If i wanted to test whether a given antimicrobial is showing bacteriocidal activity or bacteriostatic activity, which tests i would need to perform? Which techniques will it involve and how reliable and accepted the technique is? Kindly let me know.
Thank you
You should do broth dilution test (i.e. geometric dilutions of your antibiotic in broth). By this, you can determine the MIC (the well or the tube, which shows no visible growth with the unaided eye). The procedure is published in the CLSI document M07-A9. Then spread aliquots each dilution including those showing no growth to solid media without antibiotic. Those concentrations, which are bacteriostatic, end up with growth on the agar plate (the drug only inhibits but not kills the bacteria), while aliquots from concentration which have bactericidal effect show no growth (killing of > 99.9%). With this approach, only the killing concentration can be determined. However, the assay does not tell you anything about the underlying mechanism. This is just the general procedure. Depending on your compound you need to figure out the appropriate test conditions, first. Best regards, Oliver
https://en.wikipedia.org/wiki/Minimum_bactericidal_concentration
However isnt there a direct method like a biochemical test or any analytical instrument based test.
I read that Raman spectroscopy could be used and so could be FTIR, kindly throw some light
In my opinion checking for bacteria's ability to divide is a direct method for determining bactericidal or bacteriostatic ability of a chemical. Bacteria will start dividing once the bacteriostatic agent is removed while in case of treatment with a bactericidal compound you will not observe any growth following treatment. You can treat bacteria with different concentrations of chemical in question and after the desired time point you can inoculate a fixed volume inoculum in fresh media. You can now determine the growth rate of the bacteria by measuring OD at 600 nm.
Regarding use of a biochemical assay or a spectroscopic assay (Raman or FTIR) you can use those assays but they will be indicative at best. To do so you need to first establish few things
1. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of your compound
2. Mode of action of your compound
You need to incubate bacteria with MBC of your compound. You can then see how FTIR or Raman spectra is changing with time. You need to check for viability in parallel samples. This will give you a reference spectrum in which you can determine the changes in peaks according to health of bacterial cell. Once you identify the peaks which are only present in live cells and dead cells then you can follow the bactericidal action of your compound. You have to keep in mind that peaks identified by you may not be generic in nature and may be due to specific mode of action of your compound.
Thank you Vishesh, FTIR is what i was thinking of. My boss asked me to get details on how to analyze bacteria using FTIR, just looking it up online :)
Dear Pritish,
Perform growth curve study. Monitor the increase in cell density of compound-treated cells against the non treated using a spectrophotometer. The untreated cell growth produces a sigmoidal curve. Compare this to the curves obtained for the compound-treated. Deviations from the normal sigmoidal curve pattern is an indication as to the effect of your compound on the bacteria. You may get a normal sigmoidal curve but with an extended lag phase, or you may get the sigmoidal pattern but with maximum optical density (OD) attained at a lowered OD than that attained by the untreated, or you may not get any curve at all. The later indicates a bactericidal effect of the compound.
You can refer to the attached articles for the procedure.
http://bmccomplementalternmed.biomedcentral.com/articles/10.1186/1472-6882-13-342
Article Fathilah AR, ZHA Rahim, Y Othman, M Yusoff. (2009). Bacteri...
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