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After diffusion of the tetrazolium salt to membrane reducing agents of the Bacillus subtilis cells, the reduction rate is shown to be affected by the state of the succinate dehydrogenase enzyme. Based on the dependence of the initial rate of iodonitrotetrazolium chloride reduction by the cellular components of Bacillus subtilis on the concentration of malonic acid, the competitive inhibition constant for the intracellular succinate dehydrogenase enzyme is quantitatively determined.

Purpose Bacterial metabolism regulators offer a novel productive strategy in the eradication of antibiotic refractory bacteria, particularly bacterial persisters. However, the potential of amino acids in the fight against Gram-negative bacterial persisters has not been fully explored. The aim of this study is to investigate the potentiation of amino acids to antibiotics in combating Gram-negative bacterial persisters and to reveal the underlying mechanisms of action. Methods Bactericidal activity of antibiotics in the absence or presence of amino acids was evaluated through detecting the reduction of bacterial CFUs. The ratio of NAD+/NADH in E. coli B2 persisters was determined using assay kit with WST-8. Bacterial respiration and ROS production were measured by the reduction of iodonitrotetrazolium chloride and fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate, respectively. Results In this study, we found that cysteine possesses excellent synergistic bactericidal activity with ciprofloxacin against multiple Gram-negative bacterial persisters. Furthermore, the potentiation of cysteine was evaluated in exponential and stationary-phase E. coli ATCC 25922 and E. coli B2. Interestingly, cysteine significantly improves three bactericidal antibiotics killing against stationary-phase bacteria, but not exponential-phase bacteria, implying that the effect of cysteine correlates with the metabolic state of bacteria. Mechanistic studies revealed that cysteine accelerates the bacterial TCA cycle and promotes bacterial respiration and ROS production. These metabolic regulation effects of cysteine re-sensitive bacterial persisters to antibiotic killing. Conclusion Collectively, our study highlights the synergistic bactericidal activity of bacterial metabolism regulators such as cysteine with commonly used antibiotics against Gram-negative bacterial persisters.

Purification of the aerial parts of Vernonia auriculifera Hiern afforded steroids (1–2), flavonoids (3–5), and polyalcohol (6). Their structures were determined using spectral evidences as well as by comparison with reported data. Iodonitrotetrazolium chloride (INT) colorimetric assay was used to assess the antibacterial activity of the extract and isolates against 13 pathogenic strains. The crude extract showed strong antibacterial activity (MIC < 100 µg/mL) against the tested bacterial strains. When combined with an efflux pump inhibitor phenylalanine beta naphthylamide (PAβN), the inhibition potency of the extract was substantially enhanced with the lowest MIC value at 4 µg/mL. Compounds 5 and 6 showed moderate activity (MIC < 100 µg/mL) against 12/13 (92.3%), and 8/13 (61.5%) bacterial strains, respectively. A minimal bactericidal concentration (MBC)/minimal inhibitory concentration (MIC) ratio ≤ 4 indicated their bactericidal effect against Escherichia coli, Enterbacter aerogenes, Klebsiella pneumoniae, Providencia stuartii, Pseudomonas aeruginosa, and Staphylococcus aureus.

The fungus Penicillium citrinum WXP-2 and the bacterium Citrobacter freundii WXP-9 were isolated and found to have poor denitrification performance. Surprisingly, co-culture of the two strains which formed fungus–bacterium pellets (FBPs) promoted the removal efficiency of nitrate (NO3−-N; 95.78%) and total nitrogen (TN; 81.73%). Nitrogen balance analysis showed that excess degraded NO3−-N was primarily converted to N2 (77.53%). Moreover, co-culture increased the dry weight to 0.74 g/L. The diameter of pellets and cell viability also increased by 1.49 and 1.78 times, respectively, indicating that the co-culture exerted a synergistic effect to promote growth. The increase in electron-transmission system activity [99.01 mg iodonitrotetrazolium formazan/(g·L)] and nitrate reductase activity [8.65 mg N/(min·mg protein)] were responsible for denitrification promotion. The FBPs also exhibited the highest degradation rate at 2:1 inoculation ratio and 36 h delayed inoculation of strain WXP-9. Finally, recycling experiments of FBP demonstrated that the high steady TN removal rate could be maintained for five cycles.

It is demonstrated that the reduction of iodonitrotetrazolum chloride in the presence of a Bacillus subtilis suspension in a physiological solution can be described using chemical kinetics equations. The effective kinetic parameters are determined and the role of the diffusion of iodonitrotetrazolium chloride in the process of its reduction involving bacterial cell components is described.

Antibiotic resistance in bacterial species is opening new avenues to search for alternative modes of antimicrobial treatment, medicinal plant extracts being one among them. The aim of this study was to access the possibility of medicinal plant extract from Shih in the manufacture of pharmaceutical preparations for oral hygiene specifically for the prevention and treatment of dental caries due to Streptococcus mutans. Antimicrobial effects of crude organic extract of Shih on S. mutans isolated from the saliva were examined by taking S. mutans with culture media only (−ve control); S. mutans treated with the antibiotic gentamicin (+ve control) and S. mutans treated with Shih. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) were Determination by Iodonitrotetrazolium chloride (INT) colorimetric assay Time-kill dynamic assay was performed using broth microdilution method. The metabolic reason behind the bacteriostatic and bactericidal effect were studied by measuring the glucose utilization by the microbes, pH as a measure of acid production, nucleic acids quantitation to check the DNA status and inhibition of water-insoluble glucan synthesis were undertaken. Shih MIC for S. mutans was at 2.5 mg/ml and MBC was 4 mg/ml. S. mutans bacterial population started reclining within 60 min of incubation with Shih at MBC. Utilization of added glucose was very high at MIC due to bacteria overcoming the stress, whereas at MBC its utilization was less. Accordingly pH also became acidic to 2.9 with MIC and 4.03 with MBC. There was a great degree of inhibition in the formation of nucleic acids indicating this crude extract interferes with DNA replication. Inhibition of glucan synthesis was to the tune of 45% as compared to control. Thus we conclude that Shih has potentially effective antibacterial activity hence it can be proposed as a potentially effective antiplaque and anticariogenic agent in the form of mouth wash or gum paint. However, the cytotoxicity of the extract needs to be evaluated in in-vitro and in-vivo conditions before it is considered as a safe antiplaque and anticariogenic agent. Keywords: Artemisia herba-alba (Shih), Streptococcus mutans, Dental caries, Antimicrobial effect, Metabolic effects

Mushrooms produce a variety of bioactive compounds that are known to have anti-pathogenic properties with safer and effective therapeutic effects in human disease prognosis. The antibacterial activity of ethanol and methanol extracts of Pleurotus opuntiae were checked against pathogenic microorganisms viz. Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis NCIM 2300, Proteus vulgaris NCIM 5266, Serratia marcescens NCIM 2078, Shigella flexeneri NCIM 5265, Moraxella sp. NCIM 2795, Staphylococcus aureus ATCC 25923 by agar well diffusion method at different concentrations of the extracts. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the extracts was determined by INT (Iodonitrotetrazolium chloride) colorimetric assay. Extracts were standardized by thin layer chromatography (TLC) in different solvent systems. The Retention factors (Rf) of different compounds were calculated by high performance TLC (HPTLC) fingerprinting at UV 254, 366, and 540 nm before and after derivatization. The ethanol and methanol extracts of P. opuntiae showed bactericidal activity against all the test pathogens at MIC values of 15.6 to 52.08 mg/mL and 20.81 to 52.08 mg/mL respectively. Whereas the MBC values for ethanol and methanol extract of P. opuntiae against all pathogens were recorded as 26.03 to 62.5 mg/mL and 125 mg/mL respectively. Preliminary mycochemical screening of both the extracts revealed high contents of bioactive compounds. Amongst all the solvent systems used in TLC, the best result was given by chloroform + hexane (8:2) which eluted out 5 different compounds (spots). HPTLC results revealed spots with different Rf values for all the 24 compounds present. Thus, it can be inferred from the present investigation that the mycoconstituents could be an alternative medication regimen and could play a role in new drug discoveries against different infections. Further, the antimicrobial components of these mushrooms can be transformed to bioengineered antimicrobial coatings for surfaces, drug and other hybrid systems for public health implications in combating persistent infections.

The file attached to this record is the author's final peer reviewed version. Introduction: The antimicrobial activity of many Essential Oils (EOs) is well established, indicating that EOs may be a source of compounds for antimicrobial drug development. Thin Layer Chromatography-Direct Bioautography (TLC-DB) can quickly identify antimicrobial components in complex mixtures and can be applied to the screening of EOs for lead compounds. Objectives: This study aimed to identify antimicrobial components of oregano, rosewood and cumin EOs against antibiotic-sensitive and -resistant bacteria using TLC-DB and a multi-faceted approach of GC-MS, LC-MS and NMR techniques to characterise bioactive compounds. The study also aimed to quantify the antimicrobial activity of bioactive compounds in order to evaluate their potential for the development of therapies against antibiotic-resistant bacteria. Materials and Methods: EOs were eluted on TLC plates and sprayed with a suspension of Staphylococcus aureus, Enterococcus faecium, Escherichia coli or Pseudomonas aeruginosa (antibiotic-sensitive and -resistant isolates). Zones of inhibition, visualised with iodonitrotetrazolium chloride, were subject to GC-MS, LC-MS and NMR to characterise the bioactive compounds. Results: Seven compounds were identified from the three EOs using GC-MS, while LC-MS and NMR failed to detect the presence of any further non-volatile or heat labile compounds. Carvacrol was most antimicrobial compound identified, with minimum inhibitory concentrations ranging 0.99-31.62 mM. Conclusion: The identified antimicrobial compounds present in oregano, rosewood and cumin EOs including carvacrol may be candidates for the development of novel antimicrobial therapies against antibiotic-resistant bacteria.

Chromolaena odorata L (Asteraceae) is a bad invasive plant, found in the humid tropics and sub-tropics worldwide. It is used against dysentery, diarrhea, malaria, wound healing, headache and toothache in traditional medicine. In the present study, we investigated the antibacterial activities of different leaves extracts of Chromolaena odorata L. (cyclohexane, dichloromethane, ethyl acetate and butanol) against four clinical diarrheal strains (Klebsiella oxytoca, Salmonella enterica, Shigella sonnei and Vibrio cholera). We demonstrated that C. odorata leaves extracts show an antibacterial activity between 0.156 and 1.25 mg/mL. Bioassay-guided chromatography by bioautography with iodonitrotetrazolium-based colorimetric assay allowed the isolation and identification of two active compounds. After the combination of RP-HPLC, mass spectrometry analysis, 1D and 2D-NMR spectroscopy, we isolated and characterized two active molecules corresponding to 3’,4’,5,6,7-Pentamethoxyflavone (Sinensetin) and4’,5,6,7-Tetramethoxyflavone (Scutellareintetramethyl ether).

Glutamate synthase, glutamine α-ketoglutarate amidotransferase (often abbreviated as GOGAT) is a key enzyme in the early stages of ammonia assimilation in bacteria, algae and plants, catalyzing the reductive transamidation of the amido nitrogen from glutamine to α-ketoglutarate to form two molecules of glutamate. Most bacterial glutamate synthases consist of a large and small subunit. The genomes of three Pyrococcus species harbour several open reading frames which show homology with the small subunit of glutamate synthase. There are no open reading frames which may be coding for a large subunit responsible for the glutamate formation in these pyrococcal genomes.In this work, two open reading frames PH0876 and PH1873 from P. horikoshii were cloned and expressed in Escherichia coli as soluble proteins. Both proteins show NADPH-dependent oxidoreductase activity using artificial electron acceptors iodonitrotetrazolium chloride at thermophilic conditions. It is possible that these open reading frames are the products of gene duplication and that they are the early forms of an electron transfer domain in archaea which may have later contributed to many electron transfer enzymes.

The antifungal and antimicrobial activities of three pentacycloundecane (PCU) tetra-amine derivatives are reported herein. The in vitro activity of these PCU derivatives against yeasts (Candida albicans and non-albicans species) and filamentous fungi was evaluated using the Clinical and Laboratory Standards Institute (CLSI) M27-A2 and M38-A2 guidelines and the 2H-tetrazolium salt, (MTS) colorimetric method. The minimum inhibitory concentration against most of the tested clinical fungal strains for GKM8 and GKM9 derivatives ranges from 15.6 to 62.5 μg/mL while GKM11 ranged from 3.9 to 7.8 μg/mL. The GKM11 derivative was also active against fluconazole-resistant strains of fungi. The GKM11 derivative also exhibited promising activity against filamentous fungi in that it was 2.5 times more active than amphotericin B against Sporothrix schenckii. Antibacterial activity was determined using the broth microdilution method (BMM) and the iodonitrotetrazolium chloride (INT) colorimetric method. The GKM11 derivative was mainly active against Gram-positive bacteria with MIC ranging from 3.9 to 7.8 μg/mL. Activity against Gram-negative bacteria tested was limited to Escherichia coli and Elizabethkingia meningoseptica (MIC of 31 μg/mL).

The aim of this study was to characterize the phenolic composition and evaluate the bioactivity of several samples of Lavandula pedunculata (Mill.) Cav, and to compare aqueous and hydroethanolic extracts. Plant materials were obtained by growing some accessions (seed samples) of various wild populations from different regions of Portugal conserved at the Portuguese Genebank in Braga. Phenolic compounds were analised by HPLC-DAD-ESI/MSn, antioxidant potential through in vitro assays (DPPH radical scavenging activity, reducing power and inhibition of lipid peroxidation), cytotoxicity on tumor cells (MCF-7, NCI-H460, HeLa and HepG2) and non-tumor (PLP2) cells, anti-inflammatory activity in rat RAW 264.7 macrophages, by the ability to inhibit NO production and antimicrobial potential by the microdilution method with INT dye (iodonitrotetrazolium chloride). Thirteen compounds were identified, being salvianolic acid B, rosmarinic acid and luteolin-7-O-glucuronide, the main compounds present, with values ranging between 44.3⁻582, 50.9⁻550, and 24.36⁻101.5 mg/g extract, respectively. L. pedunculata aqueous extract revealed a higher antioxidant potential (EC50 values between 14 to 530 μg/mL), which could be related to its higher concentration in phenolic compounds; however, the hydroethanolic extract showed a higher anti-inflammatory (lower EC50 values than 124 μg/mL) potential and antiproliferative capacity (lower GI50 values than 34 μg/mL). Thus, this study highlights the bioactive effects of this species and opens up possibilities of uses in food and pharmaceutical formulations. However, there are potential differences in such properties according to geographical origin of plant material, as in general, the samples from Alentejo presented higher results in all the bioactivities, compared with Trás-os-Montes samples. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Program PT2020 for financial support to CIMO (UID/AGR/00690/2013), L. Barros and R. Calhelha contracts. The authors are grateful to FEDER-Interreg España-Portugal programme for financial support through the project 0377_Iberphenol_6_E. info:eu-repo/semantics/publishedVersion

Schrankia leptocarpa DC. (Mimosaceae) is an African medicinal plant, traditionally used by plants practitioners in Benin to cure malaria, diarrhea and stomach ache. There is little ethnobotanical and almost no chemical information available for this species. Eight extracts from the entire plant of Schrankia leptocarpa were prepared using diethyl ether, acetone, cyclohexane, methylene chloride, ethyl acetate, methanol, butanol and ethanol/water (20:80). Phytochemical analysis were performed and antibacterial was evaluated against four reference bacteria, Staphylococcus aureus , Enteroccocus feacalis , Escherichia coli and Pseudomonas aeruginosa , and four patient isolated bacteria using iodonitrotetrazolium microtest. The antioxidant properties were investigated using 2,2-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion radical scavenging activity. All extracts showed antimicrobial activity ranging from 0.078 to 5 mg/ml against one or more bacteria. The most potent extract was the diethyl ether extract with a minimum inhibitory concentration value of 75 μg/ml on E. faecalis . Phytochemical screening showed a wide variety of phytoconstituents such as steroids, terpenes, phenolic acid, flavonoids, tannins and alkaloids. The DPPH radical scavenging activity indicated that the radical scavenging activity ranged from 1.35 to 3.47 μg/ml. The superoxide anion radical scavenging showed inhibitory percentage ranging from 32.04 to 86%. Higher activity was observed with ethyl acetate extract. The results provide an evidence for the traditional use of S. leptocarpa for the treatment of infective diseases. Keywords: Schrankia leptocarpa , antibacterial, antioxidant, 2,2-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion

Temperature has a strong influence on the activity of living organisms. This study, involving two indoor experiments, evaluated the effects of root zone temperature (10, 15 and 23 degrees C) on the formation and development of arbuscular mycorrhizae (AM). In the first trial, greenhouse-grown sorghum [ Sorghum bicolor (L.) Moench] was either colonized by Glomus intraradices SchenckSmith or left non-mycorrhizal. Root length, root and shoot weight and root colonization were measured after 5, 10 and 15 weeks of plant growth. Although suboptimal root zone temperatures reduced growth in both mycorrhizal and non-mycorrhizal plants, mycorrhizal plants were larger than non-mycorrhizal plants after 15 weeks at 15 and 23 degrees C. At suboptimal root zone temperatures, mycorrhizal inoculation sometimes slightly reduced root development. AM colonization was more affected than root growth at suboptimal root zone temperatures. Colonization was markedly reduced at 15 degrees C compared with 23 degrees C, and almost completely inhibited at 10 degrees C. The second experiment was conducted in vitro using transformed carrot ( Daucus carota L.) roots supporting G. intraradices. Mycelium length and spore number were measured weekly for 15 weeks. Spore metabolic activity (iodonitrotetrazolium reduction), root length and percentage root colonization were measured after 15 weeks. G. intraradices sporulation was reduced at temperatures below 23 degrees C, while spore metabolic activity was significantly reduced only at 10 degrees C. Root length and in particular percentage colonization were decreased at suboptimal temperatures. A negative interaction between AM hyphal growth and root growth resulting in reduced probability of contact at suboptimal root zone temperatures is proposed to explain the greater reduction observed in root colonization than in root and hyphal growth.

Excess consumption of caffeine (400 mg/day/adult) can lead to adverse health effects. Recent introduction of caffeinated products (gums, jelly beans, energy drinks) might lead to excessive consumption, especially among children and nursing mothers, hence attracting the Food and Drug Administration's attention and product withdrawals. An "in-home" test will aid vigilant consumers in detecting caffeine in beverages and milk easily and quickly, thereby restricting its consumption. Known diagnostic methods lack speed and sensitivity. We report a caffeine dehydrogenase (Cdh)-based test which is highly sensitive (1-5 ppm) and detects caffeine in beverages and mother's milk in 1 min. Other components in these complex test samples do not interfere with the detection. Caffeine-dependent reduction of the dye iodonitrotetrazolium chloride results in shades of pink proportional to the levels in test samples. This test also estimates caffeine levels in pharmaceuticals, comparable to high-performance liquid chromatography. The Cdh-based test is the first with the desired attributes of a rapid and robust caffeine diagnostic kit.

Otimização de métodos de extração e avaliação de micélio extrarradicular ativo (MEA) e total (MET) constituem os principais passos para um estudo mais profundo da interação fungo micorrízico arbuscular (FMA) e hospedeiro, já que a absorção e translocação dos nutrientes do substrato ao hospedeiro são realizadas, respectivamente, por tais estruturas fúngicas. Métodos de extração de micélio extrarradicular de FMAs propostos por diversos autores foram reavaliados e mesclados, originando um método exclusivo e de fácil execução, juntamente com a calibração de métodos para avaliação e quantificação de MEA (fluorescência induzida com a hidrólise do diacetato de fluoresceína e redução de iodonitrotetrazólio), em substrato arenoso. Extraction and evaluation of active (AEM) and total (TEM) extraradicular mycelium is the first step in a comprehensive study of the interaction between host plant and arbuscular mycorrhizal fungi (AMF), since these hyphae are responsible for nutrient absorption and translocation from the soil to the host. Extraction methods for extraradicular mycelium in the literature were reviewed and reevaluated, and modifications were made, resulting in a new, simple technique. The evaluation and quantification method of AEM was tested using induced fluorescence following fluorescein diacetate (FDA) hydrolysis and iodonitrotetrazolium (INT) reduction in sandy soil.

As glutamate and ammonia play a pivotal role in nitrogen homeostasis, their production is mediated by various enzymes that are widespread in living organisms. Here, we report on an effective electrophoretic method to monitor these enzymes. The in gel activity visualization is based on the interaction of the products, glutamate and ammonia, with glutamate dehydrogenase (GDH, EC: 1.4.1.2) in the presence of either phenazine methosulfate (PMS) or 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium (INT). The intensity of the activity bands was dependent on the amount of proteins loaded, the incubation time and the concentration of the respective substrates. The following enzymes were readily identified: glutaminase (EC: 3.5.1.2), alanine transaminase (EC: 2.6.1.2), aspartate transaminase (EC: 2.6.1.1), glycine transaminase (EC: 2.6.1.4), ornithine oxoacid aminotransferase (EC: 2.6.1.13), and carbamoyl phosphate synthase I (EC: 6.3.4.16). The specificity of the activity band was confirmed by high pressure liquid chromatography (HPLC) following incubation of the excised band with the corresponding substrates. These bands are amenable to further molecular characterization by a variety of analytical methods. This electrophoretic technology provides a powerful tool to screen these enzymes that contribute to nitrogen homeostasis in Pseudomonas fluorescens and possibly in other microbial systems.

We investigated different procedures for using a fluorescent stain and dehydrogenase indicator technique for detection of respiring bacteria in two stream ecosystems in the southeastern United States. A modified filter-transfer-freeze (FTF) procedure was useful in detecting iodonitrotetrazolium formazan (INT) crystals in unattached bacteria. This technique did not enhance detection of INT crystals in particle-bound epilithic and epipelic bacteria. No significant differences were found between pre-stained polycarbonate filters mounted with immersion oil and those mounted with glycerol, although we preferred glycerol because of its optical clarity. In contrast to previous studies, we did not find that immersion oil dissolved INT crystals within bacterial cells even after a period of 24 h. Percentages of active bacteria in comparable habitats were not significantly different between a sandstone and a carbonate stream. However, benthic habitats (epilithon and epipelon) had significantly higher percentages of ...

The relationship between desiccation and the production of extracellular polysaccharides (EPS) by soil bacteria was investigated by using a Pseudomonas species isolated from soil. Cultures subjected to desiccation while growing in a sand matrix contained more EPS and less protein than those growing at high water potential, suggesting that resources were allocated to EPS production in response to desiccation. Desiccation did not have a significant effect on activity as measured by reduction of iodonitrotetrazolium. Purified EPS produced by the Pseudomonas culture contained several times its weight in water at low water potential. Sand amended with EPS held significantly more water and dried significantly more slowly than unamended sand, implying that an EPS matrix may buffer bacterial colonies from some effects of desiccation. We conclude that bacteria may use EPS production to alter their microenvironment to enhance survival of desiccation.

The relative contribution of two major soil microbial groups, fungi and bacteria, to soil respiration was investigated because of their importance in decomposition and nutrient cycling in agroecosystems. The bactericide streptomycin and the fungicide cycloheximide were applied singly and in combination in two glucose-amended (5 g kg−1 soil) and texturally differing agricultural soils, a Valentine sand (Typic Ustipsamment) and a Sharpsburg silty clay loam (Typic Argiudoll). Soils receiving only glucose served as controls. Total and metabolically-active fungal and bacterial microscopic counts were made at 1, 3 and 10 days after amendment. CO2 evolution was measured for 24 h before each sampling for the purpose of relating soil respiration to specific microbial metabolic activity. Intracellular reduction of iodonitrotetrazolium to formazan and accumulation of fluorescein from fluorescein diacetate were used as indicators of metabolically-active bacteria and fungi, respectively. Production of new biomass in controls and fluctuations in CO2 evolution were primarily associated with bacterial activity on the first day, indicating an advantage of bacteria over fungi in initial glucose metabolism. Fungal activity was associated with CO2 production at later sampling times. Reduced respiration, as a result of biocide treatment, occurred only on the first day whereas nontarget or indirect effects of both inhibitors were apparent at later times. Fungal activity rose two to five times above the level of the glucose-amended control on day 3 in streptomycin-treated soils, and bacterial activity was stimulated in the cycloheximide treatment of the Sharpsburg soil on day 10. The effectiveness of streptomycin inhibition on total and active bacterial densities was greater in the Sharpsburg silty clay loam than in the Valentine sand. There was no reduction of total hyphal length due to biocide treatment, and fungal activity with cycloheximide did not differ significantly from that of the control. For our experimental conditions the use of biocides alone did not effectively differentiate between bacterial and fungal activity. Selective staining of metabolically-active microorganisms, however, may offer promise in achieving this goal and appears useful in understanding the importance of bacteria and fungi to nutrient cycling in soil ecosystems.

The objective of this experiment was to quantify the extramatrical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus etunicatum (Becker & Gerdemann) grown on maize (Zea mays L. var. Piranão) provided with various levels of phosphate fertilizer and harvested at 30, 60 and 90 days after planting (DAP). Total extramatrical mycelium (TEM) was extracted from soil using a modified membrane filtration method, followed by quantification using a grid intersection technique. Active extramatrical mycelium (AEM) proportion was determined using an enzymatic method which measured dehydrogenase activity by following iodonitrotetrazolium reduction. At low levels of added P, there was relatively less TEM than at high levels of added P, but the AEM proportion at low soil P availability was significantly greater than at high soil P. O objetivo do trabalho foi quantificar o micélio extramatricial do fungo micorrízico arbuscular (AM) Glomus etunicatum (Becker & Gerdemann) e medir sua atividade 30, 60 e 90 dias após o plantio (DAP), utilizando-se a cultura do milho (Zea mays L. var. Piranão) com quatro doses de adubação fosfática. O micélio extramatricial total (TEM) foi extraído do solo pelo método da membrana de filtração modificado e foi quantificado pela técnica da grade de interseção. A proporção de micélio extramatricial ativo (AEM) foi determinada pelo método que mede a atividade das enzimas desidrogenases com coloração de iodonitrotetrazolio. Em baixas doses de P,o TEM foi menor do que a quantidade em altas doses de P, mas a proporção de AEM foi maior nas baixas doses de P.

Lyophilized Enterococcus faecium M-74 was administered to 12 adult subjects in a daily oral dose of 5 x 10(9) bacteria for six weeks. The bacterium temporarily colonized the host intestine and its excretion with stool persisted for five weeks after the last does. The mean levels of serum cholesterol and LDL showed a a biphasic effect--an elevation followed by a sharp decrease (on day 64 of investigation). The decrease corresponded in time with a significant increase in the ability to reduce iodonitrotetrazolium and superoxide production by peripheral neutrophils incubated with zymosan or phorbol myristate acetate, and also with an elevated production of IgG by peripheral blood mononuclear cells. Hence, intake of E. faecium may have a hypocholesterolemic and immunostimulatory effect. It was also demonstrated that E. faecium significantly reduced the average activity of beta-D-glucuronidase in stools.

IgG antibodies against the 2,3-di-o-acyltrehalose glycolipid of Mycobacterium tuberculosis were determined in a set of 49 sera from patients with pulmonary tuberculosis and 65 negative control subjects. We compared a conventional ELISA method using a beta-galactosidase anti-human IgG conjugate developed with ONPG, with an amplification ELISA system constituted of an anti-human IgG biotinylated conjugate, a streptavidin-alkaline phosphatase complex, and NADP as a substrate. The resulting NAD was measured by using a redox enzymatic recycling system of alcohol dehydrogenase, diaphorase and iodonitrotetrazolium as chromogen. With specificity set at 92.31% in both methods, we obtained a sensitivity of 42.86% in the conventional method and a sensitivity of 61.22% in the amplified method. We conclude that by using a more sensitive method we can detect cases that otherwise could be identified as false negatives.

Iminodiacetate (IDA) is a xenobiotic intermediate common to both aerobic and anaerobic metabolism of nitrilotriacetate (NTA). It is formed by either NTA monooxygenase or NTA dehydrogenase. In this paper the detection and characterization of a membrane-bound iminodiacete dehydrogenase (IDA-DH) from Chelatobacter heintzii ATCC 29600 is reported, which oxidizes IDA to glycine and glyoxylate. Out of 15 compounds tested, IDA was the only substrate for the enzyme. Optimum activity of IDA-DH was found at pH 8.5 and 25°C, respectively, and the Km for IDA was found to be 8mM. Activity of the membrane-bound enzyme was inhibited by KCN, antimycine and dibromomethylisopropyl-benzoquinone. When inhibited by KCN IDA-DH was able to reduce the artificial electron acceptor iodonitrotetrazolium (INT). It was possible to extract IDA-DH from the membranes with 2% cholate, to reconstitute the enzyme into soybean phospholipid vesicles and to obtain IDA-DH activity (more than 50% recovery) using ubiquinone Q1 as the intermediate electron carrier and INT as the final electron acceptor. Growth experiments with different substrates revealed that in all NTA-degrading strains tested both NTA monooxygenase and IDA-DH were only expressed when the cells were grown on NTA or IDA. Furthermore, in Cb. heintzii ATCC 29600 growing exponentially on succinate and ammonia, addition of 0.4 g l-1 NTA led to the induction of the two enzymes within an hour and NTA was utilized simultaneously with succinate. The presence of IDA-DH was confirmed in ten different NTA-degrading strains belonging to three different genera.

Morphological and functional characteristics of vaginal exudate leukocytes were examined in 47 patients with urogenital trichomoniasis. Electron microscopic morphology, viability, phagocytosis of Candida albicans blastospores and ability to undergo respiratory burst in the iodonitrotetrazolium reductase test were evaluated in these cells. Vaginal inflammatory leukocytes were almost exclusively polymorphonuclear neutrophils, and their concentration was positively correlated (r = 0.58; p less than 0.001) with the number of trichomonads in the exudate. Median leukocyte viability reached 39% and both phagocytic and tetrazolium reductase activities of these cells were significantly reduced in comparison with those of circulating polymorphonuclear leukocytes. Patients with a clinical picture of severe mucosal inflammation had significantly higher vaginal exudate leukocyte concentrations and viability than those without inflammatory signs. The possible role of vaginal leukocytes in the pathogenesis of urogenital trichomoniasis is discussed.

A thorough investigation on the long-known reactivity of spinach ferredoxin-NADP+ reductase with tetrazolium salts has allowed the setting-up of a new assay, highly specific for this enzyme. By immunological titration it has been determined that more than 92% of the iodonitrotetrazolium chloride reductase activity of spinach leaves' homogenate belongs to ferrodoxin-NADP+ reductase. It has been shown that addition of formazanbinding agents, like Triton X-100, is required for the assay of the partially purified enzyme; furthermore Triton X-100 enhanced considerably the tetrazolium salts reductase activity of all the enzyme preparations tested. The optimal condition of pH, ionic strength and Triton X-100 concentration have been determined. Kinetic experiments yielded a series of parallel lines in the double-reciprocal plots and the kinetic parameters obtained are reported. Moreover it has been shown that at least 75–80% of the activity with tetrazolium salts of ferrodoxin-NADP+ reductase is not mediated by the superoxide radical.

Levels of DNA, RNA, protein, ATP, glutathione, and radioactivity associated with [ 35 S]methionine-labeled cellular protein were estimated at various times during the starvation-survival process of a marine psychrophilic heterotrophic Vibrio sp., Ant-300. Values for the macromolecules were analyzed in terms of total, viable, and respiring cells. Electron micrographs (thin sections) were made on log-phase and 5.5-week-starved cells. On a per-cell basis, the levels of protein and DNA rapidly decreased until a constant level was attained. A second method in which radioactive sulfur was used for monitoring protein demonstrated that the cellular protein level decreased for approximately 2.5 weeks and then remained constant. An initial decrease in the RNA level with starvation was noted, but with time the RNA (orcinol-positive material) level increased to 2.5 times the minimum level. After 6 weeks of starvation, 45 to 60% of the cells remained capable of respiration, as determined by iodonitrotetrazolium violet-formazan granule production. Potential respiration and endogenous respiration levels fell, with an intervening 1-week peak, until at 2 weeks no endogenous respiration could be measured; respiratory potential remained high. The cell glutathione level fell during starvation, but when the cells were starved in the presence of the appropriate amino acids, glutathione was resynthesized to its original level, beginning after 1 week of starvation. The cells used much of their stored products and became ultramicrocells during the 6-week starvation-survival process. Ant-300 underwent many physiological changes in the first week of starvation that relate to the utilization or production of ATP. After that period, a stable pattern for long-term starvation was demonstrated.

Populations of heterotrophic bacteria were enumerated from stream and groundwater samples taken from an undisturbed catchment basin in southwestern Alberta. Direct counts using epifluorescence microscopy were compared with total viable counts using standard plate count methods, the iodonitrotetrazolium formazan method (reduction of 2-(p-iodophenyl)-3-(p-nitro phenyl)-5-phenyl tetrazolium chloride to iodonitrotetrazolium formazan), the nalidixic acid method, and the slide culture method. The nalidixic acid method gave the highest results, with total viable counts as high as 34.6% of the total direct count. Attempts to enumerate bacteria on media made from decaying leaves and algal–bacterial slime gave lower values, approximately 10% of the numbers obtained on enriched media. Stream waters were found to be dominated by Pseudomonas spp. and groundwaters were dominated by Bacillus spp. No differences were found in either numbers or species identified between tryptone – glucose – yeast extract agar, brain–heart infusion agar, nutrient agar, or casein–peptone–starch agar.

Three independent techniques, [ 3 H]thymidine incorporation, the reduction rate of p -iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA, were adapted to measure the activity of attached and unattached estuarine bacteria. In experiments employing the estuarine isolate Vibrio proteolytica , nutrient concentrations were manipulated by varying the concentration of peptone-yeast extract. In the presence of exogenous nutrients, the activity of free-living cells was greater than that of attached cells as measured by [ 3 H]thymidine incorporation and ATP/DNA ratios. In the absence of peptone-yeast extract, however, the activity of attached cells surpassed that of free-living cells as determined by [ 3 H]thymidine incorporation and INT formazan normalized to DNA. Of the three techniques, [ 3 H]thymidine incorporation was deemed most sensitive for detecting changes in activity resulting from slight differences in nutrient concentration. By this technique, attached cells were much less sensitive to changing nutrient concentrations than were free-living cells. Below a threshold concentration, attached cell activity remained constant, while the activity of unattached cells decreased as a function of decreasing nutrient concentration. The results suggest that loss of cell surface area available for substrate uptake due to attachment may be an important factor in determining the relative activities of attached and free-living cells.

The tetrazolium salt, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) was used to determine viable respiring cells in batch cultures of Saccharomyces cerevisiae. Respiring cells reduce INT to water insoluble iodonitrotetrazolium formazan (INT-formazan) which is deposited within the respiring cell. The INT-formazan granules can be observed by brightfield microscopy. This allows a rapid quantitative determination of the percentage of respiring cells and total cells within the same microscopic field.