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Centrifuging bacteria


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#1 Shanevanbreda

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Posted 17 October 2013 - 02:23 AM

Hi guys,

I am busy planning an experiment to prepare M. tuberculosis for electron microscopy analysis (ultrastructural analysis). Before fixation, I will need to centrifuge my cells to remove culture media and add fixation media. How many g's should I centrifuge my culture at to obtain a nice pellet with minimal damage to my cells?

I was thinking of using between 500 - 600 RCF's.

#2 Missle

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Posted 17 October 2013 - 04:13 AM

You could spin that slowly but you'll need to spin for longer (30min +).  I've spun at 1500 x g for 10 minutes before and gotten good results.  How important is yield (is it okay if you only recover say 70% of the cells?)?  If yield isn't an issue then it shouldn't be a problem.



#3 Shanevanbreda

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Posted 17 October 2013 - 05:07 AM

Yield shouldn't be too much of a problem. Ok cool, I'll give that a try. I was also thinking of centrifuging cells at a low speed that have grown to a McFarland 3 to get a nice pellet.

#4 El Crazy Xabi

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Posted 17 October 2013 - 07:15 PM

SEM or TEM?
If it's for SEM you may have it easier using poly-L-lysine coated slides so you don't even need to centrifuge

 

McFarland 3 is quite a lot of bugs :)

By the way, after recovering cells how do you prepare them? do you embeed in agar/agarose to make a plug ? I may do something about TEM in the future but I have limited knowledge about specific procedures



#5 Shanevanbreda

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Posted 18 October 2013 - 03:29 AM

TEM. For SEM I have found doing the whole fixation and dehydration protocol works great using 0.4um filter membranes and a 1ml syringe.

Yes, after fixation and centrifuging, remove the supernatant and add a very small amount of 1 % w/v agar dissolved in 1 x PBS. Then immediately centrifuge again and remove any excess agar from the bacterial pellet. It's important to only add the agar when it is around 50 degrees Celsius. If you can, heat up the rotor of the centrifuge to 50 as well.

If you need specific protocols for what you need to do, you can send me a message and I will help you out.




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