Hi, all!
I need to determine if my treatment causes membrane damage in bacteria (S. aureus and E. cloacae), but I cannot use a fluorescent probe such as PI and similar ones because my compound is also fluorescent and they interfere with each other (I've already tried this in many ways).
Therefore, I started trying to measure leaking DNA at 260 nm, using a few protocols I saw in previous studies (they are pretty simple: treat bacteria, centrifuge, measure the supernatant).
However, my positive control is not returning any readings; I've tried SDS 1 and 10% and ethanol 70% as membrane damagers.
Is there anything I should be aware of? Or a precise protocol I could follow? Or even a different kind of experiment I could do without any fluorescence involved?
I really appreciate any help!
There is likely to be too much interference at 260 nm from your compound, the medium, and various substances exuded by the bacteria. Another approach might be to measure the release of ATP into the medium using a luminescence assay kit. Unless your compound is also luminescent, it should not interfere.
I see, thank you for that insight.
However, I am not getting reading even without my compound: I've tried testing just a positive control (SDS or ethanol) and still was not able to detect any significant leaking, as the readings are pretty close to the untreated control
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