Chrome Azurol Research Articles

Spectrophotometric Microdetermination of Thorium(IV) and Uranium(VI) with Chrome Azurol‐S in Presence of Cationic Surfactant

Cationic surfactant, cetyldimethylethylammonium bromide (CDMEAB), sensitize the color reactions of Th(IV) and U(VI) with chrome azurol‐S(CRAS). Formation of water soluble deeply colored ternary complexes of metal ions show large bathochromic shift. Same stoichiometric composition of ternary complexes with 1:2:4 molar ratio (M‐CRAS‐CDMEAB) have been observed for both the metal ions and are responsible for enhancement in molar absorptivities and sensitivities at shifted wavelength. The ternary complexes of thorium(IV) and uranium(VI) exhibit absorption maxima at 640 and 620 nm with molar absorptivities 85500 and 69600 L.mol-1.cm-2 respectively. Beer’s law were obeyed in concentration range 0.12‐0.185 ppm for Th(IV) and 0.13‐0.162 ppm for U(VI) in presence of CDMEAB. Conditional formation constants and various analytical parameters have been evaluated and compared the results of binary and ternary complexes. Enhancement in the molar absorptivities in presence of CDMEAB clearly indicated the usefulness of these colored reactions for microdetermination.

Use of Blue Agar CAS Assay for Siderophore Detection

Schwyn and Neiland developed a universal siderophore assay using chrome azurol S(CAS) and hexadecyltrimethylammonium bromide (HDTMA) as indicators. In Schwyn and Neiland’s original paper, the procedure given for making CAS agar is written in general terms and can be difficult to follow, especially for an individual who has limited experience making more complex media. Here, we give a step-by-step protocol for making the CAS agar plates, and we discuss how this media can be incorporated in a comprehensive project in a microbiology lab course for biology majors.

Cytochrome c 4 is required for siderophore expression by Legionella pneumophila, whereas cytochromes c 1 and c 5 promote intracellular infection

A panel of cytochrome c maturation (ccm) mutants of Legionella pneumophila displayed a loss of siderophore (legiobactin) expression, as measured by both the chrome azurol S assay and a Legionella-specific bioassay. These data, coupled with the finding that ccm transcripts are expressed by wild-type bacteria grown in deferrated medium, indicate that the Ccm system promotes siderophore expression by L. pneumophila. To determine the basis of this newfound role for Ccm, we constructed and tested a set of mutants specifically lacking individual c-type cytochromes. Whereas ubiquinol-cytochrome c reductase (petC) mutants specifically lacking cytochrome c1 and cycB mutants lacking cytochrome c5 had normal siderophore expression, cyc4 mutants defective for cytochrome c4 completely lacked legiobactin. These data, along with the expression pattern of cyc4 mRNA, indicate that cytochrome c4 in particular promotes siderophore expression. In intracellular infection assays, petC mutants and cycB mutants, but not cyc4 mutants, had a reduced ability to infect both amoebae and macrophage hosts. Like ccm mutants, the cycB mutants were completely unable to grow in amoebae, highlighting a major role for cytochrome c5 in intracellular infection. To our knowledge, these data represent both the first direct documentation of the importance of a c-type cytochrome in expression of a biologically active siderophore and the first insight into the relative importance of c-type cytochromes in intracellular infection events.

The siderophore-producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium wilt and promotes the growth of pepper

In this study, the Chrome Azurol Sulphonate (CAS) agar plate assay for siderophores was used to screen bacteria from the rhizospheric soil of pepper in Hainan, China. As a result, Bacillus subtilis CAS15, which produced a large orange halo, was obtained. A dual-culture test showed that CAS15 strongly antagonized the growth of 15 plant fungal pathogens, with rates of inhibition ranging from 19.26 to 94.07%. Analysis of the siderophore production by the ESI-MS and DHB(G) assays showed that CAS15 produced the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin, and that siderophore productivity was inhibited by iron. A pot culture experiment was used to study the effects of B. subtilis CAS15 on pathogen development and plant growth. CAS15 reduced the incidence of Fusarium wilt in pepper significantly, by 12.5–56.9%, which indicated that B. subtilis CAS15 induced systemic resistance to Fusarium wilt in pepper. Iron supplementation reduced this biocontrol effect. There were significant differences in plant height at 14, 21, 28 and 40 days, with treated plants growing 27.24–54.53% taller than controls. B. subtilis CAS15 also enhanced the yield of pepper by shortening the time to 50 percent flowering to 17.26 days, increasing the average fruit weight 36.92%, and increasing the average yield per plant 49.68%. This research showed that B. subtilis CAS15 has great potential for plant growth promotion and biological control.

Plant Growth Promoting Characteristics in Some Flavobacterium spp. Isolated from Soils of Iran

Plant growth promoting rhizobacteria (PGPR) is referred to a heterogeneous group of beneficial rhizospherebacteria that could enhance plant yield through one or more mechanisms. Flavobacterium has been noted asPGPR in almost all review articles. However, there are a few studies regarding plant growth promotion imposedby them. Some of Plant growth promoting characteristics such as Phosphate solubilizing capacity, ability to useof 1-Amino Cyclopropan-1-Carboxylate (ACC) as sole nitrogen source and production of auxin, siderophore,salicylic acid, chitinase and hydrogen cyanide were evaluated in forty-four flavobacteria isolated fromrhizosphere of wheat. Results showed that none of the isolates were able to produce siderophore, salicylic acidand chitinase and they were not able to use ACC as well. Determining the siderophore showed that none isolatesdid not grow on Chrome Azurol S (CAS) Agar medium. The results of this part were further analyzed usingCAS Agar Diffusion (CASAD) method, but the results were also negative. HCN production was observed in allisolates, but in lowest limit. Thirty-four isolates were capable to solubilize insoluble inorganic Phosphate (P)sources. The average rate of P-solubilization was 3.54 ?g Pml-1, ranging from zero to 37.48 ?gPml-1. There wasa significant negative correlation (r = -0.81**) between solubilized P and the final pH of the growth medium. Inthis study, all the isolates were able to produce auxin, ranging from 0.27 to 12.03 ?gml-1 averaged by 2.03.Considering the ability of the isolates to produce auxin and for P-solubilization, it is necessary to evaluate theireffect on growth and yield of different crop plants.

Lead-Enhanced Siderophore Production and Alteration in Cell Morphology in a Pb-Resistant Pseudomonas aeruginosa Strain 4EA

A lead-resistant bacterial strain 4EA from soil contaminated with car battery waste from Goa, India was isolated and identified as Pseudomonas aeruginosa. This lead-resistant bacterial isolate interestingly revealed lead-enhanced siderophore (pyochelin and pyoverdine) production up to 0.5 mM lead nitrate whereas cells exhibit a significant decline in siderophore production above 0.5 mM lead nitrate. The bacterial cells also revealed significant alteration in cell morphology as size reduction when exposed to 0.8 mM lead nitrate. Enhanced production of siderophore was evidently detected by chrome azurol S agar diffusion (CASAD) assay as increase in diameter of orange halo, and reduction in bacterial size along with significant biosorption of lead was recorded by scanning electron microscopy coupled with energy dispersive X-ray spectrometry (SEM-EDX). Pseudomonas aeruginosa strain 4EA also exhibits cross tolerance to other toxic metals viz. cadmium, mercury, and zinc besides resistance to multiple antibiotics such as ampicillin, erythromycin, amikacin, cephalexin, co-trimoxazole, mecillinam, lincomycin, ciphaloridine, oleondamycin, and nalidixic acid.

Characterization of Pseudomonas aeruginosa RM-3 as a Potential Biocontrol Agent

Molecular characterization of rhizobacterial isolate RM-3, based on sequencing of a partial 1,313-bp fragment of 16S rDNA amplicon, validated the strain as Pseudomonas aeruginosa. The strain showed significant growth inhibition of different phytopathogenic fungi in dual plate and liquid culture assays. Maximum growth inhibition was found in case of Macrophomina phaseolina in plate assay (68%), whereas it was 93% in Dreschlera graminae in dual liquid assay. Microscopic studies (light and scanning electron) showed morphological abnormalities such as perforation, fragmentation, swelling, shriveling and lysis of hyphae of pathogenic fungi. The strain also exhibited production of siderophore and hydrogen cyanide (HCN) on chrome azurol S and King's B media, respectively. Besides, this strain also produced extracellular chitinase enzyme and an important antibiotic, phenazine. Seed bacterization with RM-3 showed a significant (P < 0.05) increase in seed germination, shoot length, shoot fresh and dry weight, root length, root fresh and dry weight and leaf area. It was also able to colonize the rhizosphere of plants and reduced percent disease incidence in M. phaseolina-infested soil by 83%. Yield parameters such as pods, number of seeds and grain yield per plant also enhanced significantly (P < 0.05) in comparison to control. Thus, the secondary metabolites producing Pseudomonas aeruginosa strain RM-3 exhibited innate potential of plant growth promotion and biocontrol potential in vitro and in vivo.

Hydrolysis and Speciation of Al Bound to Pectin and Plant Cell Wall Material and Its Reaction with the Dye Chrome Azurol S

Hydrolysis of aluminum (Al) in solution increases at pH >or= 4 and with an Al concentration. Pectin, an important anionic polysaccharide of plant cell walls, adsorbs Al, but this phenomenon is poorly understood. This study showed that Al(3+) hydrolysis results in binding of Al to pectin in excess of the stoichiometric equivalent, leading to oversaturation of the pectin with Al. However, the degree of pectin methyl-esterification did not affect the extent of Al hydrolysis. Binding of Al to purified cell wall material also resulted in Al hydrolysis in a pH- and soluble Al concentration-dependent manner, but the source of cell wall material had no effect at fixed pH. Staining of Al-treated pectin and cell wall material from wheat ( Triticum aestivum L.) and sunflower ( Helianthus annuus L.) with the Al-specific dye, chrome azurol S (CAS), resulted in the formation of a purple color, with the intensity related to the extent of Al hydrolysis.

Aggregation-induced sensitization in aqueous solutions of Al 3+–chrome azurol S complexes and ionic liquids based on 1-alkyl-3-methylimidazolium bromide

The dependences of the absorption spectra of chrome azurol S (CAS) and Al 3+–CAS complexes on the concentration of ionic liquids (ILs), 1-alkyl-3-methylimidazolium bromide ([C n Mim]Br, with n = 8, 10, 12), were measured at 298.15 K in aqueous solutions with pH = 5.6. It is found that ILs can induce the significant red-shifts of the wavelength at the absorption maximum ( λ max) of Al 3+–CAS complex from 545 to 646 nm for [C 8Mim]Br, and to 642 nm for [C 12Mim]Br and [C 16Mim]Br, respectively. Accompanying the red-shifts, the absorbance at λ max, and thereby molar absorptivity ( ɛ), changes markedly with the IL concentration, and consequently the effect of ILs on the absorbance at λ max is systematically investigated in this work. Along with the experimental results of resonance light scattering, the sensitization mechanism is analyzed from the aggregation of the ILs and the interactions of ILs with CAS and Al 3+–CAS complexes. It is shown that the hydrophobic interactions of IL subaggregates with CAS in Al 3+–CAS complex are the crucial factor for the sensitization.

Interaction of Chromium(III) with Chrome Azurol S in Water–Glycerol Media

The interaction of Cr(III) with Chrome azurol S (CAS) in water–glycerol binary mixtures has been investigated by spectrophotometry. The absorption electronic spectra of Chrome azurol S and its complex with Cr(III) in water–glycerol media with glycerol contents from 10 to 50% by vol. have been measured. The stability constant of the complex in water–glycerol binary mixtures has been determined. It has been shown that the rate of reaction increases as the organic solvent content increases.

An assay for screening microbial cultures for chalkophore production

Methanotrophs, bacteria that utilize methane as their sole carbon and energy source, are known to have high requirements for copper. These bacteria have recently been found to synthesize a copper-chelating agent, or chalkophore, termed methanobactin. To aid in screening methanobactin production by methanotrophs, a plate assay developed from the chrome azurol S (CAS) assay for siderophore production, was modified. In the typical CAS assay, a colour change from blue to orange in iron-CAS plates is observed as iron (III) ion weakly bound to CAS is sequestered by siderophores with higher affinities. In our modified assay, iron (III) chloride of the original CAS solution was substituted with copper (II) chloride, and removal of copper from CAS caused a colour change from blue to yellow. Assay results indicated that of the four tested methanotrophs (Methylosinus trichosporium OB3b, Methylococcus capsulatus Bath, Methylomicrobium album BG8 and Methylocystis parvus OBBP), only M. trichosporium OB3b, M. capsulatus Bath and M. album BG8 produced chalkophores capable of competing with CAS for copper, while M. parvus OBBP did not or did not export sufficient concentrations of methanobactin for detection by this assay. It was also found using Fe-CAS plates that at least M. trichosporium OB3b and M. album BG8 produce siderophores. These results may be expanded for the detection of chalkophores in other microorganisms as well as for screening of putative mutants of chalkophore synthesis.

Siderophore production and utilization by milk spoilage Pseudomonas species

Many bacteria respond to potentially growth-limiting availability of iron by producing low-molecular-weight iron chelators (siderophores). The aim of this work was to examine the siderophores synthesized and utilized by Pseudomonas spp. implicated in milk spoilage. Twenty isolates of Pseudomonas spp. previously shown to have significant milk spoilage potential were tested for the ability to produce siderophores. Of these, 14 produced pyoverdin and 2 of these also produced pyochelin; 1 produced only pyochelin; 1 produced only salicylate; 2 produced non-pyoverdin, hydroxamate-containing siderophore; and 2 produced chrome azurol sulfonate reactive material that was neither pyoverdin nor pyochelin. There was considerable diversity among the pyoverdins produced. All isolates were shown to utilize iron complexed with exogenous pyoverdin, but usage of particular exogenous pyoverdins differed among isolates. Interference with the iron-uptake systems of the Pseudomonas spp. may be a means by which food spoilage can be slowed, and the pyoverdin system would appear to be a potential target. However, given the diversity of pyoverdins produced and utilized, and the presence of other siderophores, successful interference with bacterial iron acquisition in this context may be challenging.

Effect of drying method as a pretreatment on CUAZ preservative impregnation in Japanese cedar logs

Green Japanese cedar logs 2 m in length and 18 cm in diameter were dried to a mean moisture content of less than 30% by either air drying or kiln drying. Dried logs were impregnated with copper azole (CUAZ) solution according to Japanese Industrial Standard (JIS) A9002. Preservative absorption was calculated from the log weight before and after preservative impregnation. Impregnated logs were then dried in the air and cut at the center to determine preservative penetration. The penetration area was determined after visualizing the preservative with chrome azurol S. The visualized area indicated that the preservative solution penetrated into the sapwood portion of almost all the air-dried logs. However, the kiln-dried logs did not show full penetration into the sapwood portion. The visualized area of some kiln-dried sapwood showed a penetration value of less than 80%, which is the minimum requirement set by the Japanese Agricultural Standard (JAS) for sawn timber. Statistical analysis showed that penetration in the air-dried sapwood was significantly better than that in the kiln-dried sapwood. It was concluded that air drying is more favorable than kiln drying as the predrying method for CUAZ impregnation. On the other hand, preservative absorption was not affected by the drying method.

Visible light photodegradation of dyes over mesoporous titania prepared by using chrome azurol S as template

Mesoporous TiO2 with anatase crystalline structure (MTiO2/CAS) has been synthesized by using chrome azurol S (CAS, 2″,6″-dichloro-3,3′-dimethyl-4′-hydroxy-3″-sulfofuchson-5,5′-dicarboxylic acid) as template. It was characterized by X-ray diffraction, nitrogen adsorption/desorption, diffuse reflectance UV–visible and FT-IR spectrometry, and transmission electron microscopy. It was found that MTiO2/CAS had substantial photocatalytic activity in the degradation of methylthionine chloride, rhodamine B, gentian violet, safranin T, methyl violet, and fuchsine basic whereas Degussa P25 (P25) had negligible photodegradation yield (<6%) under visible light irradiation.

The 58-Kilodalton Major Virulence Factor of Francisella tularensis Is Required for Efficient Utilization of Iron

We investigated the role of the 58-kDa FTT0918 protein in the iron metabolism of Francisella tularensis. The phenotypes of SCHU S4, a prototypic strain of F. tularensis subsp. tularensis, and the Delta FTT0918 and Delta fslA isogenic mutants were analyzed. The gene product missing in the Delta fslA mutant is responsible for synthesis of a siderophore. When grown in broth with various iron concentrations, the two deletion mutants generally reached lower maximal densities than SCHU S4. The Delta FTT0918 mutant, but not the Delta fslA mutant, upregulated the genes of the F. tularensis siderophore locus (fsl) operon even at high iron concentrations. A chrome azurol sulfonate plate assay confirmed siderophore production by all strains except the Delta fslA strain. In a cross-feeding experiment using medium devoid of free iron, SCHU S4 promoted growth of the Delta fslA strain but not of the Delta FTT0918 strain. The sensitivity of SCHU S4 and the Delta FTT0918 and Delta fslA strains to streptonigrin demonstrated that the Delta FTT0918 strain contained a smaller free intracellular iron pool and that the Delta fslA strain contained a larger one than SCHU S4. In contrast to the marked attenuation of the Delta FTT0918 strain, the Delta fslA strain was as virulent as SCHU S4 in a mouse model. Altogether, the data demonstrate that the FTT0918 protein is required for F. tularensis to utilize iron bound to siderophores and that it likely has a role also in siderophore-independent iron acquisition. We suggest that the FTT0918 protein be designated Fe utilization protein A, FupA.

Chelating and antioxidant activity of lignans from Chilean woods (Cupressaceae)

Abstract Wood species resistant to biodegradation by fungi contain phenolic compounds with proven fungicidal activity. Saprophytic fungi are able to colonize wood and later begin to break down its polymeric components through metal-dependent reactions. These generate free radicals at the onset of the degradation process. Later on, extracellular enzymes are active that use metallic cofactors. Five lignans were isolated from the methanolic extracts of the Chilean heartwoods (Austrocedrus chilensis, Fitzroya cupressoides, and Pilgerodendron uviferum) belonging to the Cupressaceae: isotaxiresinol, secoisolariciresinol, isolariciresinol, didemethylmatairesinol, and matairesinol. Their structures were determined spectroscopically. The antioxidant activity of the methanolic extracts and the isolated compounds was determined in vitro against the radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and their iron-chelating capacity was evaluated by the Chrome Azurol S assay. All isolated methanolic extracts and lignans exhibited antioxidant activity against DPPH and Fe-chelating capacity with values greater than 87%. This may be interpreted that the antioxidant properties and metal-chelating capacity of these compounds have a protective effect on the wood of these species when exposed to attacks by saprophytic fungi.

Purification of Legiobactin and Importance of This Siderophore in Lung Infection by Legionella pneumophila

When cultured in a low-iron medium, Legionella pneumophila secretes a siderophore (legiobactin) that is both reactive in the chrome azurol S (CAS) assay and capable of stimulating the growth of iron-starved legionellae. Using anion-exchange high-pressure liquid chromatography (HPLC), we purified legiobactin from culture supernatants of a virulent strain of L. pneumophila. In the process, we detected the ferrated form of legiobactin as well as other CAS-reactive substances. Purified legiobactin had a yellow-gold color and absorbed primarily from 220 nm and below. In accordance, nuclear magnetic resonance spectroscopy revealed that legiobactin lacks aromatic carbons, and among the 13 aliphatics present, there were 3 carbonyls. When examined by HPLC, supernatants from L. pneumophila mutants inactivated for lbtA and lbtB completely lacked legiobactin, indicating that the LbtA and LbtB proteins are absolutely required for siderophore activity. Independently derived lbtA mutants, but not a complemented derivative, displayed a reduced ability to infect the lungs of A/J mice after intratracheal inoculation, indicating that legiobactin is required for optimal intrapulmonary survival by L. pneumophila. This defect, however, was not evident when the lbtA mutant and its parental strain were coinoculated into the lung, indicating that legiobactin secreted by the wild type can promote growth of the mutant in trans. Legiobactin mutants grew normally in murine lung macrophages and alveolar epithelial cells, suggesting that legiobactin promotes something other than intracellular infection of resident lung cells. Overall, these data represent the first documentation of a role for siderophore expression in the virulence of L. pneumophila.

Novel graphical method for the simultaneous determination of stability constant and molar absorptivity of some metal - Chrome Azurol S complexes

Stability constants and molar absorptivities of complexes formed by Chrome Azurol S (tri sodium salt of 3-sulpho-2:6-dichloro-3:3'-dimethyl-4-hydroxy-fuschsone-5:5-dicarboxy acid) with metal ions like Al III , Sc, Cu II , Th IV and U VI ions have been determined by a novel graphical method in which neither the concentration of ligand has to be kept high in comparison to the concentration of metal ion nor any term has to be omitted to get the linear equation.

Application of cotton as a solid phase extraction sorbent for on-line preconcentration of copper in water samples prior to inductively coupled plasma optical emission spectrometry determination

Copper, as a heavy metal, is toxic for many biological systems. Thus, the determination of trace amounts of copper in environmental samples is of great importance. In the present work, a new method was developed for the determination of trace amounts of copper in water samples. The method is based on the formation of ternary Cu(II)–CAS–CTAB ion-pair and adsorption of it into a mini-column packed with cotton prior applying inductively coupled plasma optical emission spectrometry (ICP-OES). The experimental parameters that affected the extraction efficiency of the method such as pH, flow rate and volume of the sample solution, concentration of chromazurol S (CAS) and cethyltrimethylammonium bromide (CTAB) as well as type and concentration of eluent were investigated and optimized. The ion-pair (Cu(II)–CAS–CTAB) was quantitatively retained on the cotton under the optimum conditions, then eluted completely using a solution of 25% (v/v) 1-propanol in 0.5 mol L −1 HNO 3 and directly introduced into the nebulizer of the ICP-OES. The detection limit (DL) of the method for copper was 40 ng L −1 ( V sample = 100 mL) and the relative standard deviation (R.S.D.) for the determination of copper at 10 μg L −1 level was found to be 1.3%. The method was successfully applied to determine the trace amounts of copper in tap water, deep well water, seawater and two different mineral waters, and suitable recoveries were obtained (92–106%).

Actinomycetes isolated from medicinal plant rhizosphere soils: diversity and screening of antifungal compounds, indole-3-acetic acid and siderophore production

A total of 445 actinomycete isolates were obtained from 16 medicinal plant rhizosphere soils. Morphological and chemotaxonomic studies indicated that 89% of the isolates belonged to the genus Streptomyces, 11% were non-Streptomycetes: Actinomadura sp., Microbispora sp., Micromonospora sp., Nocardia sp, Nonomurea sp. and three isolates were unclassified. The highest number and diversity of actinomycetes were isolated from Curcuma mangga rhizosphere soil. Twenty-three Streptomyces isolates showed activity against at least one of the five phytopathogenic fungi: Alternaria brassicicola, Collectotrichum gloeosporioides, Fusarium oxysporum, Penicillium digitatum and Sclerotiumrolfsii. Thirty-six actinomycete isolates showed abilities to produce indole-3-acetic acid (IAA) and 75 isolates produced siderophores on chrome azurol S (CAS) agar. Streptomyces CMU-PA101 and Streptomyces CMU-SK126 had high ability to produced antifungal compounds, IAA and siderophores.