Quantifying number of live and dead bacteria - (Oct/08/2008 )
I'm trying to decide which method would be the best to use to quantify the number of cells in a bacterial culture. I am only interested in bacteria that adhere to the surface. The most common method to count live bacteria is CFU count. However, I was thinking of using a DNA/RNA ratio. Use total DNA (as a more stable molecule) to quantify the total (?) number of cells and use some housekeeping RNA (as less stable and indicative of cell activity) to quantify the number of live cells.
Does anybody have better ideas or suggestions to the idea I have outlined? Any thoughts will be helpful.
This mgiht be something you wanna try out.
It will not give a precise cell number or live cell count but it is a good way to compare cell adherence.
1. First pour out the medium containing the planktonic cell or u could use a pipet to be more gentle
2. wash briefly with water
3. fill in the well with 0.2% cystal violet .
4. let it stain for 10 min
5. pour out the CV, wash with water ( gently to avoid adherent cell from coming off)
6. Then dissolve CV using either ethanol or 33% acetic acid
7. Measure absorbance at 600nm
Does anybody have better ideas or suggestions to the idea I have outlined? Any thoughts will be helpful.
Simple. You can do a propiduim iodine stain and FACS to quantify live/dead bacteria. The dye stains dead bacteria, while another dye will stain live bacteria (usually included in the kit). The stain might be expensive and I don't know if you want to do FACS. But you could dilute and count under florescence microscope too.
Hope that helps.