I am working with S.aureus bacterial strain and inducing cell death in it with antibacterial treatment. I want to compare my control and treated sample in Flow cytometry and fluorescence microscopy.
Pellet down the cells at low RPM---- discard media---give PBS wash thrice gently---fix the cells with 0.1% formaldehyde---wash off--- dry for few minutes---- stain with fluorescent dye in complete dark conditions to avoid quenching---immediately go for imaging without a delay. Prepare all the reagents fresh. Don't use them if there is any precipitation or turbidity. Add the stain to every experimental group just before imaging.
Hi Divyesh.
I suppose you meant Propidium Iodide, and your automatic editing software changed it in a known word.
If I understand well your question, you have S. aureus with some antimicrobial agent, and you want to estimate the cell death by incorporation of propidium iodide on live bacteria. Is it right ?
If so, I think you should use fresh live bacterial suspensions with 0.01%-0.1% Propidium in culture medium, and analyse them directly under microscope or with flow cytometer. You will see dead bacteria.
If you really need to fix them, you will have to wash the culture twice by centrifugation before resuspending and fixing it with 2% formaldehyde (5% formalin).
Fixed bacteria in formaldehyde can be kept in the fridge for weeks, but Propidium Iodide staining will fade due to the weak interactions of the dye with the phosphate groups wihin the DNA molecule, this is why I don't think fixing stained bacteria is a good option.
You can also try the inverse strategy with DiHydroEthidium, which is a membrane-permeant blue fluorescing dye. When oxydized in living cells, it is converted in Ethidium which stains the nuclei in red (as Propidium). You will then see living bacteria.
If you combine with a green fluorescent DNA stain such as Sybr-Grenn I or II or Syto9 you will be able to see every microbe (living with DiHydroEthidium, or dead with Propidium) in your suspension.
Hope this helps
Oliv
Thanks, Olivier Catrice I will try another time with your method. I have tried to use Propidium Iodide directly on the bacterial suspension in PBS but due to some reason, it was not seen in the fluorescence microscope. infect no picture was observed in microscope so might need to see the microscope first. and do I need to use stain before the fixation or after the fixation?
do you have any slide preparation method for fluorescence microscopy?
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