I have determined MIC's for a certain antimicrobial peptide against M. tuberculosis. My starting CFU/ml is between 1 - 5 x 10^5 cells, using H37Ra. I would like to do various microscopy studies (TEM) on the cells incubated in the presence of the antimicrobial peptide at various time points. Since I have such a low concentration of cells, I struggle to obtain a pellet when centrifuging at 10000 g's for 1 min or 5000 g's for 2 min. I was wondering if anyone could give me any suggestions to obtain a decent pellet to work with? Using centrifuge speeds that won't introduce artifacts or damage my cells.
Thanks
I would try to centrifuge at lower force for much longer time. At lower RCF, cells are sedimenting less rapidly, but if you extend time, they finally should reach the bottom. Also, if you pellet smaller volume, the track they need to go to reach the bottom of tube is shorter and they will need less time and less power. I am not sure what may be the lowest RCF which would work to pelet bacteria, but you may try to start even with 500 g if you are worried about damage. If you can grow your bacteria without the peptide to higher concentrations, you may try a serial dilution and test which conditions will still give you a pellet at very low concentration of cells after e.g. 20-30 min centrifugation at 500 g.
Thanks, I will try experiment with the different conditions. Unfortunately I don't want to grow the bacteria to higher concentrations in presence of the peptide. I believe this will give me 'false images' since the bacteria that are growing have not been affected by the peptide. Ideally I would like to view them at 0, 1, 6, 12 and 24 hours.
Do you think I can take 1 x 10^5 CFU/ml, centrifuge it at very high speeds and observe if a pellet forms? If no pellet forms, then is this an indication that one won't form at very low speeds spun for an extended period?
Just curious, but can you just use a 0.22um filter and use the bacteria trapped on the surface of your filter for downstream applications ?
If you must use centrifugation you can try PEG-NaCl to help with the precipitation process as it works well with Phages as well.
I have already tried with filters, it forms an unwanted artifact, they look flatten due to the pressure. I will think of PEG NaCl or I will just increase the amount of bacteria I am using.
I had trouble seeing a consistent pellet when I centrifuged E. coli grown in Davis minimal broth (viability as low as 5 x 10^6 CFU/ml). In a regular tabletop centrifuge (14,000rpm ~ 22,000xg) the pellets were very faint, sometimes impossible to even grasp. I solved this issue by using a big Beckman Coulter one that reached 20,000rpm ~ 40,000xg during 30 min. I had no problem extracting their DNA, but if you want to minimize damage you would probably have to test different times and conditions.
Hi Shane,
I know this thread is old but, did you find a good combination to pellet low concentrations of TB cells? I am looking for the same. I would not like cells to break, but I do not care about their viability though.
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