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Troubleshooting pelleting issues w/ Acinetobacter baylyi

pellet pelleting troubleshooting issues acinetobacter baylyi

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#1 microboi3000

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Posted 05 November 2016 - 12:56 PM

Hello everyone,

 

I am currently working on experiments with Acinetobacter baylyi and I am running into issues with my cells pelleting. I am putting aliquots of liquid culture into Sorvall tubes and spinning them in a Sorvall to collect my cells. In this process, I remove the supernatant after spinning in the Sorvall, then add another aliquot of liquid culture. However, when I go to remove the supernatant, the pellet tends to "peel" off the side of the tube and I lose a significant amount of the pellet when I attempt to remove all the supernatant liquid. I've tried pouring the liquid off as well as pipetting. Both methods result in me losing many cells and a very turbid liquid waste container; not what I want. I've tried increasing the speed from 8000RPM to 12000RPM and increasing the spin time as well. In the end, the pellet does not want to stay intact and ends up sloughing off the sides of the tube. I was thinking maybe my problem is attributed to the high number of cells. On average, I've calculated the concentration of cells to be upwards of 6.0 x 1010 CFUs/mL in a single liquid culture. So, the concentration should be even higher since a good chunk of my pellet is lost in the collection and cleaning process. 

 

Does anyone have any ideas for dealing with this issue? Should I just try to remove the supernatant more gently? Let me know what you guys think!

 

-microboi3000



#2 El Crazy Xabi

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Posted 06 November 2016 - 07:48 PM

Speed in g/rcf? rpm is not reproducible and not comparable between different instruments

What times are you talking about? 5 min, 1 h?

Size of the tubes? The longer the tube axis is, the longer you need to centrifuge as the particles will need to travel a longer distance to the bottom

Do you use a slow deceleration stop? Centrifuges usually have settings to modify how fast they stop when they finish their running time. If your precipitate is not very sticky, you may want to change this setting







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