Mario, Reading your plan of attack on bacteria, made me ponder the problems I had sorting mitochondria. Would the protocol you prescribed for bacteria work on mitochondria in conjunction with the removal of the neutral density filter. Thank you in advance for any advise -----Original Message----- From: Mario Roederer [mailto:roederer@drmr.com] Sent: Wednesday, July 24, 2002 3:21 PM To: cyto-inbox Subject: Re: Bacterial Analysis Your operators should threshold (trigger) on log side scatter. If they are triggering on FS (as is customary), then most bacteria won't come close to being seen. You might also just trigger on fluorescence, and see if the counts are closer. By triggering on fluorescence, you'll also get an idea of how low to set the side scatter (log) threshold to catch all the events. You will likely have a lot of background (noise) events. Consider running a medium blank and decreasing the log SS trigger threshold until you have about 50 counts/sec (i.e., noise at 50/sec). Then run the bacteria, and see what it looks like--you can then increase the threshold until you are sure you aren't losing real events. Then, at that threshold, run blank medium again and see what the background event count is like. You should have no problems seeing all of the bacteria. mr
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