Hello to everybody, i'm new in the list. my question deals with the use of SYTO 13 (Mol. Probes) to stain heterotrophic bacteria and detect them by flow cytometry. I've stained E. coli using the procedure of Giorgio et al (1996) Limnol and Oceanogr. 41(4) 783-789, and measure them with a FacScalibur cytometer. No problem with FL1, cells are clearly visible and differences between stained and non stained cells are clearly detectable. The problem is that the stained cells also appear at FL3, i've adjustedl compensations to avoid this without succes. I use a quite high voltage on FL1 and FL3 (700), being my current settings for the detection of unicellular cyanobacteria in lakes. If i decrease FL3 voltage then i loose the autofluorescent cells in nonstained samples. My idea was to measure in one step total and autofluorescent bacteria measuring the former in FL1 and the latter in FL3, but that appears impossible actuallyf. Could somebody tell if that's usual with SYTO 13 or it's me not improving the settings? Is there any other nucleic acid stain excitable at 488 nm with less interference in FL3? Thanks in advance for your answers. -- Xavier Cristina i Pau Institut d'Ecologia Aquàtica Universitat de Girona Facultat de Ciencies E-17071 Girona SPAIN xevi@morgat.udg.es
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