Hey everyone,
I'm currently trying to determine the MIC of some samples, against Listeria, but I cannot do it by visual observation of button presence/absence since the samples are highly cloudy... I'm reading the 96-well plates in a spectrophotometer, but the samples highly skew my results. At the lowest dilutions I can't see if there is any growth, because the sample precipitates and the absorbance is huge...
Does anyone have an idea to overcome this problem?
Thanks in advance!
If you see precipitation of sample (the test compound) then you need to dissolve the compound completely and do the assay. Use DMSO or Tween 80 or 20 at 0.1% to 1.0% concentration. Prepare a stock solution of the compound you want to test in a 0.1% to 1.0% DMSO or Tween 80 or 20 solution and serially dilute the compound using culture medium and look for any precipitation, if there is no precipitation then you can add the cells.
To solve the cloudiness after the assay, use a fresh plate and dilute the samples (10 times) and read the OD and multiply the results by 10.
Add little amount of butanol to your sample, you may have know cloudiness in samples. Serial microdilution needs ten different glass tubes in a sequence, You should put 10 ml of liquid media in each tube, now add 1 ml of antimicrobial compound in first tube, mix it, take out 1 ml from it add in the next tube. In this way you will get a ten fold serial microdilution that is much enough for antimicrobial testing.
