I hope someone could help with this question. I am trying prepare bacterial cells for TEM analysis after treatment with a cationic antimicrobial peptide. I determine my MIC in 96 well microtiter plates, with a final volume of 200 ul (starting inoculum is ~ 2.5 x 10^5 CFU/ml).
When I remove the wells contents at the determined MIC, I try to centrifuge it for a few minutes (~2 - 5) in a microfuge, but I can't get a pellet to form.
Can someone suggest a method of obtaining a pellet for these low cell titers at my observed MIC?
Should I pool a volume of ~ 1 ml, then centrifuge?
What are acceptable centrifuge speeds and times for bacteria (I am working with tuberculosis), that won't introduce any artifacts or damage to the cells?
Hi Shane,
I suppose to process the treatment of bacterial cells suspension with the agent directly into polyethylene conical embedding capsules, then centrifuge, remove the liquid by thin pipette, replace them gently with the fixative without raising the sample (or centrifuge again), etc. up to embedding. There is also the possibility to give very small droplet of molten agarose (cca 1% solution in PBS, made in the 100°C water bath, then cool down to 38-40°C) over the treated and centrifuged specimen after liquid removing. After solidifying of the agarose process them for TEM changing safely the solutions over the specimen directly in the capsule.
Regarding the centrifugation, these small objects are less sensitive to centrifugation and I suppose to use for example 3000-6000 rpm for 20 minutes.
Best regards
Pavel Travnik
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