Congo Red Method Research Articles

Catalytic synthesis of sulfated polysaccharides. II: Comparative studies of solution conformation and antioxidant activities

Sulfated derivatives of Artemisia sphaerocephala polysaccharide (ASP) with high degree of substitution (DS) were synthesized using 4-dimethylaminopyridine (DMAP)/dimethylcyclohexylcarbodiimide (DCC) as catalyst. Size exclusion chromatography combined with multi-angle laser light scattering (SEC-LLS) results showed a decrease in fractal dimension (df) values of sulfated ASP (SASP). Compared to ASP and SASP with low DS (0.51–1.01), SASPcata2 exhibited an internal structure between rigid rod and random coil with a DS of 1.24. DS had greater influence on its conformation in aqueous solution. Circular dichroism (CD), methylene blue (MB) and Congo red (CR) spectrophotometric method and atomic force microscopy (AFM) results confirmed that the degradation of ASP and SO3H groups improved significantly the stiffness of the chains due to the electrostatic effect. Furthermore, antioxidant experiments revealed that high DS could enhance the scavenging activities of radicals and reducing power of SASP in vitro. The extended chain conformation was beneficial to enhance the biological activity of sulfated polysaccharides.

Characterization of Thermal Reversible Cross-Linking Agents for Flexible Poly(vinyl chloride)

In order to improve thermal stability of Poly (Vinyl Chloride), a kind of thermal reversible cross-linking agents, the end cyclopentadienyl ethanethiol salts, sodium cyclopentadienyl ethyl mercaptan (CPD-C2H4SNa) was synthesized in this paper. The DSC results proved that the prepared CPD-C2H4SNa has obvious thermal reversible characteristics and the thermal reversible temperature was at 177 °C. The effects of cross-linking agent amount, cross-linking reactive time and temperature on flexible PVC compounds were evaluated through gel content testing. It was showed that the cross-linking agent with 3 Phr is optimum for PVC compounds. Moreover, both the cross-linking reactive time and temperature has effects on gel content of PVC compounds. The gel content of CPD-C2H4SNa cross-linked PVC compounds reaches maximum at 76% for 20 min at 160 °C.The static thermal stability of flexible PVC was measured according to congo red method. The results showed that the thermal stability of PVC compounds was enhanced with increasing cross-linking agent amount.

Gum arabic capped‐silver nanoparticles inhibit biofilm formation by multi‐drug resistant strains of Pseudomonas aeruginosa

Clinical isolates (n = 55) of Pseudomonas aeruginosa were screened for the extended spectrum β-lactamases and metallo-β-lactamases activities and biofilm forming capability. The aim of the study was to demonstrate the antibiofilm efficacy of gum arabic capped-silver nanoparticles (GA-AgNPs) against the multi-drug resistant (MDR) biofilm forming P. aeruginosa. The GA-AgNPs were characterized by UV-spectroscopy, X-ray diffraction, and high resolution-transmission electron microscopy analysis. The isolates were screened for their biofilm forming ability, using the Congo red agar, tube method and tissue culture plate assays. The biofilm forming ability was further validated and its inhibition by GA-AgNPs was demonstrated by performing the scanning electron microscopy (SEM) and confocal laser scanning microscopy. SEM analysis of GA-AgNPs treated bacteria revealed severely deformed and damaged cells. Double fluorescent staining with propidium iodide and concanavalin A-fluorescein isothiocyanate concurrently detected the bacterial cells and exopolysaccharides (EPS) matrix. The CLSM results exhibited the GA-AgNPs concentration dependent inhibition of bacterial growth and EPS matrix of the biofilm colonizers on the surface of plastic catheters. Treatment of catheters with GA-AgNPs at 50 µg ml(-1) has resulted in 95% inhibition of bacterial colonization. This study elucidated the significance of GA-AgNPs, as the next generation antimicrobials, in protection against the biofilm mediated infections caused by MDR P. aeruginosa. It is suggested that application of GA-AgNPs, as a surface coating material for dispensing antibacterial attributes to surgical implants and implements, could be a viable approach for controlling MDR pathogens after adequate validations in clinical settings.

Preparation of MgAl LDHs Intercalated with Amines and Effect on Thermal Behavior for Poly(vinyl chloride)

MgAl LDHs intercalated with CO(NH2)2, NH4Cl and NH3·H2O were prepared by co-precipitation and XRD, FT-IR, TGA-DTA and SEM techniques were employed for characterization. The results indicated that the layer-layer spacing of LDHs was enlarged by 0.169, 0.285 and 0.227 A with the intercalation of CO(NH2)2, NH+4 and NH3, separately. The effects on thermal stability and degradation behavior of synthesized LDHs where mole ratios of Mg/Al/urea = 3:1:1, Mg/Al/NH4Cl = 3:1:1.5 and Mg/Al/NH3·H2O = 3:1:0.5 were investigated by Congo Red Method and thermogravimetric analysis (TGA) from 25°C - 800°C under N2. The TGA and DTA results showed, by incorporating 5% MgAl-CO(NH2)2 LDH, the dehydrochlorination temperature of PVC composites is 12°C higher than MgAl-NH+4 and MgAl-NH3 LDHs. MgAl-NH3 LDH enhances the ending temperature of the first degradation stage of PVC composites by 8°C compared with MgAl LDH. The LDH intercalated with CO(NH2)2 is proved to be an effective thermal stabilizer for PVC processing.

Comparison of Microscopic Morphology of Fungi Using Lactophenol Cotton Blue (LPCB), Iodine Glycerol and Congo Red Formaldehyde Staining.

Comparison of Microscopic Morphology of Fungi Using Lactophenol Cotton Blue (LPCB), Iodine Glycerol and Congo Red Formaldehyde Staining.

초식동물 배설물로부터 분리한 Bacillus sp. H9-1의 섬유소 분해효소생산 최적화

This study was performed to find cellulolytic strain of enzymatic saccharification for bioethanol production. Cellulolytic strains were isolated from 59 different feces of herbivores from Seoul Grand Park located in Gwacheon Gyeonggi-Do. The celluloytic strain was selected by congo red staining and DNS method. Among the isolated strains, H9-1 strain isolated from the feces of rabbit has the highest CMCase activity. H9-1 strain was identified as Bacillus sp. based on 16S rDNA gene sequencing. The optimal conditions for CMCase activity by Bacillus sp. H9-1 were at <TEX>$40^{\circ}C$</TEX> and at initial pH 8.

Defining Niagara Terroir by Chemical and Sensory Analysis of Chardonnay Wines from Various Soil Textures and Vine Sizes

An alternative method for detecting the production of slime by coagulase negative staphylococci was compared with the routinely used Christensen method on 124 isolates of coagulase negative staphylococci from carriage sites, blood cultures, and infected peritoneal dialysis fluids. The alternative method requires the use of a specially prepared solid medium--brain heart infusion broth, supplemented with 5% sucrose, and Congo red stain. Of the 124 tests, there was complete agreement between methods in 107 and only one strain was clearly negative by Christensen9s method while positive on Congo red agar. The Congo red method is rapid, sensitive, and reproducible and has the advantage that colonies remain viable on the medium. It is also not subject ot interbatch variation of media which sometimes affects the reproducibility of the Christensen method.

Influence of Polyols as a Co-Stabilizer on Stabilization Efficiency of Calcium/Zinc Stabilizers to PVC/ACS Blend

In order to prepare good thermal stabilizer for PVC/ACS blends, the Congo red methods was adopted to determine the thermal stabilizing time of blends and study the synergy effect between polyols and Ca/Zn stabilizer for PVC/ACS blends. The rheological behaviors and dynamic mechanical properties of PVC/ACS blends were investigated by rotational rheometer and dynamic mechanical analyzer (DMA). The results showed that the different polyols have different influences on the thermal stability, melt viscosity, dynamic mechanical properties of PVC/ACS blends. The pentaerythritol has a good synergy effect with Ca/Zn stability and the PVC/ACS blend has a lower melt viscosity.

Influence of Polyols as a Co-Stabilizer on Stabilization Efficiency of Calcium/Zinc Stabilizers to PVC

In order to prepare the innocuity polyvinyl chloride (PVC) products, the calcium/zinc stearate was used as the thermal stabilizer of PVC and the polyols were used as co-stabilizer of Ca/Zn stabilizer. The influence of polyols on the stabilization efficiency of Ca/Zn stabilizer to PVC was investigated by Congo red methods. The rheological behaviors and dynamic mechanical properties of PVC were also investigated by rotational rheometer and dynamic mechanical analyzer (DMA). The results showed that different polyols have different influences on the stability, but the pentaerythritol has a good synergy effect with Ca/Zn stabilizer for PVC and pentaerythritol and dipentaerythritol have a lower melt viscosity and good product color.

Effect of lanthanide (La(III))‐containing ionomer on thermal stabilization of poly(vinyl chloride)

AbstractA type of lanthanide (La(III))‐containing ionomer based on acrylate processing aid (ACR) for poly (vinyl chloride) (PVC) was synthesized, and influence of the ionomer on thermal stabilization of PVC was investigated with visual color comparison and Congo red methods. Results revealed that the ionomer with a suitable La(III) content behaved as a good costabilizer to PVC. It was able to extend static stabilization time of PVC and postpone “zinc burning.” The stabilizing efficiency of the ionomer to PVC depended on ion content, which was discussed in terms of Eisenberg–Hird–Moore model. Moreover, Fourier transform infrared test verified that this ionomer can react with zinc stearate (ZnSt2) to form some new structures, which is responsible for postponing “zinc burning.” The ionomer and epoxidized soybean oil exhibited a synergistic effect on the stabilizing efficiency of calcium stearate (CaSt2)/ZnSt2 stabilizer to PVC compounds. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Clearance of Amyloid Beta Plaques from Brain of Alzheimeric Rats by Lavandula angustifolia

An important marker in neurodegenerative Alzheimer’s disease (AD) is abnormal production of amyloid beta (Aβ) peptide leading to formation of plaques in the brain. Through decreasing Aβ aggregates, anti-inflammatory agents, phagocytosis, and proteolytic enzymes are known to decline risk of Aβ plaque formation. In the previous study we showed that aqueous extract of Lavandula angustifolia (lavender), with known anti-inflammatory effects, improves memory deficits in animal model of Alzheimer. Here, we assess if lavender play a role in clearance of Aβ plaques in the hippocampus. The Alzheimeric animals were created with intracerebroventricular injection of Aβ 1-42. To confirm formation of Aβ plaques, brain sections were stained by Congo red method. Twenty days post-injection they were administered with different doses (50, 100 and 200 mg/kg) of the aqueous extract of lavender for duration of 20 days. Our results demonstrated that 50 mg/kg of lavender not effectively influenced the Aβ plaques. On the other hand, the herbal medicine at the doses of 100 and 200 mg/kg markedly decreased the extent of Aβ aggregates. We concluded that the lavender extract dose dependently underlies elimination of Aβ plaques. The exact mechanism by which the herbal medicine removes the Aβ aggregates needs to be elucidated.

Screening of a Thermostable Cellulase-producing Strain and Its Enzymatic Properties

Using CMC Congo Red agar plate method,an actinomycetic strain producing cellulase with high temperature tolerance and pH stability was isolated from the sample collected from mushroom plantation.The strain was identified and named as Thermobifida fusca DY3 by morphological,physiological and biochemical characteristics,and 16S rDNA gene sequence analysis.The study on the cellulase showed that the extracellular thermostable cellualse exhibited the maximum activity at 65 ℃ and retained more than 75% of its maximum activity after incubation for 60 minutes at 70 ℃.The enzyme also showed its pH stability between pH 5.5 and 10.0,and retained 80% of its maximum enzymatic activity after treated at pH 10.0.The sodium carboxymethylcellulose was its best zymolyte.All the characteristics of the enzyme provide reference value for its application in biological pretreatment of lignocellulose.

Comparison of the adhesion ability of Candida albicans strains to biotic and abiotic surfaces

The purpose of the present study is to investigate the ability of oral Candida albicans strains to adhere to Caco-2 and Hep-2 epithelial cells, to produce slime using Congo red and Safranin methods and to form a biofilm on polymethylmethacrylate. A total of 20 C. albicans strains were tested in the present work. The biofilm formed by C. albicans isolates on acrylic denture was measured in vitro using the colorimetric method based on the reduction of the tetrazolium salt (XTT) and dry weight measurement. Our results showed that oral C. albicans strains were able to adhere to epithelial cell lines and biomaterials with different degree. In fact, more than 61% of the tested strains were adhesive to Hep-2 and 83% to Caco-2 cells. Strong slime production after safranin stain was found in 40% of strains. Slime producer C. albicans strains (16 strains) tested by safranin staining method were highly adhesive to Hep-2 (62.5%) and Caco-2 (87.5%) monolayers and biofilm formed on polystyrene and poly-methyl methacrylate (PMMA) surfaces (100%, respectively). Key words : Candida albicans , biofilm, polymethylmethacrylate, Hep-2, Caco-2 cells, slime production.

Evaluation of different detection methods of biofilm formation in clinical Candida isolates.

Biofilm is one of the known virulence factors of Candida, an important pathogen and commensal. Early detection of biofilm production may be useful for clinical decision because of its suggestive property for potential pathogenic capacity of Candida isolates. In this study, we simultaneously screened 411 clinical isolates of Candida spp. by microtiter plate (MTP), visual tube (TM) and congo red agar (CRA) methods for determining their ability to form biofilm and also evaluated the reliability of these methods in order to determine most suitable screening method. The accuracy of TM and CRA methods were also evaluated by using MTP as reference method. Of the 411 Candida spp., 159 (38.7%) displayed a biofilm-positive phenotype by MTP method. Sensitivity and specificity of the tube method were 68 and 98%, respectively and the positive predictive value (PPV) was 97% and the negative predictive value (NPV) was 83%. The sensitivity for congo red agar test was 79% and the specificity was 99%, and the PPV was 99% and the NPV was 88%. Tube method showed very good agreement for the isolates producing strong biofilm, whereas differentation of isolates producing weak biofilm was difficult. By the congo red method, classification of existing biofilm was problematic. Among the three methods studied, microtiter plate method may be suggested as the most sensitive method, which is easy to conduct and applicable as a routine process. Key words: Candida, biofilm, microtiter plate, tube method, congo red agar.

Morphological changes of the human purkinje cells and deposition of neuritic plaques and neurofibrillary tangles on the cerebellar cortex of Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disorder, characterized by progressive decline in memory and in social performance. The morphological hallmarks of the disease are neuronal loss, loss of dendritic spines, neurofibrillary degeneration and neuritic plaques mainly in the hippocampus and the cortex of the cerebral hemispheres. This study is based on the morphological analysis of the cerebellar cortices of eight brains, 4 patients suffered from Alzheimer's disease and 4 normal controls, by Golgi method, as well as Nissl, Gallyas', Bielschowsky's, Methenamine Silver staining and Congo red methods. Although typical neuritic plaques were not seen in the cerebellar cortex and the diffuse plaques found in the cerebellum in far smaller proportion than plaques in the prefrontal and parietal cortices of the same cases, Golgi impregnation technique revealed a loss of Purkinje cells and a marked decrease in the density of dendritic arborization.

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-l,3-1,4-beta-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone alpha-factor (MFalpha1(S)), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-betaG) was preliminarily screened by the clearing hydrolysis zone formed after the barley beta-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-l,3-1,4-beta-glucanase assay methods showed that the recombinant strain SC-betaG had high endo-l,3-1,4-beta-glucanase expression level with the maximum of 69.3 U/(h.ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-l,3-1,4-beta-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Poly(vinyl chloride)-paste/clay nanocomposites: Investigation of thermal and morphological characteristics

The paper concentrates on poly(vinyl chloride) – PVC – from the point of nanocomposite characterisation through thermal degradation of samples, evolution of the changes caused by elevating temperature using TGA, FTIR and Congo Red methods combined with morphological characterisation by XRD and TEM analyses. A novel method of PVC-paste/nanocomposite preparation while processing was used. During preparation, PVC plasticizer was mixed with clay, both natural and organophilic, and the suspension was then compounded with the other components. Two factors were followed: effect of shearing alone, and in combination with temperature. As is presented, the type of nano-filler and its chemical modifier have obvious influence on final properties either thermal or morphological. Presented contribution follows previous part of investigation and brings further information from PVC-paste/nanocomposite field.

Enhancement of Cellulase Activity by Clones Selected from the Combinatorial Library of the Cellulose-Binding Domain by Cell Surface Engineering

To improve the cellulolytic activity of a yeast strain displaying endoglucanase IotaIota (EG II) from Trichoderma reesei, a combinatorial library of the cellulose-binding domain (CBD) of EG II was constructed by using cell surface engineering. When EG II degrades celluloses, CBD binds to cellulose, and its catalytic domain cleaves the glycosidic bonds of cellulose. CBD had a flat face, composed of five amino acids for binding. It was supposed that the three hydrophobic amino acid residues of the five amino acid residues were essential for binding to cellulose. Therefore, by improving the two remaining amino acid residues, construction of mutants with a combinatorial library of the two amino acids in CBD was carried out and binding ability and hydrolysis activity were measured. In the first screening by halo assay using the Congo Red staining method, about 200 of the 2000 colonies formed clear halos, and then five colonies with the clearest halos were finally selected. In the second screening, the binding ability of the five mutants to phosphoric acid-swollen Avicel was measured. In addition, the measurement of hydrolysis activity toward carboxymethylcellulose (CMC) using the screened mutants was carried out. As a result, the mutated EG II exhibiting higher binding ability (1.5-fold) had higher hydrolysis activity (1.3-fold) compared to the parent EG II-displaying yeast cell, demonstrating that CBD has confirmatively some effect on the cellulase activity through its binding ability of the enzyme to cellulose.

Quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with Congo red histochemical stain

In the current protocol, we describe the Congo red staining method and a method for separately quantifying vascular and parenchymal amyloid deposits in brain tissue sections. Congo red staining detects amyloid deposits in brain tissue of amyloid precursor protein transgenic mice and human Alzheimer's tissue. It detects compacted amyloid in a beta-sheet secondary structure and labels amyloid in both the brain parenchyma (amyloid plaques) and blood vessels. Congophilic amyloid in blood vessels is called cerebral amyloid angiopathy (CAA). To date, analysis of CAA has largely used a severity rating scale, including both qualitative and quantitative characteristics. Here, we describe a simple method for quantifying total Congophilic staining and resolution of this staining into the parenchymal and vascular components based on morphological criteria. It is becoming increasingly important to separately quantify various components of the Alzheimer's pathology, given the advancement of amyloid-lowering therapies into clinical trials. The entire procedure for the Congo red staining can be performed at room temperature (20-25 degrees C) in a fume hood. The staining protocol should take 1 h 30 min including time for coverslipping slides. Time required for image analysis depends greatly on the number of samples being analyzed and the software being used. In our hands, 30 images can be collected per hour and quantified in a further 2 h.

Sensitivity and specificity of Congo red staining according to Romhányi. Comparison with Puchtler's or Bennhold's methods

The sensitivity and specificity of various Congo red staining methods is very important in the diagnosis of amyloidosis. When using a less sensitive staining method, some true positive cases of amyloidosis remain undetected. A more highly specific method potentially detects more cases and reveals amyloidosis in an earlier stage of deposition. In this paper, the Congo red staining method according to Romhányi is discussed in comparison with Puchtler's and Bennhold's methods. Using Romhányi's technique, there is no alcoholic differentiation, and thus no dye molecules are washed off the amyloid filaments. The binding of the oriented dye molecules is optimal for polarization microscopy. With this method, the polar hydrophilic mounting medium, gum Arabic is used. Mounted in this carbohydrate-containing, hydrophilic medium, the Congo red molecules are oriented parallel to the surface of the amyloid filaments and the sign is linear positive, corresponding to an additive character of topo-optical staining reactions. Otherwise, the Congo red molecules are oriented perpendicular to the surface of collagen, reducing the intensity of birefringence and even inducing an inversion of the original sign of the collagen birefringence. With alcoholic differentiation, Congo red dye molecules are extracted and this decreases the birefringence of amyloid deposits, i.e. minimal amyloid deposits may be missed. Using the apolar hydrophobic mounting medium, Canada balsam, an axis-parallel arrangement of Congo red dye molecules on the surface of collagen fibers and amyloid will occur, resulting in an additive topo-optical reaction with a green polarization color and a false positive diagnosis of amyloidosis ("phantom amyloidosis").