Upon filtering (0.2um) or ultrafiltering (3kDa) an exponential culture of Salmonella enteritidis in M9 I can measure fluorescence at the wavelengths expected for NADH. However, if I heat these same samples (75°C, 20 min) I observe a big increase in fluorescence. This does not happen with pure NADH in M9.
Does someone have similar experiences or an idea why could this be?
Thank you very much,
Karen
Hola Pedro!
Gracias por tu interés pero lamentablemente ya hemos finalizado el estudio y los resultados están procesándose para su publicación: la serie de apendicitis, de hecho, es probable que prontamente salga publicada en la revista Medicina de Buenos Aires.
No obstante, te cuento que desde la comisión de Uso Adecuado de Recursos de la Sociedad Argentina de Infectología intentamos hacer estudios epidemiológicos que nos ayuden a responder las preguntas respecto a gérmes y sensibilidad a antibióticos que nos hacemos a diario con nuestros pacientes e intentamos involucrar centros de la mayor parte posible de las provincias y regiones del país.
Actualmente estamos llevando a cabo un estudio de infecciones urinarias no complicadas, si te interesase participar, decime y te contacto con alguna de las coordinadoras del mismo.
Te mando un abrazo.
Pablo
Is it possible that the filtrate contains the oxidized form of the cofactor NAD+ which gets reduced at the higher temperature?
Hej. Thanks a lot for your suggestion Radek. I have already tested this using a conducting polymer electrode (which worked with pure NADH) but I could not see any difference in electrochemical potential between the heated and non heated samples. Of course it could be possible that I was measuring several things within bacterial culture that have masked NADH but judging by the amplitude of the fluorescent vs electronic signal this should not be the case. Maybe I should buy an NAD+/NADH kit to find a real answer to this though. : )
Do you think it is possible that something is quenching its fluorescence in bacterial culture filtrates and this something dissociates at higher temperatures?
Thanks!
Yes, that could of course be the case. Fluorescence lifetime could through some light on it - provided it is collisional and not static quenching.
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