I try to make antimicrobial test for plant extract with disk diffusion method. I macerated grinded plant in ethanol 96%. This ethanol extract is tested for antimicrobial activity against E.coli. Surprisingly, I got zone of inhibition of pure ethanol as control 23 mm and ethanol plant extract was less than 23 mm (faint haze zone) where clear zone i got is around 13 mm. Could anyone tell my why?
Dear Ayu Mutiara Haspari
Kindly repeat the whole setup of your experiment including the control (ethanol), sometime unusual results occurs but it is better to repeat such results and especially the antimicrobial activities should be determined in triplicate to avoid error chances
A quick check of total phenolic content (TPC) and antioxidant activity (DPPH) is recommended before disk diffusion assay. Compare the activity of control (ethanol) and extracts. Replicate the experiments for concordant results.
Remember: Use have used 96% ethanol which is close to absolute ethanol. Pure ethanol have bactericidal properties as they tend to lyse the cell wall.
In my opinion, try extracting the plant phenolics with 80% ethanol.
Overall pure ethanol extraction is fine, but you do need to make sure all ethanol is evaporated before reconstituting the extract. Also, your extract might diffuse slower than pure ethanol, which could have an effect. You should do a few more repetitions. The disk method is notoriously inexact, and many journals do not accept papers based on this method only.
After evaporating the ethanol, dissolve the weighed residue in certain % of DMSO and then do disc diffusion assay.
Try repeating your experiment but using the the agar well diffusion method. Experiment must be replicated usually for a minimum of three times. I would prefer you use a certain % of DMSO instead of ethanol in carrying out your experiment.
your result which you have got for absolute ethanol is correct because you are using alcohol which have antibacterial activity. you should not use it as control.
try to repeat your experiment by dissolving your sample in a low concentration of solution or buffer. and check wither this concentration have any antibacterial activity. use negative and positive control.
regards
Did you have a measurement of alcohol content in your plant extract?, alcohol is hydrosoluble and catch the environmental water, 96% alcohol denatures proteins and precipitates other compounds, what media do you use for E coli culture?
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