Silver Staining Technique Research Articles

Rapid detection of nucleic acid sequences of pathogenic bacteria based on a series piezoelectric quartz crystal sensor with transcription activator-like effectors

Accurate and rapid detection of pathogenic bacteria is of great importance in the field of clinical diagnosis and food safety. Current methods for pathogenic bacteria detection have some problems in accurate, rapid and universal application. Here we proposed a pathogenic bacteria series piezoelectric quartz crystal (SPQC) sensor for achieving highly specific and sensitive detection of pathogenic bacteria. The universal sequences of common clinical pathogens screened by our group were used as detection targets. A new TALEs nuclease was synthesized as a recognition element, which recognizes double-stranded DNA at the level of a single base mismatch in the range of 17–19 bases. Targets could be specifically recognized by TALEs, resulting in the change of electrode surface, which would be further amplified by hybridization chain reaction and silver staining technique. Finally, the changes would be detected by SPQC system. This strategy was demonstrated to have excellent performance, enabling sensitive detection of targets with a detection limit of 25 cfu/mL in less than 3 h. What's more, the identification of single base mismatch could be achieved when the target ranging in length between 17 and 19 bases. The proposed method is rapid, accurate and easy universal application and expected to be applied in clinical diagnosis and food safety.

A point-of-care detection platform for Escherichia coli O157:H7 by integration of smartphone and the structural colour of photonic microsphere

A smartphone-based sensitive, rapid, label-free and high-throughput detection platform for Escherichia coli O157:H7 was established. The specific recognition capability of this platform was dependent of the aptamer modified on the silica photonic microsphere (SPM), whose structural colour was utilized for the quantification of the target bacterium. Gold nanoparticles and silver staining technique were employed to improve the sensitivity of the detection platform. Such smartphone-based detection platform gave a wide linear detection range of 102 ∼ 108 CFU/mL with a low limit of detection (LOD) of 68 CFU/mL and high specificity for Escherichia coli O157:H7. Moreover, the recovery rates of the detection method were measured in the range of 99 ∼ 108% in the milk, pork and purified water samples. Furthermore, the developed detection platform did not require complex sample pretreatment and could be easily manipulated, displaying great application potential in the fields of food safety, environmental monitoring and disease diagnosis.

Effect of lime water processing of Pinelliae Rhizoma Praeparatum on toxic component lectin protein

The present study investigated the effect of immersion in the excipient lime water on the toxic component lectin protein and explained the scientific connotation of lime water detoxication during the processing of Pinelliae Rhizoma Praeparatum. Western blot was used to investigate the effects of immersion in lime water with different pH(pH 10, 11, and 12.4), saturated sodium hydroxide, and sodium bicarbonate solution on the content of lectin protein. The protein compositions of the supernatant and the precipitate after immersing lectin protein in lime water of different pH were determined by the SDS-PAGE method combined with the silver staining technique. The MALDI-TOF-MS/MS technique was used to detect the molecular weight distribution of peptide fragments in the supernatant and precipitate after immersing lectin protein in lime water of different pH, and circular dichroism spectroscopy was used to detect the ratio changes in the secondary structure of lectin protein during the immersion. The results showed that immersion in lime water at pH>12 and saturated sodium hydroxide solution could significantly reduce the content of lectin protein, while immersion in lime water at pH<12 and sodium bicarbonate solution had no significant effect on lectin protein content. The corresponding lectin protein bands and molecular ion peaks were not detected at the 12 kDa position in the supernatant and precipitate after immersing the lectin protein in lime water at pH>12, which was attributed to the fact that lime water immersion at pH>12 could significantly change the ratio of the secondary structure of lectin protein, resulting in irreversible denaturation, while lime water immersion at pH<12 did not change the ratio of the secondary structure of lectin protein. Therefore, pH>12 was the key condition for the detoxication of lime water during the processing of Pinelliae Rhizoma Praeparatum. Lime water immersion at pH>12 could cause irreversible denaturation of lectin protein, resulting in a significant decrease in the inflammatory toxicity of Pinelliae Rhizoma Praeparatum, which played a key role in detoxification.

Determination of reticular fibers in tissues fixed with sugarcane molasses

Reticular fiber consists of one or more types of very thin and delicately woven type III collagen strands, which form a highly ordered cellular network and provide a supportive network. Since most of these collagen types are combined with carbohydrates, they react with silver stains and periodic acid-Schiff reagent, but are not shown by ordinary histological stains like those using hematoxylin. In this study, it was aimed to determine the reticular fibers in tissues fixed with sugarcane molasses and formol using silver staining technique (Gordon Sweet –GS-). The reticular fibers in the tissues fixed with sugarcane molasses were compared with those in the tissues fixed with formol. According to the findings, the staining quality of the liver and spleen tissues fixed with sugarcane molasses showed similar characteristics with the tissues fixed with formol. Very weak staining was observed in kidney and testicle tissues. The fact that the tissues fixed in the same fixation solution show different results in the Gordon Sweet (GS) staining method shows us that this issue needs to be supported by more detailed studies.

The Effect of Final Irrigation Protocols on the Apical Sealing Ability of Epoxy Resin-based and Bioceramic-based Root Canal Sealers

Aim: This study was designed to investigate the effect of different final irrigation protocols on the apical sealing ability of bioceramic and epoxy resin-based sealers.
 Materials and methods: Thirty human single-rooted mandibular premolars were instrumented using ProTaper Next rotary files. Teeth were randomly divided into three groups according to the final irrigation regimen; Group I: 5 ml 0.2% CNPs/3 min, Group II: 2.5 ml 0.2% CNPs/1.5 min followed by 2.5 ml 17% EDTA/1.5 min, and Group III: 5 ml 17% EDTA/3min. All groups were subdivided into two subgroups based on the obturation material; Subgroup A: gutta-percha/Sure-Seal Root BC Sealer and Subgroup B: gutta-percha/AH Plus. All canals were obturated using single cone obturation technique. The apical sealing ability was assessed using modified silver staining technique with ammoniacal silver nitrate tracer solution. Samples were sectioned longitudinally and examined using scanning electron microscope.
 Results: Sure-Seal Root BC sealer showed significantly lower nanoleakage compared to AH Plus (p < 0.001). No significant difference was recorded in the nanoleakage of Sure-Seal Root BC sealer among the three groups (p = 0.284), while AH Plus showed a significantly higher nanoleakage in the EDTA group (p = 0.002). The depth of silver nitrate penetration into the dentinal tubules was significantly higher in AH Plus subgroup with the three different irrigation protocols (p < 0.001). For both sealers, the highest penetration depth for silver nitrate tracer solution was recorded in the EDTA group (p < 0.001).
 Conclusions: The apical sealing ability of bioceramic sealers is better than that of epoxy resin-based sealers. The type of the final irrigating solution seems to affect the post-obturation seal of both AH Plus and Sure-Seal Root BC sealer.
 Keywords: Bioceramic, AH Plus, Chitosan nanoparticles, EDTA, Sealing ability.

Pneumocystis Jirovecii Pneumonia Diagnosis via Metagenomic Next-Generation Sequencing.

The incidence of non-HIV-infected Pneumocystis Jirovecii Pneumonia (PJP) is increasing. The prognosis for non-HIV PJP is poor and diagnostic tests are of lower sensitivity in non-HIV patients. Metagenomic next-generation sequencing (mNGS) was compared with routine detection assays, including Gomori methenamine silver (GMS) stain and polymerase chain reaction (PCR) technique. Specimens of 4 bronchoalveolar lavages (BAL) and 1 lung tissue samples were obtained from 4 non-HIV patients from our hospitals. Although both GMS and mNGS were positive for P. jirovecii with PCR as positive control, the testing time of mNGS was obviously shorter than GMS. Compared with the traditional GMS method, mNGS has absolute advantages. However, the issue with PJP presentations having atypical symptoms and ambiguous imaging features persists. Hence, the disease can easily be ignored. Secondly, PJP progresses rapidly in non-HIV-infected patients and can cause severe respiratory failure with unfavorable prognosis. This study affirms that mNGS can be used to quickly and accurately diagnose PJP, but a combination of clinical judgement of symptoms, laboratory testing, and imaging examination is required to make a comprehensive judgment along with mNGS test results.

Risk factors for bovine rotavirus infection and genotyping of bovine rotavirus in diarrheic calves in Bangladesh.

Bovine rotavirus (BRV) is considered the leading cause of calf diarrhea worldwide, including Bangladesh. In this study we aimed to identify risk factors for BRV infection and determine the G and P genotypes of BRV strains in diarrheic calves. Fecal samples were collected from 200 diarrheic calves in three districts between January 2014 and October 2015. These samples were screened to detect the presence of BRV using rapid test-strips BIO K 152 (RTSBK). The RTSBK positive samples were further tested by polyacrylamide gel electrophoresis and the silver staining technique to detect rotavirus dsRNA. Risk factors were identified by multivariable logistic regression analysis. The G and P genotypes of BRV were determined by RT-PCR and sequencing. A phylogenetic tree was constructed based on the neighbor-joining method using CLC sequence viewer 8.0. About 23% of the diarrheic calves were BRV positive. The odds of BRV infection were 3.8- (95% confidence interval [95% CI]: 1.0-14.7) and 3.9-times (95% CI:1.1-14.2) higher in Barisal and Madaripur districts, respectively, than Sirrajganj. The risk of BRV infection was 3.1-times (95% CI: 1.5-6.5) higher in calves aged ≤ 5 weeks than those aged >5 weeks. Moreover, the risk of BRV infection was 2.6-times (95% CI:1.1-5.8) higher in crossbred (Holstein Friesian, Shahiwal) than indigenous calves. G6P[11] was the predominant genotype (94.4%), followed by G10P[11] (5.6%). The BRV G6 strains were found to be closest (98.9-99.9%) to Indian strains, and BRV G10 strains showed 99.9% identities with Indian strain. The VP4 gene of all P[11] strains showed >90% identities to each other and also with Indian strains. The most frequently identified BRV genotype was G6P[11]. About 23% of calf diarrhea cases were associated with BRV. To control disease, high-risk areas and younger crossbred calves should be targeted for surveillance and management. The predominant genotype could be utilized as the future vaccine candidate or vaccines with the dominant genotype should be used to control BRV diarrhea in Bangladesh.

The Correlate between Argyrophilic Nucleolar Organizer Region Stain and Ki-67 Immnochistochemistry in Diagnostic Breast Cancer

Introduction: This experimental study gauged the value of argyrophilic nucleolar organizer region (AgNOR) staining as a possible technique for the estimation of cell kinetics in conventional histology sections, in benign and malignant breast lesions. Methods: With a silver staining technique and immunohistochemistry, we associated the numbers of AgNORs and Ki67 scores in 30 breast carcinomas and 10 benign breast lesions. Results: The mean values of Ag NORs silver stain dots count for normal, benign, grade II and III were 1.28±0.17, 2.83±0.68, 5.23±0.87 and 7.32±0.92, respectively. There was a statistically significant difference (p≤0.001) was noticed between the all individual groups, among the normal and breast lesion as well as among the GII and GIII. Immunohistochemical Results of Ki-67 protein exhibited homogenous golden-brown color in control case and a positive brown granules or diffuse dark brown color in the nuclei of both benign and malignant cases under the 400X magnify examined under the light microscope. Discussion: AgNOR counts performed on routine formalin-fixed paraffin sections could provide substantial kinetic evidence. Additionally, the difference in AgNOR counts between benign and malignant tumors is such that they may be of diagnostic worth.

Oral leukoplakia-epidemiological survey and histochemical analysis of 107 cases in Brazil.

To perform an epidemiological survey comparing the cell proliferative activity of 107 cases of oral leukoplakia with their clinical and histopathological characteristics. A cross-sectional, observational, and histological-histochemical study. The cases came from the Histopathological Diagnostic Service of UPF/RS and the School of Dentistry of Araçatuba FOA/UNESP/SP (1986-2016). The histopathological sections were stained using the silver staining (AgNOR) technique and the nuclei of 100 epithelial cells selected randomly were recorded to count the number of nucleolar organizer regions (NORs). The mean NORs per lesion were correlated to clinical and histological characteristics using ANOVA, at 5% significance. Most of the cases included men (62.62%), white (64.49%), and with an average age of 53.94 years. The most probable etiological factors were smoking (44.7%) and alcohol consumption (9.85%). The evolution time of most lesions was fast (33.65%), manifesting mainly in the form of plaques (70.37%) and without symptoms (58.88%). They were located mainly in the cheek mucosa (26.62%) and presented white color (66.35%), well-defined edges (59.81%), firm consistency (47.5%), and keratinized surface (49.53%). Etiological factor (p = 0.003), evolution time (p = 0.006), symptoms (p = 0.029), location (p = 0.020), consistency (p = 0.047), histopathological characteristics (p = 0.004), and superficial keratinization (p = 0.001) were statistically significant regarding the mean NORs of the leukoplakias studied. Oral leukoplakias caused by alcohol consumption and/or tobacco use, considering an evolution time of fewer than 12 months, asymptomatic, located in the lower lip or tongue, and with a firm consistency and increased superficial keratinization should be treated more aggressively by the clinician to avoid cancerization.

A Study on Keratin-Associated Protein (KAP) 3.2 Gene and Its Polymorphism in Sandyno Breed of Sheep

The current study investigates the polymorphic patterns of keratin-associated protein (KAP) 3.2 gene in Sandyno breed of sheep. Genomic DNA was isolated from blood samples of 51 numbers of Sandyno breed. Ovine specific primer associated PCR amplification of KAP 3.2 gene revealed product at 393 bp and genotyped by PCR-SSCP (Single Strand Conformation Polymorphism) method and visualized under silver staining technique. KAP 3.2 gene locus revealed 3 genotypes, viz. AA, AB and BB with a frequency of 0.84, 0.16 and 0 in Sandyno breed with allele frequencies of A(0.92) and B(0.08). Regarding population genetic indices, the effective number of alleles (Ne) for KAP 3.2 in Sandyno breed of sheep was found to be 1.1716. The PIC values was 0.1356 and FIS values was negative (– 0.0864) in this breed. The result revealed that the selected population of Sandyno breed of sheep was in Hardy-Weinberg equilibrium without any significant deviation from the population mean and was monomorphic for KAP 3.2 gene.

The Neuropathological Diagnosis of Alzheimer's Disease-The Challenges of Pathological Mimics and Concomitant Pathology.

The definitive diagnosis of Alzheimer’s disease (AD) rests with post-mortem neuropathology despite the advent of more sensitive scanning and the search for reliable biomarkers. Even though the classic neuropathological features of AD have been known for many years, it was only relatively recently that more sensitive immunohistochemistry for amyloid beta (Aβ) and hyperphosphorylated tau (HP-tau) replaced silver-staining techniques. However, immunohistochemistry against these and other proteins has not only allowed a more scientific evaluation of the pathology of AD but also revealed some mimics of HP-tau pathological patterns of AD, including age-related changes, argyrophilic grain disease and chronic traumatic encephalopathy. It also highlighted a number of cases of AD with significant additional pathology including Lewy bodies, phosphorylated TDP-43 (p-TDP-43) positive neuronal cytoplasmic inclusions and vascular pathology. This concomitant pathology can cause a number of challenges including the evaluation of the significance of each pathological entity in the make-up of the clinical symptoms, and the threshold of each individual pathology to cause dementia. It also raises the possibility of underlying common aetiologies. Furthermore, the concomitant pathologies could provide explanations as to the relative failure of clinical trials of anti-Aβ therapy in AD patients.

Two Freshwater Flagellates from the Mahananda Wildlife Sanctuary, West Bengal

During the study of the diversity of free living protozoan ciliates from the Mahananda Wildlife Sanctuary, West Bengal, two free-living flagellates were identified, namely Rhabdomonas costata (Korshikov) Pringsheim, 1951 and Entosiphon sulcatum Skvortzov and Noda 1969. Brief description, based on live observation and silver stained preparations has been provided for both species. The attempt to stain the specimens using the silver staining technique resulted in impregnation of the nuclear apparatus and flagella rendering easy morphological measurements and studying variations.

Histochemical and immunohistochemical detection of pathogenic leptospires serovars in tissues of some captive wildlife from a University zoo in Nigeria

ABSTRACTThe detection and documentation of pathogenic Leptospira serovars in wild captive and zoological garden animals are scarce in literature from Nigeria. The knowledge of the prevalence of prevalence of pathogenic Leptospira serovars in these animals as a zoonotic risk to workers, zoo visitors and the general public is essential. This investigation was carried out on archival kidney and liver samples of captive and Zoological Garden animals (66) of an institutional facility, submitted for necropsy to the Department of Veterinary Pathology between the periods of 2010–2015. The gross diagnosis reports were obtained from the necropsy records, detection of pathogenic Leptospira serovars was by Warthin Starry silver staining and immunohistochemistry techniques using standard methods. Six samples out of the sixty-six samples were positive for leptospira four samples were positive by silver stain method, while two samples were positive by immunohistochemistry. In this study, serovar Pomona and grippotyphosa were detected in the foxes while serovar Pomona was detected in the horse. This study has revealed the presence of pathogenic leptospires in some captive wild and zoological garden animals.

Microwave assisted silver stain for pap smear to improve the identification of coccobacillary flora

Introduction: Pap smear has been utilized since a long time in detecting cervical pre-cancers and cancers. Their potential in diagnosing pathogenic organisms however is underestimated. Reporting organisms morphologically consistent with Candida species, in cervicovaginal smears, is easier, but coexisting coccobacillary flora is often missed in the bargain. Such co-existing infections make the treatment difficult for clinicians, prolonging the distressing symptoms of patients. We evaluated modified silver staining technique using a microwave - JONES MARRES STAIN (JM) and tried comparing the results with conventional Pap smears to find any additional flora. Aim: To identify co-existing organisms in addition to Candida in a Pap smear stained with Jones Marres stain. Materials and methods: This retrospective prevalence study that included 82 consecutive conventional Pap smears routinely stained with Pap stain was then subjected to silver stain in a microwave. Inclusion criteria: Pap smears reported as showing Candida group of organisms. Results: In the 82 smears evaluated, JM stain helped in easier picking up of additional coccobacillary flora in 37(45%) of cases in comparison to 4(4.87%).These coexisting infections were missed in conventional Pap smear reporting. Conclusion: Modified JM stain helped in diagnosing additional flora thus reducing the chances of missing out co-infections and hence is useful for providing effective treatment Keywords: Microwave Silver stain, Co-infection, Pap smear, Coccobacillary flora.

History of Pick's disease

History of Pick's disease

Colonization of Pneumocystis jirovecii in Patients who Received and not Received Corticosteroids Admitted to the Intensive Care Unit: Airborne Transmission Approach

Pneumocystis pneumonia (PCP) is responsible for pulmonary infection in immunocompromised patients. The current study aimed at investigating the frequency of Pneumocystis colonization in patients hospitalized in the intensive care unit (ICU) and evaluating the relationship between PCP and Pneumocystis colonization. The current cross sectional study was conducted on bronchoalveolar lavage (BAL) fluids of 100patients collected from surgery and neurosurgery ICUs with different underlying corticosteroid therapy conditions. Patients were divided into 2 groups (patients receiving and not receiving corticosteroids). Direct examination on BAL fluids was performed by the Gomori methenamine silver and Giemsa staining techniques. Additionally, 2 filtered air samples of the 2 above mentioned units were collected. A nested-PCR targeted mtLSUrRNA gene and sequencing were used to identify Pneumocystis spp. In direct microscopy, 31 out of 100 hospitalized patients (31%) showed positive results. Twenty-three (46%) of smear positive patients were from the group of patients receiving corticosteroid, the other 8(16%) were from the group of patients not receiving corticosteroids (P= 0.001). Pneumocystis jirovecii DNA was detected in 77 out of 100 BAL samples by nested-PCR (77%) in which 40 (52%) and 37 (48%) samples were obtained from the patients receiving and not receiving corticosteroids, respectively. Pneumocystis genome was observed in 1 of the 2 filtered air samples. A significant number of patients receiving corticosteroids were also colonized by P. jirovecii that may be predisposed to PCP or be transmitted to susceptible patients. A significant relationship was observed between the mean hospital stay and detection rate.

The characteristic of protein biosynthesis in brain neurons with chronic alcohol intoxication

The objective of the present study was to evaluate the possibilities for the use of the changes in the AgNOR staining patterns in the neurons of the dorsal raphe nucleus (DRN) for the purposes of the medical differential diagnostics of the cases of death from chronic alcohol intoxication. We elucidated the characteristics of the activity of protein biosynthesis including the number and the area of the nucleoli in the nuclei of the neurons of the individuals who had died from chronic alcohol intoxication (n=20) in comparison with the subjects of the control group (n=13). To reveal the morphological structures associated with protein biosynthesis in the nucleoli of the serotoninergic neurons of the dorsal raphe nucleus in the brain, the histological preparations were stained with the use of the silver-staining technique for nucleolar organizer regions (AgNOR). The comparative statistical analysis of the results thus obtained with the calculated confidence coefficients was carried out. The aggregated analysis of all the dorsal raphe subnuclei revealed the impairment of the AgNOR staining characteristics in the neurons of the subjects who had died from chronic alcohol intoxication in comparison with those of the subjects comprising the control group. It is concluded that the results of the study can be used for differential diagnostics of deaths from chronic alcohol intoxication and other causes.

Utility of a modified silver staining technique for detection of Leptospira

Background: Leptospira are spiral thin and highly motile pathogenic bacteria that are best visualized by dark ground microscopy. Although these bacteria are not stained by the Gram’s stain, the Fontana stain, which is a silver impregnation staining method, can be used successfully for light microscopy. It is important to investigate the usefulness of Fontana stain method for direct demonstration of Leptospira in human body fluids. Objectives: To determine the usefulness and sensitivity of a modified Fontana silver staining method for microscopic detection of Leptospira in clinical specimens. Methodology: 6×108 organisms/ml of Leptospira interrogans serovar Icterohaemorrhagiae and Canicola were spiked into PBS (Phosphate Buffered Saline), alkalinized urine and serum in triplicate and serial dilutions were made (6×106 to 6×101 organisms/ml). Smears were prepared using 10 µl of each dilution. In addition, centrifuged sediment of urine were also used to prepare smears. Slides were stained by modified Fontana method as reported by Gangadhar et al.(1998) and examined. Numbers of leptospires per field (×100) were recorded. Results: Leptospira spiked in PBS and urine appeared as thin slender bacteria with characteristic hooked ends after Fontana staining under the light microscope. Serum could not be used for the detection of Leptospira by this method. Leptospires could be detected by staining the spiked PBS and urine at 6x103 – 6x106 organisms/ml. Conclusion: Leptospires could be detected by Fontana staining in spiked PBS, urine (uncentrifuged and sediment). Serum was not suitable for detection of leptospires by Fontana staining. The detection limit of leptospires in PBS and urine by Fontana stain was found to be 6000 organisms/ml.

Christmas-tree Derived Amplification Immuno-strategy for Sensitive Visual Detection of Vibrio parahaemolyticus Based on Gold Label Silver Stain Technology.

A developed Christmas-tree derived immunosensor based on a gold label silver stain (GLSS) technique was fabricated for a highly sensitive analysis of Vibrio parahaemolyticu (VP). In this strategy, captured VP antibody (cAb) was immobilized on a solid substrate; then, the VPs were sequentially tagged with a signal probe by incubating the assay with a detection VP antibody (dAb) conjugated gold nanoparticles (AuNPs)-labeled graphite-like carbon nitride (g-C3N4). Finally, the attached signal probe could harvest a visible signal by the silver meal deposition, and then followed by homebrew Matlab 6.0 as a grey value acquisition. In addition, the overall design of the biosensor was established in abundant AuNPs and g-C3N4 with a two-dimensional structure, affording a bulb-decorated Christmas-tree model. Moreover, with the optimized conditions, the detection limit of the as-proposed biosensor is as low as 102 CFU (Colony-Forming Units) mL-1, exhibiting an increase of two orders of magnitude compared with the traditional immune-gold method. Additionally, the developed visible immunosensor was also successfully applied to the analysis of complicated samples.

The historical background of Jules and Augusta Dejerines’ work

The historical background of Jules and Augusta Dejerines’ work