Dear Murugan,

Go for cell culture. Take normal healthy cell lines and add your extract and then after incubation see whether its toxic to normal cell or not

I agree, go to cell culture, perhaps L929 because it is easy to work, and referred on official methods, perhaps using neutral red or other vital dye.

If you are still interested in the compound after testing for mammalian cell toxicity, a next step is to try to confirm the mode of action in bacteria. A typical way to do this is to select for resistant cells and sequence the gene for the supposed target. Mutations in the target are a good sign that you have the right mode of action. This doesn't always work, however, because there could be resistance due to non-target-based mechanisms. An alternative is to overexpress the supposed target and see whether doing so confers resistance. If the target is involved in biopolymer synthesis (RNA, DNA, protein, peptidoglycan, fatty acids), then you could look for inhibition by the compound of incorporation of radioactive precursors into the biopolymer in whole cells.

Dear Murugan

You need to edit your question. Either it should be antibacterial activity or bactericidal activity. Antibactericidal activity conveys that your compound has no activity against the tested bacteria.

Thank you very much Dr. Rajeswara Rao sir, for your correction, its a typo error, i changed it.

Can anyone suggest me a list of  human cell lines that could be used for my cytoxicity study.

I would like to mention here my targeted protein involves in cell wall synthesis, what kind of assays i could do to confirm the mode of action is good.

Thank you very much for your suggestions Dr. Adam B shapiro...

awaiting for some more suggestions.

I suggested that preliminary brine shrimp leathality bioassay assessment is done by the (Mayer1982)  procedure:

For the preparation of sea water 38 g of sodium chloride was weighed, dissolved in distilled water to make 1 liter solution and then filtered off to a get clear solution. This simulated seawater was used for hatching of brine shrimp. The shrimp

were allowed for two days to hatch and mature as nauplii (larvae). In a small beaker, measured amount of the sample was accurately weighed and dissolved in DMSO (Dimethylsulfoxide) to give a final concentration of 10 mg/ml (10 μg/μl). From the test tube containing brine shrimp nauplii, 9 test tubes were taken for the sample where each contained 5ml of seawater and 10 nauplii. These test tubes were marked from 1 to 9 for the sample. To these test tubes different concentrations (160 μg/ml,120 μg/ml, 80 μg/ml, 60 μg/ml, 40 μg/ml, 20 μg/ml, 10

μg/ml, 5 μg/ml and 2.5 μg/ml) of the sample were added. Then the samples were subjected to brine shrimp lethality bioassay (Meyer et al., 1982). In this case, only 50 μl DMSO was added in 5 ml sea water containing 10 nauplii. No extract was added to prepare control solution. Measured amount of the vincristine sulphate (Techno Drugs Ltd., Bangladesh) was dissolved in DMSO to get an initial concentration of 0.512 μg/μl. 9 test tubes for the standard sample were taken where each contained 5 ml of seawater and 10 nauplii. These test tubes were marked from 1 to 9. In test tube no 1, 160 μl of the vincristine sulphate solution was added to give a concentration of 10μg/ml. In a similar way, 120 μl, 80 μl, 60 μl, 40 μl, 20 μl,10 μl, 5 μl and 2.5 μl of the sample were added to test tubes 2, 3, 4, 5, 6, 7, 8 & 9 to give the concentration of 160 μg/ml,120 μg/ml, 80 μg/ml, 60 μg/ml, 40 μg/ml, 20 μg/ml,10 μg/ml, 5 μg/ml and 2.5 μg/ml of the standard sample solution respectively. After 24 hours, test tubes were observed and the number of dead nauplii were counted and the LC50 values were calculated.

You can used your desired concentration

By Choosing the target cell line and testing your extract on it , after incubation you will observe  if itis cytotoxic or not.

I suggested that preliminary brine shrimp cytotoxcity,  leathality bioassay assessment is done by the (Meyer alber1992) procedure well explain by abdul hasanat..

Article Pharmacological activities of selected wild mushrooms in Sou...

Briefly, to confirm in in vitro cytotox assays, you should test your compound in a battery of cell lines. But it depends you want. you could use HepG2, L929, Vero, H9c2... but sometimes using HepG2 is enough for the first step in your studies. In other assays, you could run metabolism assays (HepG2 with ratS9 or HepaRG...) Remember, 48h treatment, dose response curves, a good redout and test other compounds from the family to check if cytotox (or not) is due to chemical structure family or not.... I agree with Sapiro's comments.

Hello Nandagopal

As I understood, you still have not done cytotoxicity tests.

As wrote Pedro, I would test against several typical target cell lines, e.g. detox. organs like liver (HepaRG, THLE; I would not use the famous HepG2 as those cells do not have all detoxification enzymes) and kidney (HK-2). Next interesting would be contact organs, for internal use (and resportion studies) an intestine cell line (Caco-2) or for topical use a skin cell line (HaCaT).

With those cells first I would look for survival/metabolic activity with Tetrazolium salts (MTT, WST) and Neutral Red Uptake. If you find an effect, then I would look for necrotic cell damage, mostly membrane disintegration, which can be easily seen by trypan blue staining and quantified by LDH-release. You have to measure the effects with different techniques (as mentioned) at different time points (e.g. 6, 24, 48, 72 h) and different concentration in order to draw dose response curves. The (low) cytotoxic concentration (EC20) should be higher than the effective antibiotic concentration.

I hope it helps.

Hello Nandagopal

It is a good area of study. From your question you have not indicated the type of Bacteria you tested your plant compound against. I will suggest that if its an enteric bacteria choose a mammalian cell line of the GIT, if the pathogen affects the skin use the skin Cell line to perform the cytotoxity of the compound. I will recommend the use of the MTT assays to determine the survival or metabolic activity at different concentrations it will give you a clear indication on the next move as you can even deduce the LD50, LD75 and since you have the MIC for this Plant compound its easier to deduce its selectivity index

Practice Aimes's test or test on paramecia or test on cell culture (skin cell mathel mytillus cell 

I am in accord with Eric Omwenga

Do MTT assay with your cell lines using increasing concentrations of your compound. This will let you know at what concentration your compound becomes cytotoxic.

I agree Leonor's response

I agree with Lenora

MTT assays with lymphocytes can also be done

Trypan blue dye exclusion method to determine the viable and non viable cell. It is an easy way to determine the cytotoxicity primarily.

Hi there, I think it may depend on what practical application you want your antimicrobial to be used in, ie: will it be used topically? or be incorporated to be used in vivo? A good place to start would be to take a look at ISO standards, maybe ISO 10993-05 (this one is for medical devices but it will give you a guideline of acceptable levels of cytotoxicity and what methods to use to test it.)

I agree with what most people have said using an MTT assay and test for a dose dependant response (if any on cells) and check your cells microscopically for morphological changes. 

But a good place to start is pick a cell line that will model what your intended application, ie: if it is to be used topically use an dermal epithelial cell line.

hopefully this can help. 

http://www.mddionline.com/article/practical-guide-iso-10993-5-cytotoxicity