Aislamiento enzimático de protoplastos a partir del mesófilo de vitroplantas de Gynerium sagittatum Aubl. Beauv. cv. "Criolla"

Gynerium sagittatum es una gramínea ampliamente utilizada en la costa Caribe colombiana como fuente de fibra natural para la elaboración de artesanías, particularmente por la comunidad Zenú. En la presente investigación se evaluó el efecto de diferentes concentraciones de enzimas: celulasa y macerozima a diferentes tiempos de incubación y sus interacciones en el aislamiento de protoplastos. Los protoplastos se obtuvieron del mesófilo foliar de vitroplantas de G. sagittatum expuesto a combinaciones enzimáticas de celulasa (1.5 y 2.0%), con macerozima (0.3, 0.6 y 0.9%), durante 3, 6 y 9 horas de incubación, para un total de 18 tratamientos con 5 réplicas cada uno. Los mayores números de protoplastos aislados correspondieron a T18 (2.0% celulasa, 0.9% macerozima), T12 (2.0% de celulasa, 0.3% macerozima), T3 (1.5% de celulasa, 0.3% de macerozima) y T6 (1.5% de celulasa, 0.6% de macerozima) por 9 horas de incubación cada uno, con valores de 88.625, 83.000, 75.000 y 53.375 protoplastos/mL respectivamente. El tiempo de incubación fue significativo en el aislamiento de los protoplastos (p<0.05). Las predicciones entre factores mostraron que una interacción de 2.0% de celulasa y 0.9% de macerozima permite obtener 44.302 protoplastos/mL, mientras que las interaciciones tiempo de incubación-celulasa y tiempo de incubación-macerozima mostraron que es posible obtener 72.073 y 71.212 protoplastos/mL con 2.0% de celulasa y 0.9% macerozima por 9 horas de incubación cada una respectivamente. Los resultados indican que la aplicación de estas enzimas permite obtener cantidades considerables de protoplastos de G. sagittatum a partir de explantes cultivados in vitro.

Gynerium sagittatum is a graminaceous plant widely used in the Caribbean coast of Colombia as a natural fiber source for the elaboration of handicrafts, particularly by the Zenú community. In the present investigation, the effect of different concentrations of cellulase and macerozyme enzymes at different incubation times and their interaction in the isolation of protoplasts was evaluated. Protoplasts were obtained from leaf mesophyll of G. sagittatum vitroplants exposed to enzymatic combinations of cellulase (1.5 and 2.0%), with macerozyme (0.3, 0.6 and 0.9%), for 3, 6 and 9 hours of incubation, for a total of 18 treatments with 5 replicates each. The highest numbers of isolated protoplasts corresponded to T18 (2.0% cellulase, 0.9% macerozyme), T12 (2.0% cellulase, 0.3% macerozyme), T3 (1.5% cellulase, 0.3% macerozyme) and T6 (1.5% cellulase, 0.6% macerozyme); at 9 hours incubation. The protoplast number for these treatments were: 88.625, 83.000, 75.000 and 53.375 protoplasts/mL respectively. Incubation time was significant in the isolation of protoplasts (p<0.05). The predictions between the factors showed that with an interaction of 2.0% cellulase and 0.9% macerozyme it is possible to obtain 44.302 protoplasts/mL, likewise, the incubation time-cellulase and incubation time-macerozyme interactions showed that it is possible to obtain 72.073 and 71.212 protoplasts/mL with 2.0% cellulase and 0.9% macerozyme for 9 hours of incubation respectively. The results indicate that the use of these enzymes and time, allows the isolation of of protoplasts from G. sagittatum in vitro plants.

Effect of different incubation time of aminolevulinic acid on photodynamic inhibition of Propionibacterium acnes biofilms / 中华皮肤科杂志

Objective:To investigate the effect of different incubation time of aminolevulinic acid (ALA) on photodynamic inhibition of Propionibacterium acnes biofilms. Methods:Propionibacterium acnes biofilms were formed in 24-well plates with pre-placed cell slides and 96-well plates. The formation of the biofilm structure was observed by confocal laser scanning microscopy (CLSM) , and the growth activity of the biofilm was assessed by the tetrazolium salt XTT assay. The in vitro successfully constructed biofilm models were divided into 6 groups: negative control group receiving neither ALA treatment nor LED radiation, ALA group incubated with ALA alone for 30 minutes, LED group receiving LED radiation alone, ALA-PDT1 group, ALA-PDT2 group and ALA-PDT3 group incubated with ALA for 15, 30 and 60 minutes respectively followed by LED radiation. After the treatment, CLSM was performed to observe the biofilm structure, as well as to determine the dead/living bacteria ratio, and XTT assay to assess the growth activity of the biofilm. Differences among groups were analyzed using one-way analysis of variance and least significant difference- t test. Results:CLSM showed that the Propionibacterium acnes biofilm model was successfully constructed in vitro. The dead/living bacteria ratios were 0.90 ± 0.16, 1.75 ± 0.19, and 2.57 ± 0.32 in the ALA-PDT1 group, ALA-PDT2 group and ALA-PDT3 group respectively, which were significantly higher than the dead/living bacteria ratio in the negative control group (0.31 ± 0.01; t= 55.56, 138.62, 74.64, respectively, all P<0.001) ; the biofilm viability value was significantly lower in the ALA-PDT1 group, ALA-PDT2 group and ALA-PDT3 group (0.35 ± 0.02, 0.26 ± 0.02, 0.18 ± 0.01, respectively) than in the negative control group (0.43 ± 0.00; t= 35.66, 2.64, 110.96, respectively, all P < 0.001) . CLSM showed that the structure of the Propionibacterium acnes biofilm was destroyed under the action of ALA-PDT, and the destruction was aggravated with the prolongation of incubation time of ALA. Conclusion:The prolongation of incubation time of ALA can enhance the inhibitory effect of ALA-PDT on Propionibacterium acnes biofilms.

Performance of MALDI Biotyper for Species Identification of Carbapenem-Resistant Enterobacteriaceae by Media Types and Incubation Time

BACKGROUND: This study was conducted to evaluate the impact of the media type used for direct identification of colonies on the surveillance culture of carbapenem-resistant Enterobacteriaceae (CRE) by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). METHODS: CRE surveillance culture isolates were subjected to species identification using the MALDI Biotyper (Bruker Daltonics, Germany) for 2 months starting in March 2017. Four types of media were evaluated: blood agar (BA), Mueller Hinton agar (MH), MacConkey agar (Mac), and MacConkey agar containing imipenem of 1 µg/mL (IMP-Mac). CRE-like colonies on IMP-Mac and their subculture colonies on the other media were tested after overnight incubation and extended incubation for one additional day. The percent identification and score value were analyzed for each media types and incubation time when the identification was correct at the genus level. RESULTS: A total of 117 isolates were identified as 84 Klebsiella pneumoniae, 12 Escherichia coli, 9 Enterobacter cloacae, 5 Klebsiella oxytoca, 4 Enterobacter aerogenes, and 2 Raoultella ornithinolytica. The successful identification rates (SIR) for BA and MH were 98.3% and 97.4% (P=0.9), respectively, while those for Mac and IMP-Mac were 82.1% (P < 0.001) and 70.9% (P < 0.001), respectively. After extended incubation, SIRs were decreased to 96.6%, 96.6% (P=1.0), 61.5% (P < 0.001), and 58.1% (P < 0.001) on BA, MH, Mac, and IMP-Mac, respectively. The average score values were significantly lower for Mac (2.017±0.22) and IMP-Mac (1.978±0.24) than for BA (2.213±0.16) (P < 0.001). CONCLUSIONS: The low performance of the MALDI Biotyper applied directly to the colonies grown on Mac or IMP-Mac indicates that subculture on BA or MH is preferable before identification by MALDI-TOF MS.

Efectos de la temperatura, la actividad de agua y el tiempo de incubación en el crecimiento fúngico y la producción de aflatoxina B1 por aislados toxicogénicos de Aspergillus flavus en sorgo / Effects of temperature, water activity and incubation time on fungal growth and aflatoxin B1 production by toxinogenic Aspergillus flavus isolates on sorghum seeds

Sorghum, which is consumed in Tunisia as human food, suffers from severe colonization by several toxigenic fungi and contamination by mycotoxins. The Tunisian climate is characterized by high temperature and humidity that stimulates mold proliferation and mycotoxin accumulation in foodstuffs. This study investigated the effects of temperature (15, 25 and 37 °C), water activity (a w, between 0.85 and 0.99) and incubation time (7, 14, 21 and 28 d) on fungal growth and aflatoxin B1 (AFB1) production by three Aspergillus flavus isolates (8, 10 and 14) inoculated on sorghum grains. The Baranyi model was applied to identify the limits of growth and mycotoxin production. Maximum diameter growth rates were observed at 0.99 a w at 37 °C for two of the isolates. The minimum a w needed for mycelial growth was 0.91 at 25 and 37 °C. At 15 °C, only isolate 8 grew at 0.99 a w. Aflatoxin B1 accumulation could be avoided by storing sorghum at low water activity levels (≤0.91 a w). Aflatoxin production was not observed at 15 °C. This is the first work on the effects of water activity and temperature on A. flavus growth and AFB1 production by A. flavus isolates on sorghum grains.

El sorgo, que se consume en Túnez como alimento humano, puede sufrir la colonización severa de varios hongos toxicogénicos, con la consiguiente bioacumulación de micotoxinas. Además, el clima de Túnez, caracterizado por las altas temperaturas y humedad, estimula el crecimiento fúngico y la acumulación de micotoxinas en los productos alimenticios. Este estudio investigó los efectos de la temperatura (15, 25 y 37 °C), la actividad de agua (a w) (entre 0,85 y 0,99) y el tiempo de incubación (7, 14, 21 y 28 días) sobre el crecimiento y la producción de aflatoxina B1 (AFB1) de 3 aislados de Aspergillus flavus (designados como 8, 10 y 14) que se inocularon sobre granos de sorgo. El modelo Baranyi se aplicó para identificar los límites del crecimiento y la producción de micotoxinas. Las tasas máximas de crecimiento para 2 de los aislados se observaron en la combinación 0,99 a w y 37 °C. La a w mínima necesaria para el crecimiento del micelio fue de 0,91 a 25 °C y 37 °C. A 15 °C, solo el aislado 8 creció a 0,99 a w, pero fue incapaz de producir la aflatoxina B1. Es posible evitar la acumulación de aflatoxina B1 en el sorgo almacenándolo a baja actividad de agua (≤ 0,91 a w). Este es el primer trabajo que ha estudiado el efecto de la actividad del agua y la temperatura sobre el crecimiento de aislados de A. flavus y su producción de aflatoxina B1 en granos de sorgo.

Serum fingerprint detection based on surface-enhanced Raman scattering / 军事医学

Objective To optimize the experiment conditions of surface-enhanced Raman spectroscopy detection of serum fingerprint spectra.Methods Normal human serum was used as the sample and Ag nanoparticles as the active substrate.The enhanced signals of different optimized experiments were obtained , including serum dose(2.5 to 500 μl), incubation time(10 to 30 minutes) temperature(4℃,room temperature and 37℃),and different treatment(extraction and protein removal).Results and Conclusion Serum doses should not exceed 50μl.The ratio should range from 1∶1 to 5∶1, the incubation time is from 10 to 30 minutes, and the incubation temperature from 4℃ to 37℃.The signals of samples directly mixed with an active substrate are stronger than those of samples which are extracted or protein removed .

Spectroscopic Characterization of PEG-DNA Biocomplexes by FTIR.

Understanding the mode involved in the binding of certain molecules to DNA is of prime importance, and PEG offers wide-ranging applications in biological, medical and pharmaceutical contexts. FTIR spectroscopy has been used to characterize how the formed biocomplexes bind or dissociate to/from each other between PEG400-ctDNA under different conditions. Characterization and investigation of the effect of incubation time on PEG400-ctDNA biocomplexes formation were studied through spectroscopic technique FTIR. The influence of time duration and incubation on intermolecular interaction was analysed at three different selected times (Zero, 1hr, and 48 hrs.) at 1:1 PEG400-ctDNA monomer to nucleotide ratio. The experiment was carried out at room temperature 22 ºC, with prior vortex stirrer of biocomplex for 10 min to improve homogeneity of sample. The results showed that the binding reaction of PEG400-ctDNA proceeds rapidly through DNA base pairs and phosphate DNA backbone, and complexation was reached after a maximum 1hr after mixing PEG400 and ctDNA at 1:1 ratio. FTIR spectroscopy results suggest that PEG400 binds with ctDNA by weak to moderate biocomplexes formation, with both hydrophilic and hydrophobic contact through DNA base pairs, with minor binding preference towards phosphate backbone of DNA helix. The mode of interaction most likely referred to an interaction through outside groove binding or electrostatic binding modes. FTIR highlighted the significant effect of incubation time on the stable biocomplexes of non-ionic PEG400 and ctDNA. Moreover, FTIR spectroscopy technique was rapid, showed good stability, and is a valuable tool for studying the biological properties of biocomplexes of PEG400 and ctDNA.

Qualidade da água do rio das pedras, oeste do Paraná, utilizando macroinvertebrados bentônicos como bioindicadores / Water quality at rio das pedras, western Paraná, using benthic macroinvertebrates as indicators / Calidad de las aguas del rio das pedras, oeste del Paraná, utilizando macroinvertebrados bentónicos como bioindicadores

Os macroinvertebrados bentônicos são amplamente utilizados como bioindicadores de qualidade da água em todo o mundo, devido, as suas características fisiológicas e morfológicas. O objetivo do estudo foi avaliar a qualidade da água do rio das Pedras,no município de Matelândia, Oeste do Estado do Paraná, na estação seca, utilizando-se de macroinvertebrados bentônicos como bioindicador, e identificar o tempo ideal de incubação dos litter bags. Sessenta litter bags foram confeccionados com folhas de árvores nativas provenientes da floresta ripária e incubadosno rio das Pedras. Oito litter bags foram coletados nos intervalos de três, sete,14, 28, 35 e 42 dias. Os macroinvertebrados bentônicos foram identificados em nível de família e foi aplicado o índice Biological Monitoring Working Party Score System. Com este estudo concluiu-se que o rio das Pedras possui águas não poluídas, demonstrando que o sistema é perceptivelmente não alterado. O tempo de incubação dos litter bags para obtenção do resultado quanto à avaliação da qualidade de água pelo índice Biological Monitoring Working Party Score System foi de sete dias e para analisar a estrutura da comunidade de macroinvertebrados bentônicos foi de 28 dias.

Benthic macroinvertebrates are widely used as bioindicators of water quality due to their physiological and morphological characteristics. The aim of this study was to evaluate the water quality at Rio das Pedras, in the city of Matelândia, Western Paraná, in the dry season, using benthic macroinvertebrates as bioindicators, and to identify the optimal time for the incubation of litter bags. Sixty litter bags were prepared with leaves from native trees from the riparian forest and incubated in Rio das Pedras river. Eight litter bags were collected at intervals of 3, 7, 14, 28, 35 and 42 days. The benthic macroinvertebrates were identified at the family level and the Biological Monitoring Working Party Score System index was applied. With this study, it can be concluded that the Rio das Pedras river has unpolluted waters, demonstrating that the system has hardly been changed. The litter bag incubation period in order to obtain the results for evaluating the water quality index for the Biological Monitoring Working Party Score System was of seven days, and the period to analyze the structure of the benthic macroinvertebrate community was of 28 days.

Los macroinvertebrados bentónicos son ampliamente utilizados como bioindicadores de calidad del agua en todo el mundo, debido sus características fisiológicas y morfológicas. El objetivo de este estudio ha sido evaluar la calidad de las aguas del Rio das Pedras, en el municipio de Matelândia, oeste del Estado de Paraná, en la estación seca, utilizándose de macroinvertebrados bentónicos como bioindicador, e identificar el tiempo ideal de incubación de los litter bags. Sesenta litter bags han sido confeccionados con hojas de árboles nativas procedentes de la floresta ribera e incubados en el Rio das Pedras. Ocho litter bags fueron recogidos en intervalos de tres, siete, catorce, veintiocho, treinta y cinco y cuarenta y dos días. Los macroinvertebrados bentónicos fueron identificados en nivel de familia y aplicado el índice Biological Monitoring Working Party Score System. Con esta investigación se ha concluido que el Rio das Pedras no tiene aguas contaminadas, demostrando que el sistema no es alterado. El tiempo de incubación de los litter bags para obtención del resultado cuanto a la evaluación de la calidad del agua por el índice Biological Monitoring Working Party Score System fue de siete días, y para analizar la estructura de la comunidad de macroinvertebrados bentónicos fue de 28 días.

Time-dependence of Ferric Reducing Antioxidant Power (FRAP) index in Chilean apples and berries / Influencia del tiempo de incubación sobre la capacidad antioxidante medida según FRAP, en manzanas y berries chilenos

We hypothesize that the Ferric Reducing Antioxidant Power (FRAP) assay that follows the reaction of Fe3+-TPTZ at 593 nm underestimates the antioxidant capacity of fruits, since the standardized time of the reaction (4 min) is not enough to titrate all the reducing compounds available. We measured FRAP, total phenolics and anthocyanins content in a variety of Chilean berry fruits (blueberries, blackberries, raspberries and strawberries) and apples (cv. Fuji, Granny Smith, Pink Lady, Red Delicious and Royal Gala). Taking into account the dependence of FRAP on the time course of the reaction, we propose to measure FRAP indexes after 1 min (FRAP-1), 30 min (FRAP-30) and 120 min (FRAP-120) of incubation. Most fruit extracts showed significant correlations between the antioxidant capacity and the incubation time, although in some cases the FRAP indexes did not correlate with the total phenolics and/or anthocyanins content. In fact, in apples and berries the correlation between anthocyanins content and FRAP indexes decreased with the incubation time. It is concluded that the fruit extracts analyzed require an incubation period higher than the established in the original experimental protocol to reach the equilibrium, due to the presence of a complex mixture of antioxidant compounds. In addition, a kinetic profile should be realized in each sample studied to establish the most suitable incubation period to titrate all the reactive antioxidant species.

Se plantea que el ensayo de la capacidad antioxidante de frutas, medido según el poder reductor de hierro (FRAP), que sigue la reacción de Fe3+-TPTZ a 593 nm, subestima la capacidad antioxidante, debido a que el tiempo de reacción (4 min) no sería suficiente para que reaccionen todos los compuestos reductores disponibles en las muestras. Se analizó la capacidad antioxidante FRAP, el contenido de fenoles y de antocianinas en diversos berries (arándano, mora, frambuesa y frutilla) y manzanas (cv. Fuji, Granny Smith, Pink Lady, Red Delicious y Royal Gala). Tomando en cuenta la dependencia del tiempo de incubación en el valor FRAP, se propone medir los índices FRAP después de 1 min (FRAP-1), 30 min (FRAP-30) y 120 min (FRAP-120). Diversos extractos de las frutas analizadas mostraron una correlación significativa entre la capacidad antioxidante y el tiempo de incubación; sin embargo, en algunos casos los índices FRAP no se correlacionaron con el contenido de fenoles totales y/o antocianinas. En efecto, en manzanas y berries la correlación entre el contenido de antocianinas e índices FRAP disminuyó con el tiempo de incubación. Se concluye que los extractos analizados requieren un tiempo de incubación mayor al que establece el protocolo analítico original para alcanzar el equilibrio, debido a la presencia de una compleja mezcla de compuestos antioxidantes. Además, el perfil cinético de cada muestra debería ser estudiado para establecer el periodo de incubación más adecuado para titular todas las especies antioxidantes reactivas.

Assessment of biofilm formation in device-associated clinical bacterial isolates in a tertiary level hospital.

Background: Biofilm formation is a developmental process with intercellular signals that regulate growth. Biofilms contaminate catheters, ventilators, and medical implants; they act as a source of disease for humans, animals, and plants. Aim: In this study we have done quantitative assessment of biofilm formation in device-associated clinical bacterial isolates in response to various concentrations of glucose in tryptic soya broth and with different incubation time. Materials and Methods: The study was carried out on 100 positive bacteriological cultures of medical devices, which were inserted in hospitalized patients. The bacterial isolates were processed as per microtitre plate method with tryptic soya broth alone and with varying concentrations of glucose and were observed in response to time. Results: Majority of catheter cultures were positive. Out of the total 100 bacterial isolates tested, 88 of them were biofilm formers. Incubation period of 16-20 h was found to be optimum for biofilm development. Conclusions: Availability of nutrition in the form of glucose enhances the biofilm formation by bacteria. Biofilm formation depends on adherence of bacteria to various surfaces. Time and availability of glucose are important factors for assessment of biofilm progress.

Diferentes pré-inóculos, temperaturas e tempos de incubação na produção aflatoxina B1 em arroz / Different previus inoculum, temperature and incubation time in the production of aflatoxin B1 in rice

Estudos indicam que um dos problemas mais sérios que afetam os manejos pré e pós-colheita do arroz é a presença de fungos das espécies Aspergillus ou Penicillium, potencialmente produtores de micotoxinas. Os objetivos deste trabalho foram avaliar a capacidade produtora de aflatoxina B1 de cepas isoladas do arroz e observar o efeito do pré-inóculo nas mesmas. Foram utilizados três isolados de Aspergillus flavus, conhecidamente já produtores, que foram testados nas temperaturas de 20 e 25°C, combinadas com tempos de incubação 11, 14 e 21 dias. Os pré-inóculos utilizados foram Yeast Extrat Sucrose (YES) e Czapeck Yeast Extrat (CYA). Todas as cepas retiradas do pré-inóculo em meio YES e inoculadas no arroz, em temperatura de 25°C/18 dias e 20°C/14 dias, produziram aflatoxina B1. O meio CYA apresentou menor desempenho, uma vez que as três cepas testadas não produziram aflatoxina B1 na combinação 20°C/14 dias. A 25°C/11 dias de incubação a aflatoxina B1 não foi detectada.

The production of aflatoxin in rice by three isolates of Aspergillus flavus was investigated for different culture conditions (temperature and incubation time) and previous inoculum (YES- Yeast Extrat Sucrose and CYA- Czapeck Yeast Extrat). All strains withdrawn from the previous inoculum medium YES and inoculated in the rice in temperature of 25°C/18 days and 20°C/14 days, produced aflatoxin B1. The medium CYA had lower performance since the three strains tested did not produce aflatoxin B1 in the combination 20°C/14 days. At 25°C/11 days of incubation time aflatoxin was not detectable.

Effect of Different Media on MPN Estimation of Ammonia-oxidizing Bacteria in Lakes / 微生物学通报

The MPN method was used to enumerate ammonia-oxidizing bacteria (AOB) in water and sediments of several shallow lakes. The suitable incubation time, medium types and substrate (ammonium sulphate) concentrations were studied. The results showed that, MPN values increased with the incubation time, reaching a stable maximum at some time stages, which was 40 days in all the samples for MSF medium. Among the three media used (XZ-AOB、MSF、SW), MSF give the highest MPN value. In addition, am- monium sulphate concentration in medium was an important factor affecting MPN estimation of AOB. Compared to AOB in lake sediments, AOB in lake water was more sensitive to ammonium sulphate concentration.