In a broth microdilution antibacterial test, In some cases, I can observe the wells containing the tested bacteria mixed with a high concentration of the extract turn red after using TTC-based pectrophotometry, meaning the extract did not have any antibacterial activity. But in order to be accurate, I am wondering whether there are bacteria which are inhibited by the extract (and cannot grow anymore in the culture media) but are still metabolically active and thus they can reduce TTC to form formazon. How can I detect these types of bacteria?
Flowcytometry can be more accurate in that it can detect dead and viable bacterial cells. can it be more useful?
Best regards
Hamid
Could plating a specific volume of the mixture on agar plates solve your problem? If you find bacterial growth after incubation, then the bacteria were not killed by the extract.
@Jameel. Thank you very much for your valuable comment. Yes it would be good method to validate the results of TTC particularly when the extract has completely killed the bacteria. But it might not be very accurate where for example the extracts kills 50% of the bacteria as quantification of colonies on the agar media is not very easy.
To solve the quantitation problem when there are a large number of bacteria plated, the standard method is to prepare 10-fold serial dilutions of the cell suspension and spread each dilution separately onto agar plates. That way, at least one of the dilutions will have a countable number of colonies.
Hamid Pourianfar Have you solved this question? I have searched about the same reason because I have tried another assay based in ATP in which my extracts showed inhibitory activity but with TTC everything turned red. So I would like to know if you have tested another 96well assay like MTT (I don't know if there will be difference), because I was thinking if the problem maybe is that TTC is a toxicity assay and not inhibitory that's why the false negative...
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