I'm trying to determine growth rates of 6 bacterial species. I will be following growth with OD600, but I want to have absolute cell numbers for several time points during the exponential phase - which I will get from a flow cytometer (based on size, BD Trucount beads, BD FACSMelody). My question is if I could freeze or somehow preserve samples obtained at different time points, and then later run them through a flow cytometer? I suppose doing that shouldn't interfere with results. What procedure could be applied?
Bacterial cells rarely occur as single discrete cells readily analyzable by flow cytometers. Even if you resuspend them thoroughly, you cannot say for sure what you see are true singlets. Under a confocal microscope or in EM (electron microscopy), you will see how exactly your cell samples are like in suspension. The best and most fundamental method for enumerating bacterial cells is serial dilution to appropriate densities, followed by agar plating (3-4 times replicates), and counting the colonies formed (CFU). For nearly all bacteria, a dilution factor of 2.5x10^5 to 5x10^5 would give you countable colonies.
If you freeze or even fix your cells, they will lump together, and that will preclude the possibility of FACS analysis.
Hello, if you want to establish method of counting number of bacterial cells in suspension based on OD at 600 you shuld create calibration curve dependence OD at 600nm from CFU/ml in cell suspension. For this you need to measure OD at 600 of at least 3 suspension with known CFU/ml.
Easy method for establishing CFU/ml of suspension is method of decimal dilutions.
Hope this info will help you.
Good luck.
It is better to use serial dilution method and don't freeze.
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