Steve Hilliard writes- >I was just approached by a student who is looking at Mycoplasma >pneumoniae, and he's got an interesting question. He has a mutant that >seems to have an altered chromatin condensation, which he has been looking >at using image analysis, but he would like to use flow. His >approach is to stain the DNA w/ DAPI, and the membrane with another >dye, and then analyze the area associated with each color. The ratio >of nucleoid area to total cellular area differs btwn wild type and >mutant. > >He considers the microscopy a pain, and would like to use flow, but >this type of spatial analysis is the type of thing that imaging is perfect >for. For one thing, these things are tiny, and bad about clumping. > Has anyone had any success with M. pneumoniae or relatives in a flow >system?? Should I be giving this guy a pitch for a laser scanning >system? I'm sure the microscopy is a pain. Mycoplasma pneumoniae, if I remember correctly, has about 7 KBP of DNA and is below 0.5 um in size; this would put it near or below the detection limit of conventional flow cytometers in both scatter and fluorescence. A slow flow system could give better signals, but I don't see how the "area" of either the nucleoid or the membrane could be measured even with slow flow. Your student must have a pretty good image analysis system, because you need really high optical resolution to get enough pixels for a meaningful estimate of any of the subcellular dimensions of an organism the size of M. pneumoniae. -Howard A slow flow system
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