Hello, I have done 96 well plate assay for detection of MIC of antibiotic for particular organism. I took the O.D of the plate and I understood the concentration of antibiotic which inhibits the growth. But i'm not getting how to interpret the results. Is anyone can help me to sought out this problem.
Dear Dhanshree
To interpret the MIC you have to see the absorption of blank/media control in comparison to growth control. And you have to make sure that when you added the inoculum there was zero or absorption must be equivalent to blank media. Now see the absorption of compound MIC line which must have difference of 90% (MIC90) and 99% (MIC 99).
Say if growth control is giving an absorption of 1.0 O.D and MIC well is giving 0.1 then there is 90% difference (MIC90) and if there is 0.01O.D then this is MIC99.
I hope this would help.
Dear Dhanshree
To interpret the MIC see the difference between blank/media control and growth control.
Now see the absorption of MIC well line. the well giving the absorption nearly equal to blank with more then 90% or 99% difference as compared to growth control is MIC90 and MIC99 respectively.
Hope this would help.
Thank you samsher. I got the idea.
Kindly check the following and help me out to find out one.
For example following are the O.D of
Control- 0.3485
1 0.2430
2 0.0985
3 0.0116. How will you correlate for MIC 90 and MIC 99
Better is that go through the CLSI Guidelines and try to follow you got the answer of ur question.
Dr. Sharma is correct, please use the CLSI guidelines. Also there are specific descriptions for MIC90.
Hai everyone thank you for your valuable answers.
I got MIC % like 69% and 28%.. nnow how can i represent as samsher said it's nnot showing 99 annd 90% MIC.
Hi, you got important Infos above just to add some points. If your suspension is clear (colorless) you can also visually determine MIC values as the growing microbes form white (depending on the type of microbes) spots. And then you make sure with OD values and calculate. For the interpretation, I would also say either CLSI or EUCAST.
As mentioned by some colleges above CLSI guidelines can assist u a lot
I agree with response as given by Tewodros Debebe.
Actually you were looking for MIC by absorption value.
You may take the visual values for MIC. For this you have to see where the growth stops coming, means which well is clear is MIC (like your control without bacteria.)
Hai,Samsher
Thank you for yur valuable time
I got it whatever yu mentioned earlier but my doubt is how to mention MIC in 99% and 90% wen the value is not coming like that. I'm getting like 48% ,70%..etc kindly clarify it
Dear Dhanshree
I suggest you to follow the guidelines as given in CLSI.
Or you can give me the full protocol and bacteria you are using. Because in your case there is difference is too low for growth and control. It seems your growth is not full or you are using low inoculum level.
Which method you are following please send me link or publication. I would see. Please send whole details of your plate with absorbance.
please go through this publication you get idea
Article Comparative Study of Hydroalcoholic Extracts of Momordica ch...
Hello Samsher.
I'm here by attaching the paper I had referred, and even I used same bacteria.
In the protocol, they have referred to take mid log phase cells and the reading of the bacteria measured @ 650 nm. (In my work I had taken the O.D.@ 600nm and when the bacteria reached 1 O.D considered as mid-log phase,) And remaining I followed exactly same as in the paper. Kindly help me out regarding the same.
Regards
http://aac.asm.org/content/51/1/169
Actually your results are promising but as I said MIC90 and MIC99 then there must be some quantifying idea in percentage.
As you are following the method descirebed in attached paper. So adhere to that prototcol. As MIC by that "The MIC was the lowest concentration of the tested agent giving complete inhibition of growth (an OD equal to the OD of the blank). The
microplate assay was repeated four times for each antimicrobial-bacterium combination, and the MIC was determined as the median of the four repetitions." as described in MS itself
They are taking the blank reading equivalent as zero. Means MIC well which have absorbance equivalent to media control is MIC.
Or you may take media control as 100% and growth control as 0% to calculate.
Hope it would help.
Good luck
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