Production of low-molecular weight toxins by marine bacteria

Toxicology

Gallacher, Susan

University:University of Glasgow

关键词: Toxicology;   

Others  :  http://theses.gla.ac.uk/72972/1/10647327.pdf

来源: University of Glasgow

【 摘 要 】

A tissue culture assay was developed which could detect sodium channel-blocking (SCB) toxins in as little as 0.1 ml of bacterial culture supernate. The assay was based on the principle that when ouabain (oub) and veratridine (ver) are added to a culture of mouse neuroblastoma (MNB) cells, an influx of Na+ ions into the cell occurs which, if left unchecked, eventually leads to cell lysis. However, if SCB toxins are present, the effect of the above two neurotoxins is blocked and the cells survive. Cell survival was measured with the vital stain, neutral red, and the absorbance of the pink colour, which developed after treatment, with citrate-buffered alcohol, was measured with a microtitre plate reader. This allowed the cell protection, due to SCB toxins, to be calculated. Standardisation of batches of chemicals, serum and culture conditions was vital to provide reproducible results. The sensitivity of the assay was increased by stimulating differentiation of the MNB cells by growth in low concentrations of serum, or by addition of 10 mM HMBA (N,N'-Hexamethylene-bis-acetamide), or rat glioma medium to the culture medium. SCB activity was found in both bacterial cell extracts and culture supernates. However, these samples also contained an inhibitor which, in the cell extracts, was heat sensitive, but in the culture supernates was heat-stable. The inhibitor in the culture supernates was identified as NaCl, and arose from the high salt concentrations in the marine bacterial culture medium. This inhibitory effect could be reduced by several procedures such as; the use of growth media containing less NaCl, initial dilution of the sample in water, assaying the sample at dilutions of 1/15 or by charcoal extraction of the supernate. Heating and ultrafiltration of the bacterial supernate and the TTX control yielded unexpected results, in that SCB activity decreased during the first 20 min of heating at 100