Eleven media were studied for their suitability in the selective isolation ofAeromonas hydrophila. Preliminary results showed that five of them (inositol-brilliant green-bile salts agar, bile salts-brilliant green agar, Rimler-Shotts agar, xylose-sodium deoxycholate-citrate agar, and dextrin-fuchsin-sulfite agar) allowed the growth of several microorganisms that are usually present in the same samples in which A. hydrophila is found. Six media (mA agar, modified Rimler-Shotts agar, DNase-toluidine blue-ampicillin agar, Pril-xylose-ampicillin agar, MacConkey-trehalose agar, and starch-bile salts agar) were selected for evaluation as recovery selective media on the basis of their efficiency in the isolation of A. hydrophila from natural water samples. mA agar showed the best recovery rate and also an acceptable specificity, but its selectivity was low. Another medium that can be considered is DNase-toluidine blue-ampicillin agar, which showed good accuracy, but its specificity was low.
<p>En el presente trabajo se estandarizó una técnica de criopreservación en nitrógeno líquido, usando como crioprotector dimetil sulfóxido al 5%, para la preservación de aislados nativos bacterianos de <em>Fibrobacter succinogenes</em>. El respectivo protocolo de criopreservación se evaluó determinando la viabilidad bacteriana por el transcurso de un año. La escala de MacFarland, ampliamente conocida en la estimación de poblaciones bacterianas aerobias, fue validada como técnica de estimación de bacterias anaerobias, usando la técnica de conteo bacteriano por <em>roll-tube </em>(R2 = 0.95). Se utilizaron dos aislados nativos, C7 y C9, de <em>Fibrobacter succinogenes</em>, obtenidos del rumen de bovinos en pastoreo de <em>Brachiaria decumbens</em>, en los Llanos Orientales de Colombia. Quince días después de la exposición de los cultivos al proceso de criopreservación, se apreció una reducción en el número de bacterias viables con relación a la población inicial de cada uno de los aislados (C7 = 9.25 x 108 UFCml-1 vs. 6.15 x 108 UFCml-1 y C9 = 12.51 x 108 UFCml-1 vs. 9.26 x 108 UFCml-1). Sin embargo, no se presentaron diferencias en la población de bacterias viables en los muestreos posteriores (C7 = 6.13 – 6.16 x 108; C9 = 9.26 – 9.35 x 108 UFCml-1). Esta técnica permite mantener la viabilidad bacteriana y puede considerarse como un procedimiento eficiente y de fácil aplicación para la preservación de bacterias ruminales. Además, constituye una herramienta fundamental para el establecimiento de bancos de germoplasma de microorganismos anaerobios ruminales en Colombia. El uso conjunto de las técnicas de criopreservación y la escala de MacFarland, posee ventajas sobre los métodos de preservación y estimación de poblaciones por cultivos activos, por su confiabilidad, eficiencia y bajo costo.</p><p> </p><p><strong>Cryopreservation of native isolates </strong><strong>of ruminal bacteria <em>Fibrobacter </em></strong><strong><em>succinogenes</em></strong><strong>.</strong></p><p>In this study, the technique of cryopreservation in liquid nitrogen to preserve native isolates of the fibrolytic bacteria <em>Fibrobacter succinogenes </em>was standardized. The technique efficiency was evaluated by establishing the bacterium viability during one year. The Mac-Farland scale, widely used to estimate aerobic bacterial populations, was validated by the roll tube technique (R2=0.95). Two native <em>F. succinogenes </em>strains, C7 and C9, were used. These strains were obtained from cattle fed on <em>Brachiaria decumbens </em>in the eastern lowlands of Colombia. Fifteen days after the initiation of the cryopreservation process, a reduction in the number of viable bacteria (CFU ml-1) was registered (C7 from 9.25 x 108 to 6.15 x 108 and C9 from 12.51 x 108 to. 9.26 x 108). However, the bacterial populations did not present differences in subsequent samplings (C7=6.13 – 6.16X108, C9=9.26 – 9.35X108 CFU ml-1, range over a year). Results indicated that the standardized cryopreservation technique is an effective and easy procedure to preserve ruminal bacteria, which would be a very useful tool to create a germplasm bank of Colombian ruminal anaerobic microorganisms. The cryopreservation technique, and the MacFarland scale have an advantage over other methods of preserving bacteria and methods of estimating populations of bacteria by active cultures. The procedures used in this research are reliable and economical techniques using minimal storage spaces and laboratory resources.</p>
<p>The capacity to degrade the cellular wall of Bouteloua repens of 12 isolates of Ruminococcus flavefaciens and 17 isolates of Fibrobacter succinogenes was determined. A procedure was developed to standardize both, the inoculum and the substrate concentration for in vitro incubations of pure culture isolates.The ruminal bacteria were isolated from either Bos indicus cattle from the Alto Magdalena region of Colombia, grazing Bouteloua repens, and Sanmartinero cows (a Colombian criollo breed) from the eastern lowlands (Llanos Orientales) of Colombia, grazing Brachiara spp. The isolates identity was confirmed using molecular methods. All isolates of F. succinogenes showed a high capacity to degrade cell wall preparations of Bouteloua repens, than those of R. flavefaciens (P<0.0001).The effect of individual isolated on cell wall digestibility was also highly significant (P<0.0001). Native isolates surpassed considerably the reference strains of R. flavefaciens ATCC 19208 (12.83% vs. 6.83%) and F. succinogenes ATCC 19169 (13.77% vs. 7.94%) on their capacity to digest Bouteloua repens cellular wall.This difference was highly significant (p<0.05) for all isolates. Findings suggest that in tropical conditions native ruminal bacteria has developed a high capacity to degrade lignocellulose. Further research about the potential usefulness of specific inocula or enzyme extracts developed from such isolates in improving the degradability of low quality forages by cattle or what industrial applications can be improved by the use of these products.</p><p><em><br /></em></p><p><strong>Degradación in vitro de </strong><strong><em>Bouteloua repens </em></strong><strong>por cultivos de </strong><strong><em>R. flavefaciens </em></strong><strong>y </strong><strong><em>F. succinogenes </em></strong><strong>aislados de ganado alimentado con pastos tropicales en Colombia</strong></p><p>Se determinó la capacidad de degradación de la pared celular de <em>Bouteloua repens </em>de 12 aislados de <em>Ruminococcus flavefaciens </em>y 17 de <em>Fibrobacter succinogenes</em>. Se desarrolló un procedimiento para estandarizar la concentración, tanto del inóculo como de los sustratos, de las incubaciones <em>in vitro </em>de cultivos puros de los aislados. Las bactérias fueron aisladas del ganado <em>Bos indicus</em>, procedente de la región del Alto Magdalena, que pastoreaban Teatino (<em>Bouteloua repens</em>) y del ganado Sanmartinero (una raza nativa) procedente de los Llanos Orientales de Colombia, que pastoreaban <em>Brachiaria spp</em>. La identificación del aislado fue confirmada usando métodos moleculares. Todos los aislados de <em>F. succinogenes </em>mostraron mayor capacidad de degradación de las preparaciones de pared celular de <em>Bouteloua repens </em>que los de <em>R. flavefaciens </em>(P<0.0001). La capacidad de los aislados individuales para degradar la pared celular también difirió significativamente (P<0.0001). Los aislados nativos sobrepasaron considerablemente la capacidad para digerir la pared celular de <em>Bouteloua repens </em>de las cepas de referencia de <em>R. flavefaciens</em>ATCC19208 (12.83% vs. 6.83%) y <em>F. succinogenes </em>ATCC19169 (13.77% vs. 7.94%), diferencia que fue altamente significativa (p<0.05) para todos los aislados. Los resultados sugieren que, en condiciones tropicales, las bacterias ruminales nativas tienen una alta capacidad para degradar la lignocelulosa. Se requiere mayor investigación sobre la utilidad potencial del inóculo o de extractos enzimáticos desarrollados a partir de tales aislados, a fin de aumentar la degradabilidad por el ganado de forrajes de baja calidad o para definir las aplicaciones industriales pueden ser mejoradas por el uso de estos productos.</p><p> </p>
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