Oral exfoliative cytology may be a more appropriate adjunctive diagnostic tool in conditions like diabetes mellitus, where the invasive techniques lose viability.Aims:The purpose of this study is to analyze the cytomorphometric changes in the exfoliated cells of the oral mucosa, as an adjunct to the diagnosis of diabetes.Materials and Methods:Smears were taken from the buccal mucosa of 30 diabetes patients (study group) and 30 healthy individuals (control group). All the smears were stained with rapid Papanicolaou stain (PAP). In the PAP smears, the nuclear area (NA), cytoplasmic area (CA), and cytoplasmic-to-nuclear ratio (CNR) were evaluated for 50 cells in each smear, using the Image Analysis Software (Magnus Pro™) and research microscope (Lawrence and Mayo™).Results:The results showed that the mean NA was significantly higher (P < 0.001) in the study group, whereas, the mean CA did not exhibit a statistically significant difference (P > 0.001). The mean CNR was significantly lower in the study group (P < 0.001).Interpretation and Conclusion:The results associated with the clinical observations suggest that diabetes can produce morphological and functional alterations in the oral epithelial cells, detectable by microscopic and cytomorphometric analysis using exfoliative cytology, which can be used in the diagnosis of the disease.

A simple and rapid staining method for detection of hemozoin pigment by methylene blue stain.

A simple and rapid staining method for detection of hemozoin pigment by methylene blue stain.

Combining microtomy and confocal laser scanning microscopy for structural analyses of plant–fungus associations

The serious problem of extended tissue thickness in the analysis of plant-fungus associations was overcome using a new method that combines physical and optical sectioning of the resin-embedded sample by microtomy and confocal microscopy. Improved tissue infiltration of the fungal-specific, high molecular weight fluorescent probe wheat germ agglutinin conjugated to Alexa Fluor® 633 resulted in high fungus-specific fluorescence even in deeper tissue sections. If autofluorescence was insufficient, additional counterstaining with Calcofluor White M2R or propidium iodide was applied in order to visualise the host plant tissues. Alternatively, the non-specific fluorochrome acid fuchsine was used for rapid staining of both, the plant and the fungal cells. The intricate spatial arrangements of the plant and fungal cells were preserved by immobilization in the hydrophilic resin Unicryl™. Microtomy was used to section the resin-embedded roots or leaves until the desired plane was reached. The data sets generated by confocal laser scanning microscopy of the remaining resin stubs allowed the precise spatial reconstruction of complex structures in the plant-fungus associations of interest. This approach was successfully tested on tissues from ectomycorrhiza (Betula pendula), arbuscular mycorrhiza (Galium aparine; Polygala paniculata, Polygala rupestris), ericoid mycorrhiza (Calluna vulgaris), orchid mycorrhiza (Limodorum abortivum, Serapias parviflora) and on one leaf-fungus association (Zymoseptoria tritici on Triticum aestivum). The method provides an efficient visualisation protocol applicable with a wide range of plant-fungus symbioses.

Improved staining of phosphoproteins with high sensitivity in polyacrylamide gels using Stains‐All

An improved Stains-All (ISA) staining method for phosphoproteins in SDS-PAGE was described. Down to 0.5-1 ng phosphoproteins (α-casein, β-casein, or phosvitin) can be successfully selectively detected by ISA stain, which is approximately 120-fold higher than that of original Stains-All stain, but is similar to that of commonly used Pro-Q Diamond stain. Furthermore, unlike the original Stains-All protocol that was time consuming and light unstable, ISA stain could be completed within 60 min without resorting to protect the gels from light during the whole staining procedure. According to the results, it is concluded that ISA stain is a rapid, sensitive, specific, and economic staining method for a broad application to the research of phosphoproteins.

A comparison of ABAcard® p30 and RSID™-Semen test kits for forensic semen identification

The screening and confirmatory tests available to a forensic laboratory allow evidence to be examined for the presence of bodily fluids. With the majority of evidence being submitted involving sexual assaults, it is important to have confirmatory tests for the identification of semen that are straightforward, quick, and reliable. The purpose of this study was to compare two commonly used semen identification kits utilized by forensic laboratories: ABAcard® p30 and Rapid Stain Identification of Human Semen (RSID™-Semen). These kits were assessed with aged semen stains, fresh and frozen post-vasectomy semen, post-coital samples collected on different substrates, post-vasectomy semen mixed with blood, saliva, and urine, a series of swabs collected at increasing time intervals after sexual intercourse, and multiple non-semen samples. The test kits were compared on the basis of sensitivity, specificity, and the cost and time effectiveness of each protocol. Overall, both semen identification tests performed well in the studies. Both kits proved specificity for identifying semen, however the ABAcard® p30 test surpassed the RSID™-Semen test in sensitivity, cost per test, and simplified test protocol.

Design and synthesis of a novel fluorescent protein probe for easy and rapid electrophoretic gel staining by using a commonly available UV‐based fluorescent imaging system

A new fluorescent molecular probe, methyl 3-(3,5-bis((bis(pyridin-2-ylmethyl)amino)-methyl)-4-hydroxyphenyl)-2-(5-(dimethylamino)naphthalene-1-sulfonamido) propanoate, dizinc(II) chloride salt (Dansyl-1-Zn(II)), which possesses Zn(II) complexes and a dansyl group, was designed and synthesized to enable the detection of proteins in solution and in high-throughput electrophoresis by using a UV-based detection system. Dansyl-1-Zn(II) exhibited weak fluorescence in the absence of proteins and strong green fluorescence at approximately 510 nm in the presence of BSA upon irradiation with light at a wavelength of 345 nm. Compared with conventional protocols for in-gel SDS-PAGE protein staining (e.g. silver staining, SYPRO Ruby, and Oriole), the operating times of which range from 90 min to overnight, Dansyl-1-Zn(II) allowed 1-step protein staining (SDS-PAGE →Staining →Detection) and shortened the operating time (35 min) with high sensitivity (LOD: 1 ng or less) under 312-nm or 365-nm light excitation with orange or red emission filters, respectively. Moreover, Dansyl-1-Zn(II) was successfully applied to protein identification by MS via in-gel tryptic digestion, Western blotting, and Native-PAGE. Accordingly, Dansyl-1-Zn(II) may facilitate highly sensitive and high-throughput protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.

Evaluation of Rapid Stain IDentification (RSID™) Reader System for Analysis and Documentation of RSID™ Tests

The ability to detect the presence of body fluids is a crucial first step in documenting and processing forensic evidence. The Rapid Stain IDentification (RSID™) tests for blood, saliva, semen and urine are lateral flow immunochromatographic strip tests specifically designed for forensic use. Like most lateral flow strips, the membrane components of the test are enclosed in a molded plastic cassette with a sample well and an observation window. No specialized equipment is required to use these tests or to score the results seen in the observation window; however, the utility of these tests can be enhanced if an electronic record of the test results can be obtained, preferably by a small hand-held device that could be used in the field under low light conditions. Such a device should also be able to read the lateral flow strips and accurately record the results of the test as either positive, i.e., the body fluid was detected, or negative, i.e., the body fluid was not detected. Here we describe the RSID™ Reader System—a ruggedized strip test reader unit that allows analysis and documentation of RSID™ lateral flow strip tests using pre-configured settings, and show that the RSID™ Reader can accurately and reproducibly report and record correct results from RSID™ blood, saliva, semen, and urine tests.

Tunable filter-based multispectral imaging for detection of blood stains on construction material substrates part 2: realization of rapid blood stain detection

Based on the blood stain detection method and criteria established in part 1 of this article, we combine and organize all necessary tasks to realize the multispectral imaging-based rapid blood stain detection system. To rapidly detect blood stains on the test surface, the developed system automatically captures the spectral images, extracts their spectral data, determines the positions of blood stains, and accurately highlights the positions of blood stains on the display. To achieve such a system, several tasks are newly introduced, including adjustment of camera exposure times to prevent image saturation or excessive darkness, the search for the sampled clean positions of the substrate to determine the substrate reflectance spectrum, and suitable detection procedures and proper arrangement of criteria to eliminate unnecessary calculations. Parallel processes between image capturing and blood stain identification help shorten the time for blood stain identifications despite a large amount of spectral data to be processed. The developed system can identify blood against several other reddish brown stains on several substrates. The measured average identification times on different test surfaces range from only 23.3 to 28.7 s, including the image capturing process.

Advantages of the rapid double-staining method for intraoperative detection of micrometastasis in sentinel lymph nodes

For rapid intraoperative diagnosis of lymph node micrometastasis, we refined the rapid immunohistochemistry method by combining anti-cytokeratin antibody-labeled nanocrystal beads with rapid hematoxylin and eosin (H&E) staining on the same section, referred to as the rapid double staining (RDS) technique. Two frozen-section slices each were obtained from 372 lymph nodes of 100 breast cancer patients. We performed RDS for 1 slide and rapid H&E staining for the other. The results were compared with the corresponding final pathological data obtained from the permanent specimens. For specimens from patients with pN1(mi) as determined by final pathological examination, the false‑negative rate was 33.3% for rapid H&E staining and 16.7% for RDS. For specimens from patients with pN0(i+) as determined by final pathological examination, the false‑negative rate was 80% for rapid H&E staining and 0% for RDS. These results indicate that RDS is superior to conventional rapid H&E staining for intraoperative diagnosis of nodal micrometastasis and isolated tumor cells.

Four weeks lithium treatment alters neuronal dendrites in the rat hippocampus

A large body of evidence from molecular, cellular and human studies suggests that lithium may enhance synaptic plasticity, which may be associated with its therapeutic efficacy. However, only a small number of studies have directly assessed this. To determine whether lithium treatment alters structural synaptic plasticity, this study examined the effect of 4wk lithium treatment on the amount and distribution of dendrites in the dentate gyrus (DG) and hippocampal area CA1 of young adult rats. Following 4wk lithium or control chow feeding, animals were decapitated, the hippocampi were prepared and stained using a rapid Golgi staining technique and the amount and distribution of the dendritic branching was evaluated using Sholl analyses (method of concentric circles). In the DG, lithium treatment increased the amount and distribution of dendritic branches in the proximal half of dendritic trees of the granule cells and reduced branching in the distal half. In area CA1, the same treatment also increased the number of dendritic branches in the proximal half of apical dendritic trees of CA1 pyramidal cells and reduced branching in the distal half of apical dendritic trees but had no effect on basilar dendritic trees. The lithium treatment altered the total density of dendritic trees in neither the DG nor area CA1. These findings suggest that, in the DG and apical CA1, chronic lithium treatment rearranges neuronal morphology to increase dendritic branching and distribution to where major afferent input is received.

Rapid staining method to detect and identify downy mildew (Peronospora belbahrii) in basil.

• Premise of the study: Demand for fresh-market sweet basil continues to increase, but in 2009 a new pathogen emerged, threatening commercial field/greenhouse production and leading to high crop losses. This study describes a simple and effective staining method for rapid microscopic detection of basil downy mildew (Peronospora belbahrii) from leaves of basil (Ocimum basilicum).• Methods and Results: Fresh leaf sections infected with P. belbahrii were placed on a microscope slide, cleared with Visikol™, and stained with iodine solution followed by one drop of 70% sulfuric acid. Cell walls of the pathogen were stained with a distinct coloration, providing a high-contrast image between the pathogen and plant.• Conclusions: This new staining method can be used successfully to identify downy mildew in basil, which then can significantly reduce its spread if identified early, coupled with mitigation strategies. This technique can facilitate the control of the disease, without expensive and specialized equipment.

Quantified High-Throughput Screening of Escherichia coli Producing Poly(3-hydroxybutyrate) Based on FACS

Here, we report on a highly sensitive method for the detection of P(3HB) accumulation in Escherichia coli cells based on the automated flow cytometry system using fluorescent dyes. E. coli containing P(3HB) were stained with either BODIPY or Nile red fluorescent dye, and their staining properties were analyzed under a variety of conditions. Compared with Nile red, BODIPY was much more sensitive in staining P(3HB) and overall demonstrated a more rapid staining of cells, a greater resistance to photobleaching, and greater cell viability. In addition, we also successfully monitored heterogeneity in P(3HB) accumulation within a cell population using BODIPY staining and flow cytometry. We believe this optimized staining method using BODIPY in combination with screening by high-speed flow cytometer will be helpful in the engineering of host cells toward an enhanced production of bioplastics.

Highly Selective and Fast Diagnosis of Alzheimer's Disease Hallmark Lesions using Congo Red in Isopropyl Alcoholic Solution

A highly selective, rapid, inexpensive, simple, and immunocytochemical compatible fluorescence staining method for Alzheimer's disease hallmark lesions applicable to sections of human specimens embedded in paraffin is described. Human necropsy material was fixed in buffered formalin, sectioned at 10 μm, mounted on slides, deparaffinized, and partially hydrated (70% ethanol). After partial hydration, sections were stained for 10 min in a solution of 0.2% Congo red in 70% isopropanol. After washing in 70% isopropanol and rehydration, auto-fluorescence of sections were quenched (optional) and processed for immunocytochemistry (optional). Finally, sections were mounted in an adequate mounting medium. Amyloid deposits appear pink at light microscopy and all Alzheimer's disease hallmark lesions appear orange or red under fluorescence microscopy using blue or green exciting light, respectively. The present method can be used in combination with all pre- or post-immunocytochemical techniques.

Guided Bone Regeneration for Socket Preservation in Molar Extraction Sites: Histomorphometric and 3D Computerized Tomography Analysis

Guided Bone Regeneration for Socket Preservation in Molar Extraction Sites: Histomorphometric and 3D Computerized Tomography Analysis

Vergleich von vier zytologischen Färbeverfahren für Ohrabstriche beim Hund

Cytological examination is crucial for the diagnosis and classification of canine otitis externa. Staining should reveal micro-organisms as perpetuating factors of otitis externa. The aim of the study was to compare four different staining methods (Diff-Quik®, Diff-Quik® after dipping in acetone, Gram Quick stain® and a commercial rapid stain for otitis externa) for ear cytology of dogs with otitis externa and to investigate the agreement of cytology and culture. In a study evaluating dogs with otitis externa, five ear swabs (one for culture and four for cytology) were taken from the horizontal part of the external auditory canal of 224 affected ears and compared semi-quantitatively. Diff-Quik® with and without prior dipping in acetone as well as the Gram Quick stain® displayed a high degree of agreement in the detection of micro-organisms (cocci p = 0.2366; rods p = 0.4832; yeasts p = 0.1574), while the commercial otitis rapid stain revealed significantly less micro-organisms (p < 0.001 for all comparisons). The results of the first three stains corresponded to the culture results by > 70%; the agreement was lower with the commercial otitis rapid stain. The quickest and easiest method was staining with Diff-Quik®. Diff-Quik® with or without prior dipping in acetone and the Gram Quick stain® had a high agreement in the detection of microorganisms and can thus be considered nearly equivalent for the diagnosis of otitis externa infectiosa. The commercial otitis rapid stain is less reliable. Based on this study Diff-Quik® can be recommended for the routine cytology of ear swabs. Additionally, a culture may be indicated and must be interpreted in the context of the cytology.

全身性破傷風患者の創傷部から分離した &lt;i&gt;Clostridium tetani&lt;/i&gt; の 1 症例

Clostridium tetani is widely distributed in ground or mud, especially in field and pond-shore surface layers. C. tetani is rarely isolated from specimens of patients with tetanus, and is generally diagnosed based on clinical symptoms such as trismus or general tonic spasms. This means that positive C. tetani infection is rarely diagnosed bacterially. Using gram straing, we identified C. tetani in specimens from patients suspected of C. tetani infection brought to the Kitasato University Hospital emergency center. Rapid gram staining information in the bacteriology laboratory is expected to improve recovery from C. tetani infection. It is therefore necessary to ensure clinical specimen quality control, and to keep standard strains of rare bacteria for isolation and identification.

Confocal microscopy and exfoliative cytology

Context:Early detection of potentially malignant lesions and invasive squamous-cell carcinoma in the oral cavity could be greatly improved through techniques that permit visualization of subtle cellular changes indicative of the neoplastic transformation process. One such technique is confocal microscopy. Combining rapidity with reliability, an innovative idea has been put forward using confocal microscope in exfoliative cytology.Aims:The main objective of this study was to assess confocal microscopy for cytological diagnosis and the results were compared with that of the standard PAP stain.Settings and Design:Confocal microscope, acridine orange (AO) stain, PAP (Papanicolaou) stain. The study was designed to assess confocal microscopy for cytological diagnosis. In the process, smears of patients with (clinically diagnosed and/or suspected) oral squamous cell carcinoma as well as those of controls (normal people) were stained with acridine orange and observed under confocal microscope. The results were compared with those of the standard PAP method.Materials and Methods:Samples of buccal mucosa smears from normal patients and squamous cell carcinoma patients were made, fixed in 100% alcohol, followed by AO staining. The corresponding set of smears was stained with PAP stain using rapid PAP stain kit. The results obtained were compared with those obtained with AO confocal microscopy.Results:The study had shown nuclear changes (malignant cells) in the smears of squamous cell carcinoma patients as increased intensity of fluorescence of the nucleus, when observed under confocal microscope. Acridine orange confocal microscopy showed good amount of sensitivity and specificity (93%) in identifying malignant cells in exfoliative cytological smears.Conclusion:Confocal microscopy was found to have good sensitivity in the identification of cancer (malignant) cells in exfoliative cytology, at par with the PAP method. The rapidity of processing and screening a specimen resulted in saving of time. It added a certain amount of objectivity to the process of arriving at a diagnosis.

Gross Morphological Features of the Organ Surface Primo-Vascular System Revealed by Hemacolor Staining

The primo-vascular system (PVS), which consists of primo-vessels (PVs) and primo-nodes (PNs), is a novel thread-like structure identified in many animal species. Various observational methods have been used to clarify its anatomical properties. Here, we used Hemacolor staining to examine the gross morphology of organ-surface PVS in rats. We observed a sinus structure (20–50 μm) with a remarkably low cellularity within PNs and PVs and several lines of ductules (3–5 μm) filled with single cells or granules (~1 μm) in PV. Both sinuses and ductules were linearly aligned along the longitudinal axis of the PVS. Such morphology of the PVS was further confirmed by acridine orange staining. In PN slices, there was a honeycomb-like structure containing the granules with pentagonal lumens (~10 μm). Both PVs and PNs were densely filled with WBCs, RBCs, and putative mast cells (MCs), which were 90.3%, 5.9%, and 3.8% of the cell population, respectively. Granules in putative MCs showed spontaneous vibrating movements. In conclusion, the results show that Hemacolor, a simple and rapid staining system, can reveal the gross morphological features reported previously. Our findings may help to elucidate the structure and function of the PVS in normal and disease states in future studies.