We are using a Thermo Fisher Attune on blue and violet lasers for bacterial viability studies and complementary physiology analysis. SYTO9 is probably our workhorse dye. The cytometer
HI Timothy,
Although I am not doing bacterial FCM, I have been struggling a lot for standardizing FCM-based analysis and counting of (cell-derived) sub-micron particles (aka MPs, MVs, EVs, Nanoparticles ...) and I know one of the difficulty is in setting-up scatter detection in a reproducible manner. In your case, the availability of DNA may allow efficient , clear-cut, fluorescent staining of single bacteria for triggering based on fluorescence. Absolute size calibration for bacteria may not be mandatory. However, QC tools to evaluate (and monitor) instrument size-related resolution on a day-to-day basis may be very useful. We routinely utilize ready-to-use cocktails of calibrated beads with sizes spanning a range of 0.1 to 0.9 µm (Megamix-Plus or Gigamix beads) to help setup the machines. See my RG page for more examples. Hope that helps. Philippe
Hi Timothy,
A very fast and reliable analysis of bacterial viability and other cell characteristics is provided by impedance flow cytometry (IFC). The technology is label-free which means that there is no need for incubation with chemical dyes or stains. Another advantage is that the sample can be re-used.
The instrument is portable and for field use it can be run on batteries. Up to about a thousand cells per second can be analyzed. IFC is particularly useful in cases where optical dyes do not work, e.g. in mixtures with nanoparticles. See attached publications or www.amphasys.com for further information.
Let me know if you are interested in additional information, Marcel
Article Viability and membrane potential analysis of Bacillus megate...
Article Impedance Microflow Cytometry for Viability Studies of Microorganisms
Conference Paper A Microfluidic Chip-Based Impedance Flow Cytometry Method fo...
Hi Timothy, is there a reason you are choosing FC as your method of analysis? I spent about 3 years trying to get bacteria analysis to work on a portable flow cytometer and it was not fun at all. You have to first make sure the size of your bacteria of interest is of a good size for detection. Secondly, the concentration you are using needs to be in the accurate range of the machine. You also need to have enough material to allow the machine to reach steady state, which can take up to a minute or so. Lastly, you need to assess the background from the dye. Are you able to distinguish the viable cells just by counting fluorescing cells against total cells? May I ask what your aim is? I would avoid trying to bacteria analysis on flow cytometer if that is an option.
Just to make sure that there is no misunderstanding: There are different types of flow cytometers. I was talking about Impedance Flow Cytometry (IFC). There are no labels, no fluorescent dyes or background fluorescence with IFC. Cells are analyzed based on their electrical properties in a high frequency field. In many ways it works similar as a security scanner at the airport.
- Badges
- Science method