I am investigating the effect of a cationic peptide on M. tuberculosis. I am using various microscopy techniques such as TEM, SEM, AFM and CSLM. Which of these two methods would be the best?
1) incubate the cells at their MIC 90 and MIC 50 for 24 hours, fix and view?
Or
2) incubate the cells at their MIC90 and take samples for viewing at 0, 1, 6, 12, 24 hours?
Due to the nature of my experiments, I would prefer 1.
Thanks in advance.
Hello Shane,
if your exploring the disruptive ability of these molecules its better to start screening with functional assays such as LDH or viability given that you can screen in a 96 well plate several conditions and concentrations of your peptide very rapidly. We have done a few experiments using TEM to evaluate the effect of cathelicidin, defensins and other peptides. Here are a few references, maybe you might find them helpfull. If you are interested in the protocol details please let me know. Best regards
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0059119
http://www.ncbi.nlm.nih.gov/pubmed/23141114
Thank you, I have already determined the MIC using the 96 well format. Now I am interested in the protocol that will best suit my microscopy investigations.
I would guess that it will be very difficult to see any substantial changes with either of the techniques as you expect the peptides to damage the membrane, not the cell wall. At the best, you will observe secondary changes as the result of cell death. Sorry for pessimistic feedback. What peptide are you investigating?
Julio's publications above is exactly what I would like to do. So I will give it a try. Thanks.
Did you determine MIC and MBC? They may differ and that may influence the concentrations you end up using. Good luck with the microscopy!
Yes, I have data for both. I'm not interested in the effects using MBC concentrations. For now I will stick to MIC. Thanks
Makes sense, MBC concentrations may actually prevent you from seeing any morphological changes, if the cells get lysed completely you won't see much.
generally, I tested the mode of action of cationic peptides on different Gram-positive and Gram-negative bacteria using TEM. Each bacterial type was grown in nutrient broth supplemented with cationic peptide in a concentration of MIC and incubated at the optimum temperature for 24 h. The cells were collected after centrifugation at 4000 rpm / 10 minutes. The other steps were performed by specialist in microscopic imaging using TEM.
I feel that you incubate the cells at MIC 90 and view at different time interval. However, with M. tuberculosis, the number of serial passages that the culture has undergone will give different results. REMEMBER this and when you present your results do not forget to mention the number of passgaes of the culture and the medium used.
Another way is to use LIVE/DEAD®Bac light kit for bacterial viability in the microscope
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