I need to separate the bacteria from the broth (LB) without rupturing of cell wall. Further, I need to inoculate it in meniral salt media to study growth kinetics.
actually I used to centrifuge it 8000 rpm, 20 C degree, 10 min, but my cells lysis, and precipitate was formed >
what temp, time , and rpm should i use ??
thanks all
Hi Fadi. This is a very good question, and one that I have not seen much discussion on, and no definitive answer.
Here are two articles in which this question has been addressed:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255633/
https://www.researchgate.net/post/What_is_the_maximum_speed_and_duration_to_centrifuge_bacteria_to_obtain_viable_cells
For questions relating to centrifugation it's better to work in relative centrifugal force (RCF,g) as these values are applicable to any centrifuge.
8000 RPM in most small centrifuges, like the eppendorf 5424, is roughly 6000 x g. I would have thought that 10 minutes at this force is unlikely to damage your bacteria. This means that your bacteria are particularly sensitive to damage by centrifugation, OR they were not going to grow in the minimal media. Have you checked that the bacteria do actually grow in it?
My practice, and the one I recommend, is to use the minimum force and time so as to achieve a clear supernatant and a firm enough pellet. I suggest 5000 x g, and check at 1 minute increments if the supernatant is clear and the pellet is firm. Also please ensure the bacteria will grow if inoculated directly into the minimal media, from colonial growth on solidified media.
Best regards
Roger Latham
Hi Fadi,
I think you can go for centrifugation in the range of 1800-2500 for 10 mins.
Hi Fadi. This is a very good question, and one that I have not seen much discussion on, and no definitive answer.
Here are two articles in which this question has been addressed:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255633/
https://www.researchgate.net/post/What_is_the_maximum_speed_and_duration_to_centrifuge_bacteria_to_obtain_viable_cells
For questions relating to centrifugation it's better to work in relative centrifugal force (RCF,g) as these values are applicable to any centrifuge.
8000 RPM in most small centrifuges, like the eppendorf 5424, is roughly 6000 x g. I would have thought that 10 minutes at this force is unlikely to damage your bacteria. This means that your bacteria are particularly sensitive to damage by centrifugation, OR they were not going to grow in the minimal media. Have you checked that the bacteria do actually grow in it?
My practice, and the one I recommend, is to use the minimum force and time so as to achieve a clear supernatant and a firm enough pellet. I suggest 5000 x g, and check at 1 minute increments if the supernatant is clear and the pellet is firm. Also please ensure the bacteria will grow if inoculated directly into the minimal media, from colonial growth on solidified media.
Best regards
Roger Latham
Dear Fadi,
I support Latham Sir's view that the answer for the question may not be definite. Yet I would like to make mention that I have also faced similar problem while separating bacterial cells from LB broth. Finally, after successive trials, I made a conclusion that they could be separated at 3000rpm for 10min at 10 °C without lysis. You can also try this for once.
Good luck
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