Centrifuging low amount of bacteria

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Yeditepe
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Centrifuging low amount of bacteria

Post by Yeditepe » Wed Dec 07, 2011 9:21 am

Hi collegues,

I need to change liquid media with fresh and modified one in the sixth hour of inoculating 10^6 cfu/ml bacteria in 50 ml falcon. I'm planning to centrifuge and change the media with fresh modified one. However, as the number of bacteria is low it is not possible to have visible pellet. Therefore, I'm not sure that I'll collect and keep all microorganism while changing media. Is there any suggestion or the way I planned is useful or not?

Thank you all

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JackBean
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Post by JackBean » Wed Dec 07, 2011 1:13 pm

keep it centrifuging for longer time, that should cause more cells to pellet.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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canalon
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Post by canalon » Thu Dec 08, 2011 1:32 am

As fast as possible for 5 minutes more than you would normally have. Do not brake at full force and change medium quickly. Or keep the last ml at the bottom of the tube, use it to rinse the sides of your tube, quick spin and transfer to a microtube, centrifuge at 14000rpm and pipette carefully. Use that to inoculate the new medium.
Patrick

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any proof. (Ashley Montague)

JorgeLobo
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Post by JorgeLobo » Sat Dec 10, 2011 2:13 pm

Suggest you approach this as a scientist. Rather than spinning "as fast as possible", test your apporach so that you can validate. Over specific Xg and time, determine the counts in supernatant and the pellet resuspended to a specific volume.

Cat
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Post by Cat » Sat Dec 10, 2011 5:48 pm

I would use 3000 g at 4C for 15-30 min (similar to what is usually used when making competent cells). Draw a line on the side of the tube and place it in the centrifuge so your pellet would be expected to be on the line. Carefully transfer old media to new tube. Vortex that media (to resuspend pellet if you transferred it accidentally) and check it for bacteria. If nothing is there, than you pellet is in the original tube and you can resuspend that in new media...

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canalon
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Re:

Post by canalon » Mon Dec 12, 2011 4:08 am

JorgeLobo wrote:Suggest you approach this as a scientist. Rather than spinning "as fast as possible", test your apporach so that you can validate. Over specific Xg and time, determine the counts in supernatant and the pellet resuspended to a specific volume.


Sure, if you have to do that on a regular basis. But:
- bacteria will take whatever speed you throw at them, so no problems here.
- Speed will vary in function of equipment available (tubes and centrifuge), so I cannot really comment here
- The point is to harvest as much as possible, with my method, you might lose some time, but probably not many more bacteria than any other validated method, and time is not usually a massive problem (I am not talking days here)

So yes, sure, it would be better, but does that matter? I am being pragmatic here.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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