Microdot method: used with chromogenic agar is a useful procedure for sanitary monitoring in aquaculture

Abstract

ABSTRACT.The microdot method is a downscaling methodology of traditional tenfold serial dilution procedure used in microbiology. The microdot method uses 100 µL for serial dilution and count colonies in a spot of 10 µL. In this study we counted colonies directly in a chromogenic agar plate to determine, at the same time, the presence and cell concentration of target bacteria required for sanitary monitoring of Chilean export fishery products. Due to among importers countries the most concerning bacteria included in sanitary monitoring are Escherichia coli, Listeria monocytogenes and Staphylococcus aureus, we used the chromogenic agar; CHROMagar ECC, CHROMagar Listeria and Baird Parker agar, respectively. The results shows no differences between quantitative results obtained with microdot and traditional method during the quantification of a culture of Escherichia coli (1.5 L). The sensitivity and specificity of the microdot method in association with each chromogenic agar was demonstrated in vitro with reference strains. In addition, the usefulness in sanitary monitoring of aquaculture procedures was evaluated in Artemia salina tanks. This method did not detected sanitary problems in surface water. Although other colonies grown in the chromogenic agar plate, their morphological and chromogenic properties not correspond to Escherichia coli, Listeria monocytogenes and Staphylococcus aureus, being identified as Salmonella enterica subsp. enterica, Microbacterium sp., Bacillus sp. and Staphylococcus pasteuri by 16S rRNA gene sequence analysis. Hence, we propose the microdot chromogenic method as a low cost, specific and reliable procedure for sanitary monitoring of aquaculture procedures. Keywords: microdot method, sanitary monitoring, aquaculture, chromogenic agar.Método de la microgota: usado con agar cromogénico es un procedimiento útil para el monitoreo sanitario en acuicultura RESUMEN. El método de la microgota es una versión a menor escala del procedimiento tradicional de dilución en serie en base diez utilizados en microbiología. El método realiza diluciones en 100 µL y cuenta colonias crecidas en una gota de 10 µL. En este estudio se cuentan colonias directamente en placas cromogénicas para determinar densidad celular y presencia de bacterias requeridas en vigilancia sanitaria de productos pesqueros chilenos de exportación. Entre los requisitos de países importadores, la vigilancia sanitaria involucra frecuentemente a Escherichia coli, Listeria monocytogenes y Staphylococcus aureus, por lo que se utilizan los agares cromogénicos; CHROMagar ECC, CHROMagar Listeria y agar Baird Parker para su identificación. La comparación entre el método de microgota y el método tradicional no muestra diferencias al evaluar un cultivo de Escherichia coli (1,5 L). La sensibilidad y especificidad del método de microgota junto a cada agar cromogénico se demostró in vitro con cepas de referencia. Además, en estanques de Artemia salina se evaluó la utilidad de este método para el monitoreo sanitario. Este método no mostró problema...

Microbial Diversity of Psychrotolerant Bacteria Isolated from Wild Flora of Andes Mountains and Patagonia of Chile towards the Selection of Plant Growth-Promoting Bacterial Consortia to Alleviate Cold Stress in Plants

“…The psychrotolerant potential was evaluated with a bacterial cell survival test at subzero temperatures described by Sun et al [ 17 ], with modifications. The isolates were cultured in 200 µL TSB in a 96-well microplate for 72 h at 20 °C and then subjected to −20 °C for 24 h. Colony-forming units (CFU) per mL were counted using the microdroplet method [ 47 ], before and after the cell freezing treatment. The bacterial cell survival percentage (BCS%) was calculated.…”

Selenocysteine Formation by Enterococcus faecium ABMC-05 Follows a Mechanism That Is Not Dependent on Genes selA and selD but on Gene cysK

“…This solution was sterilized through a 0.22 µm sterile syringe filter (Millipore) and supplemented with MRS medium in the tubes to incubate them at 37 • C for 36 and 48 h under anaerobic conditions. The viable count was carried out by a plate count on MRS agar (BD Difco, MD, USA) using the micro-drop technique [24]. The control consisted of MRS supplemented with Na 2 SeO 3 without bacteria.…”

Evaluación de las propiedades fisicoquímicas y microbiológicas del jugo de betabel (Beta vulgaris L.) termoultrasonicado.

“…El índice de estabilidad se valoró determinando el porcentaje de partículas sedimentadas (p/p) después de la centrifugación (Hamilton Bell, V6500, USA) (Strahsburger et al, 2016).…”