[Fungal biomass estimation in soils from southwestern Buenos Aires province (Argentina) using calcofluor white stain]. / Estimación de la biomasa fúngica en un suelo del sudoeste de la provincia de Buenos Aires (Argentina) con una tinción directa con blanco de calcoflúor.

Soil microorganisms are vital for ecosystem functioning because of the role they play in soil nutrient cycling. Agricultural practices and the intensification of land use have a negative effect on microbial activities and fungal biomass has been widely used as an indicator of soil health. The aim of this study was to analyze fungal biomass in soils from southwestern Buenos Aires province using direct fluorescent staining and to contribute to its use as an indicator of environmental changes in the ecosystem as well as to define its sensitivity to weather conditions. Soil samples were collected during two consecutive years. Soil smears were prepared and stained with two different concentrations of calcofluor, and the fungal biomass was estimated under an epifluorescence microscope. Soil fungal biomass varied between 2.23 and 26.89µg fungal C/g soil, being these values in the range expected for the studied soil type. The fungal biomass was positively related to temperature and precipitations. The methodology used was reliable, standardized and sensitive to weather conditions. The results of this study contribute information to evaluate fungal biomass in different soil types and support its use as an indicator of soil health for analyzing the impact of different agricultural practices.

Estimación de la biomasa fúngica en un suelo del sudoeste de la provincia de Buenos Aires (Argentina) con una tinción directa con blanco de calcoflúor / Fungal biomass estimation in soils from southwestern Buenos Aires province (Argentina) using calcofluor white stain

Los microorganismos del suelo son vitales para el correcto funcionamiento de los ecosistemas, principalmente por su papel en el ciclado de nutrientes. La intensificación del uso del suelo y las prácticas agrícolas alteran negativamente la actividad microbiana. La biomasa fúngica es uno de los parámetros más utilizados para estudiar el impacto de las actividades agrícolas en la estructura y el funcionamiento del suelo. El objetivo del presente trabajo fue estimar la biomasa fúngica en un suelo del sudoeste bonaerense con el fin de obtener valores de referencia que permitan usar este parámetro como un indicador de cambios en el ecosistema y, por otro lado, demostrar que la metodología empleada es sensible a las variaciones en las condiciones climáticas. Se colectaron muestras de suelos durante 2 años consecutivos. Se prepararon frotis de suelo y se tiñeron con soluciones de distintas concentraciones de blanco de calcoflúor y luego se estimó la biomasa fúngica observando los frotis con microscopio de epifluorescencia. Los valores de biomasa fúngica estimados variaron entre 2,23 y 26,89 μg Cfúngico/g de suelo y estuvieron dentro del rango esperable para el tipo de suelo estudiado. La biomasa fúngica mostró una relación positiva con la temperatura y las precipitaciones. La metodología empleada resultó ser confiable, repetible y sensible a cambios en las condiciones climáticas. Los resultados podrían usarse como valores de referencia para estudiar la biomasa fúngica de suelos bajo distintas condiciones y emplearse como indicadores del impacto de las distintas prácticas agrícolas sobre el ecosistema.

Soil microorganisms are vital for ecosystem functioning because of the role they play in soil nutrient cycling. Agricultural practices and the intensification of land use have a negative effect on microbial activities and fungal biomass has been widely used as an indicator of soil health. The aim of this study was to analyze fungal biomass in soils from southwestern Buenos Aires province using direct fluorescent staining and to contribute to its use as an indicator of environmental changes in the ecosystem as well as to define its sensitivity to weather conditions. Soil samples were collected during two consecutive years. Soil smears were prepared and stained with two different concentrations of calcofluor, and the fungal biomass was estimated under an epifluorescence microscope. Soil fungal biomass varied between 2.23 and 26.89 μg fungal C/g soil, being these values in the range expected for the studied soil type. The fungal biomass was positively related to temperature and precipitations. The methodology used was reliable, standardized and sensitive to weather conditions. The results of this study contribute information to evaluate fungal biomass in different soil types and support its use as an indicator of soil health for analyzing the impact of different agricultural practices.

Bacterias autótrofas y heterótrofas asociadas a nieve marina lodosa en arrecifes con escorrentía continental / Autotrophic and heterotrophic bacteria associated to muddy marine snow in reefs with terrestrial runoff / Bacterias autótrofas e heterotróficas associadas a neve marinila lodosa em recifes com escoamento continental

En agregados de nieve marina influenciada por escorrentía continental, la densidad de bacterias heterótrofas es mayor que la de autótrofas. Esta premisa fue puesta a prueba en cuatro zonas arrecifales localizadas a diferente distancia del Canal del Dique; principal fuente de aportes continentales a los arrecifes de coral del Archipiélago Nuestra Señora del Rosario, Cartagena, Caribe colombiano. Mediante epifluorescencia se determinó densidad promedio de microorganismos presentes en agregados de nieve marina. Los resultados mostraron mayor densidad de bacterias heterótrofas (3.63 x 10(4) ± 1.6 x 10(4) SE células mL-1) que de autótrofas (6 x 10³± 1.3 x 10³ SE células mL-1), principalmente en arrecifes cercanos a descarga de escorrentía continental (bacterias heterótrofas con densidad 8.9 x 10(4) células mL-1 y 3 x 10(4) células mL-1 para Isla Arena y Tesoro, respectivamente). La densidad de microorganismos encontrada es típica de zonas con alto contenido de materia orgánica particulada; por lo cual podría servir como potencial indicador de escorrentía continental. Estudios futuros deben enfocarse en determinar la composición de comunidad bacteriana asociada a nieve marina y posible virulencia sobre organismos arrecifales.

Em aglomerados de neve marinha influenciados por escoamento continental, a densidade de bacterias heterotróficas é maior que a de autótrofas. Esta premissa foi posta a prova em quatro zonas de recifes localizadas em diferentes distancias do Canal de Dique; principal fonte de contribuic.ao continental aos recifes do coral do Arquipélago Nossa Senhora do Rosàrio, Cartagena, Caribe colombiano. Mediante epifluorescencia determinou-se densidade de micro-organismos presentes em aglomerados de neve marinha. Os resultados mostraram maior densidade de bacterias heterotróficas (3.63 x 10(4) ± 1.6 x 10(4) SE células mL-1) que de autótrofas (6 x 10³ ± 1.3 x 10³ SE células mL-1), principalmente em recifes próximos a descarga de escoamento continental (bacterias heterotróficas com densidade 8.9 x 10(4) células mL-1 y 3 x 10(4) células mL-1 para a Ilha Arena e Tesoro, respetivamente). A densidade de micro-organismos é típica de zonas com elevado conteúdo de matéria orgànica particulada; pela qual pode servir como potencial indicador de escoamento continental. Estudos futuros devem enfocar-se em determinar a composicäo da comunidade bacteriana associada a neve marinha e possível virulencia sobre organismos recifais.

The density of heterotrophic bacteria is greater than autotrophic in marine snow aggregates influenced by continental runoff. Four coral reef areas at different distances from the Canal del Dique served to evaluate this premise; this canal is the main source of inland resources for the coral reefs of the Nuestra Señora del Rosario archipelago in Cartagena in the Colombian Caribbean. The average density of microorganisms in marine snow aggregates was determined using epifluorescence. The results showed higher density of heterotrophic bacteria (3.63 x 10(4) ± 1.6 x 10(4) SE cells mL-1) than autotrophic (6 x 10³± 1.3 x 10³ SE cells mL-1), mainly in reefs near continental runoff discharges (heterotrophic bacteria density of 8.9 x 10(4) cells mL-1 and 3 x 10(4) cells mL-1 Isla Arena and Tesoro, respectively). The density of microorganisms found is typical of high-particulate organic matter areas and, therefore, could be a potential indicator of continental runoff. Future studies should focus on determining the composition of the bacterial community associated with marine snow and its potential virulence on reef organisms.

Simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes in ram sperm by fluorescent probes / Avaliação simultânea das membranas plasmática, acrossomal e mitocondrial de espermatozoides de carneiros por sondas fluorescentes

In this experiment, it was defined a protocol of fluorescent probes combination: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and JC-1. For this purpose, four ejaculates from three different rams (n=12), all showing motility >80 percent and abnormal morphology <10 percent, were diluted in TALP medium and split into two aliquots. One of the aliquots was flash frozen and thawed in three continuous cycles, to induce damage in cellular membranes and to disturb mitochondrial function. Three treatments were prepared with the following fixed ratios of fresh semen:flash frozen semen: 0:100 (T0), 50:50 (T50), and 100:0 (T100). Samples were stained in the proposal protocol and evaluated by epifluorescence microscopy. For plasmatic membrane integrity, detected by PI probe, it was obtained the equation: v=1.09+0.86X (R²=0.98). The intact acrosome, verified by the FITC-PSA probe, produced the equation: v=2.76+0.92X (R²=0.98). The high mitochondrial membrane potential, marked in red-orange by JC-1, was estimated by the equation: v=1.90+0.90X (R²=0.98). The resulting linear equations demonstrate that this technique is efficient and practical for the simultaneous evaluations of the plasmatic, acrosomal, and mitochondrial membranes in ram spermatozoa.(AU)

Neste experimento, foi definida uma combinação de sondas fluorescentes: iodeto de propídio (PI), aglutinina de Pisum sativum conjugada ao isotiocionato de fluoresceína (FITC-PSA) e JC-1. Para esta proposta, quatro ejaculados de três carneiros (n=12), que apresentavam motilidade >80 por cento e alterações morfológicas <10 por cento, foram diluídos em meio TALP e divididos em duas alíquotas. Uma alíquota foi submetida a três ciclos de flash frozen e descongelação, para induzir danos nas membranas celulares e distúrbios na função mitocondrial. Três tratamentos foram preparados com as seguintes proporções preestabelecidas de sêmen fresco: sêmen submetido a flash frozen: 0:100 (T0), 50:50 (T50) e 100:0 (T100). As amostras foram coradas no protocolo proposto e avaliadas por microscopia de epifluorescência. Para integridade de membrana plasmática, detectada pela sonda PI, foi obtida a equação: v=1,09+0,86X (R²=0,98). O acrossomo intacto, verificado pela sonda FITC-PSA, produziu a equação: v=2,76+0,92X (R²=0,98). O alto potencial de membrana mitocondrial, marcada em vermelho-alaranjado pelo JC-1, foi estimado pela equação: v=1,90+0,90X (R²=0,98). As equações lineares resultantes demonstraram que a técnica é eficiente e prática para avaliação simultânea das membranas plasmática, acrossomal e mitocondrial em espermatozoides de carneiros.(AU)

Simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes in ram sperm by fluorescent probes / Avaliação simultânea das membranas plasmática, acrossomal e mitocondrial de espermatozoides de carneiros por sondas fluorescentes

In this experiment, it was defined a protocol of fluorescent probes combination: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and JC-1. For this purpose, four ejaculates from three different rams (n=12), all showing motility >80 percent and abnormal morphology <10 percent, were diluted in TALP medium and split into two aliquots. One of the aliquots was flash frozen and thawed in three continuous cycles, to induce damage in cellular membranes and to disturb mitochondrial function. Three treatments were prepared with the following fixed ratios of fresh semen:flash frozen semen: 0:100 (T0), 50:50 (T50), and 100:0 (T100). Samples were stained in the proposal protocol and evaluated by epifluorescence microscopy. For plasmatic membrane integrity, detected by PI probe, it was obtained the equation: v=1.09+0.86X (R²=0.98). The intact acrosome, verified by the FITC-PSA probe, produced the equation: v=2.76+0.92X (R²=0.98). The high mitochondrial membrane potential, marked in red-orange by JC-1, was estimated by the equation: v=1.90+0.90X (R²=0.98). The resulting linear equations demonstrate that this technique is efficient and practical for the simultaneous evaluations of the plasmatic, acrosomal, and mitochondrial membranes in ram spermatozoa.

Neste experimento, foi definida uma combinação de sondas fluorescentes: iodeto de propídio (PI), aglutinina de Pisum sativum conjugada ao isotiocionato de fluoresceína (FITC-PSA) e JC-1. Para esta proposta, quatro ejaculados de três carneiros (n=12), que apresentavam motilidade >80 por cento e alterações morfológicas <10 por cento, foram diluídos em meio TALP e divididos em duas alíquotas. Uma alíquota foi submetida a três ciclos de flash frozen e descongelação, para induzir danos nas membranas celulares e distúrbios na função mitocondrial. Três tratamentos foram preparados com as seguintes proporções preestabelecidas de sêmen fresco: sêmen submetido a flash frozen: 0:100 (T0), 50:50 (T50) e 100:0 (T100). As amostras foram coradas no protocolo proposto e avaliadas por microscopia de epifluorescência. Para integridade de membrana plasmática, detectada pela sonda PI, foi obtida a equação: v=1,09+0,86X (R²=0,98). O acrossomo intacto, verificado pela sonda FITC-PSA, produziu a equação: v=2,76+0,92X (R²=0,98). O alto potencial de membrana mitocondrial, marcada em vermelho-alaranjado pelo JC-1, foi estimado pela equação: v=1,90+0,90X (R²=0,98). As equações lineares resultantes demonstraram que a técnica é eficiente e prática para avaliação simultânea das membranas plasmática, acrossomal e mitocondrial em espermatozoides de carneiros.

Dinámica de crecimiento de tubos polínicos en poblaciones de Pachira quinata (Jacq.) W. Alverson (Bombacaceae) / Dynamics of pollen tube growth in Pachira quinata W. Alverson (Bombacaceae) populations

La dinámica de crecimiento de tubos polínicos fue estudiada en dos poblaciones de Pachira quinata, incluyendo los factores que la afectan como: el tipo de cruce, autopolinización y polinización cruzada intra e interprocedencia. El tiempo de cosecha de las flores afecta el número de óvulos penetrados, pero no afecta la distancia máxima alcanzada por los tubos, ni el número de tubos polínicos a lo largo del estilo. El número de tubos polínicos y la proporción de óvulos penetrados fue significativamente mayor cuando el polen fue almacenado a temperatura ambiente, comparado con los valores de polen refrigerado. El número de tubos polínicos a lo largo del estilo es independiente del tipo de cruce utilizado, asi mismo, la proporción de óvulos penetrados es menor en las polinizaciones interprocedencia que en cruces intraprocedencia y autopolinización. La alta proporción de óvulos penetrados en autopolinización indica que la incompatibilidad en Pachira quinata es una reacción de acción tardía y ocurre a nivel del ovario. El mayor número de óvulos penetrados en cruces intraprocedencia comparado con cruces interprocedencia, sugieren un efecto de depresión en la fecundación entre individuos de distintas áreas geográficas.

The dynamics of pollen tube growth was studied in populations of Pachira quinata, as well as the factors that affect it, such as: the type off crossing, self-pollination and cross-pollination among and inside populations. It was found that the time ofgathering of the flowers determines the number of penetrated ovules, but it does not determine the maximum distance reached by the tubes, neither the number of pollen tubes along the style. The number of pollen tubes and the proportion of ovules penetrated were significantly bigger when the pollen was stored at environment temperature, compared with the values of refrigerated pollen. The number of pollen tubes along the style is independent of the type of crossing used; likewise, the proportion of penetrated ovules is smaller in the pollinations among populations than in crossing among populations and self-pollination. The high proportion of ovules penetrated in self-pollination indicates that the incompatibility in Pachira quinata is an auto-incompatibility of late action that occurs at the ovary zone. The biggest number of ovules penetrated throughout crossing inside populations compared with crossing among populations suggests the existence of an out-breeding depression effect among individuals from different geographical areas.

Enumeração de bactérias em leite cru, utilizando a técnica de contagem direta por microscopia de epifluorescent (Direct Epifluorescent Filter Technique - DEFT) / Enumeration of bacteria in raw milk, utilizing the straight technique of tally by microscopy of epifluorescência (Direct Epifluorescent Filter Technique - DEFT)

Avaliou-se o método rápido de análise quantitativa de bactérias por microscopia de epifluorescência (DEFT) no leite cru tipo C recebido pelo Laticínios Escola da Universidade Federal de Viçosa, Viçosa-MG. Um total de 190 amostras foram quantificadas pelo método DEFT e pelo método oficial de contagem padrão de mesófilos aeróbios em placa (CPP). Pôde-se observar a distinguir células coradas de alaranjado (consideradas viáveis) e células coradas de verde ( consideradas mortas), sendo que apenas as alaranjadas foram enumeradas. O resultado da análise de regressão línea apresentou um coeficiente de correção r = 0,83, sendo que a equação de regressão ficou Y = 0,68X + 1,96; onde Y = log10 UFC/mL (CPP). O limite de detecção apresentado pelo método DEFT ficou em 5,3 x 10³ CME/mL. O método DEFT apresentou um CV de 1,6 por cento. Baseando-se nas análises comparativas, pode-se inferir que o método DEFT tem um grande potencial para ser usado no Brasil para avaliar o nível de contaminação do leite cru tipo C. Além da possibilidade da sua aplicação em outros estudos na área de alimentos.(AU)

Quick and reliable methods to count bacterial contamination in foods are required for the industrial quality control. The Direct Epifluorescent Filter Technique (OEFT) can analyse samples in less than 30 minutes, with the advantage 10 differentiate viable and dead cells. A total of 190 samples of grade C raw milk were sampled at a dairy processing plant and analyzed for the Standard Plate Count and DEFT to compare the two methods. The time required to obtainli results by the DEFT method was 20 minutes. 1t IO as possible to distinguish viable cells, fluorescing orange, and dead bacteria, fluorescing green. The correlation coefficient (r) was 0.83. The precision measured by the coefficient of variation was good (CV = 1.62%). The results confirmed that the DEFT is method that gives accurate results rapidly, needs small place, and has a relatively low cost. (AU)