I carried out a microdilution test for extract and fraction samples of 80 mg samples with DMSO 300 µl and distilled water 700 µl. For the negative control I used DMSO 300 µl and distilled water 700 µl without sample. First I put 50 µl of media into each well, then 50 µl of the sample on the 1st well, and the sample was resuspended. Finally I put 50 µl of bacterial suspension on each well. After 18 hours of incubation, I did some resazurin staining.
[based on my calculation I think the percentage of DMSO in the 1st well is 75 µl/1000 µl=0,075 so it is 7,5% (v/v)] and the literature said that the E. coli can tolerate 10% DMSO
300 µl/ml x 50 µl = C1 x 100 µl (50 µl media + 50 µl sample)
C1 = 150 µl/ml
150 µl/ml x 50 µl (after resuspension) = C2 x 100 µl (+ 50 µl bacteria suspension)
C2 = 75 µl/ml =75 µl/1000 µl = 7,5%
On microdilution with S aureus ATCC 25923 bacteria, the negative control did not inhibit the bacteria. However, microdilution with the E coli ATCC 25922 was inhibited in well 1 and 2 in the negative control (it's on the photo). I used the same negative control on both bacteria.
I don't know why this is happening, is there any explanation?
Hello,
The issue could be the difference in tolerance levels to DMSO. E.coli doesn't tolerate more than 10% DMSO whereas S.aureus has a higher value, I believe this explains the difference you observed. You can sort out this issue by preparing the active in lower concentration of DMSO. Hope this is helpful. You can even look up to reference values from published articles.
DMSO is potentially toxic. keep DMSO amount to minimum. You are using too high. Check literature for further reference.
As Thulasi Mohan S wrote, you are using much too much DMSO! 30% is a huge concentration! Dilute your compounds 10-fold!
And the way the question is written "Why my E. coli is inhibited by DMSO when the DMSO is in the negative control?" suggest you miss understood the purpose of "negative control". It is meant to check if you have an issue and clearly you have one (too much DMSO). Be thankful too negative controls!
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