I am working with the microbial metabolites. I need to separate the bacteria (Enterobacter) from the nutrient broth (LB) without rupturing its cell wall. Further, I need to lyse the bacteria in suitable buffer or solvent to recover the intracellular product.
Please tell me an optimal condition and reagents to carry out this process.
Actually, I want to analyze different proteins-peptides, carbohydrates and fatty acids produced by microbes (both intracellular and extracellular products).
***Using REMI Centrifuge !!
Dear Chandan,
You can centrifuge at either 6000 rpm 10 mins at RT or 20 degree celcius or 8000 rpm 5 mins at RT or 20 degree celcius. Conditions and reagents will vary according to the intracelluar product (DNA, RNA or total proteins) you want to recover.
Santosh ! Thanks!
One of my colleagues has suggested to proceed with 10,000 RPM centrifugation at 4 degree C for 5-10 minutes (depending on cell type).
Further for lysing the cell PBS (Phosphate Buffered Saline) is to be used. I am currently following this protocol.
Any comments/ suggestions ?
From my experience, a mere centrifugation at 5000 g, 10 min, 20°C is sufficient to separate cells from broth. You should obtain two well separate phases (do not shake !) If not, increase the speed rather the time. The matter is to not disrupt signicant number of cells which therefore would release their intracellular metabolites. If you want to freeze metabolic reactions (for a kinetic study), put your fractions in melting ice and centrifuge at 4°C and seprate supernatant form pellet rapidly.
good luck !
Refrigeration depends on whether the product you are recovering is readily metabolized. Cooling the cells will prevent changes. PBS is what one puts cells in when you don't want them harmed. Do you mean that you are going to sonicate them/french press them/add lysozyme/etc. in a PBS solution? PBS alone will keep them nicely intact.
We have used centrifugation at 6000 g, 15 min, 10-15°C to separate cells from broth,
anyway you should set the conditions for the type of cells, it is best to try two or three levels above and below of this.
I centrifuge at 5000 rpm, 5 min for Eppendorf tubes, 15-20 min for Falcon tubes (depending on culture volume), temp. 20C
Fatty acid is intact means intracellular you cant get extracellular except in bio-transformation studies. For fatty acid extraction you can do it at 6000 g for 10 minutes is enough. after that was the pellet with PBS and add chloroform and methanol in the ratio of 2:1 and PSB buffer. Refer Folch method for lipid extraction in detail.
Thanks everyone for your valuable suggestions.
yes ! David, I am going to sonicate the cells in PBS to obtain the intracellular products.
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