I have a few substances that resemble curcumin, and I need to conduct antimicrobial tests, especially MIC experiments in a 96-well plate, with these substances. However, these substances are highly colored, making it difficult to determine if there is inhibition or turbidity. I've checked the literature, and some studies have used growth indicators like MTT.
Despite this, it remains unclear in which one growth has completely stopped.
Do you have any advice on assessing the antimicrobial effect of such highly colored substances?
Do you think agar dilution is the only solution? If you recommend agar dilution, should I initiate the experiment in a manner similar to MIC testing in a 96-well plate and then, after 24 hours, spread each 100 µL solution onto agar plates? Alternatively, should I conduct this study initially in a 24-well plate or Eppendorf tubes and then proceed to agar dilution?
Thank you in advance for your responses.
I don't think it matters whether you do your assays in 96 well plates, 24 well plates or tubes. But if you can not measure optical density or turbidity, then the easiest thing may be to measure surviving CFUs. You can plate out a sample of the culture after treating, but you might need to also do a dilution series. You can just do a series of 10 fold dilutions and spot 5 ul of each onto your plates (you can usually get a number of spots per plate). Otherwise plating directly after 24 hours is likely to have too high a cell density to count directly, unless the compound is highly inhibitory.
Hi, an initial plating technique can be employed, wherein the contents of the 96 wells are aspirated following the specific treatment. Subsequently, centrifugation is performed to facilitate cellular pelleting, and supernatant is discarded. The resulting cellular pellet is then resuspended in a minimal volume of fresh culture medium, typically ranging from 5 to 10 microliters. The resuspended cell culture is then carefully spotted onto agar plates. Subsequent monitoring for growth, typically over a 24-hour period, facilitates the discrimination of compounds exhibiting either bacteriostatic or bactericidal effects (when bacterial cultures are used). This rapid screening approach serves as an efficient method for assessing the antimicrobial activity of test samples. To enhance precision, quantification of colony-forming units (CFUs) can be conducted selectively on concentrations of interest as per serial dilution methods.
Hope this helps!
Hi, good day. Simple is that for initial screening in comparison with positive and negative control, to find out MIC, you can use the manual method of Well diffusion method, pouring dilutions in the wells with compulsory care so that it may not overflow, you prepare the extract for use and measure the inhibitory clear zone around each well. Colour doesn't matter of any plant material as bacteria don't care for any colour, if it is not effective against it, if effective it will vanish all the bacteria. If you use DMSO as solvent, it may dissolve the extract showing no colour at all.
Hello dears, I would like to invite you to read my ebook "The fantastic microbiological world" for free on Amazon (https://www.amazon.com.br/dp/B0CF1VKKK8)
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays