To enumerate the total amount of bacteria per blood volume, I would like to selectively remove host cells from mouse blood while retaining bacteria.
Size filtration is unlikely to work as filters will immediately get clogged by erythrocytes.
Does anyone know whether either erythrocyte lysis by Ammonium-Chloride-Potassium buffer or RBC removal by Ficoll gradient would retain intact bacteria? I guess after RBC removal, leukocytes could then be removed by size filtration.
Hello, Jakob.
First withdraw blood into anticoagulant-containing needle and Eppen-tube. To separate bacteria without losing them in the clot, the first step is critical. Dilute the blood into known volume of buffer after taking a known portion of blood into a 13 ml Falcon tube. add more known volume of buffer to reach over 8 ml gradated vol (the higher vol, the better separation). Gentle mix with 3 upside/down without forming foam. Spin them at around 400-500rpm for 6 min in cell culture lab. Cells will precipitate at the bottom and on wall of the tube. At this speed, bacteria are still in the suspension. Take the tube out without disturbing the weakly sitting on the side wall and bottom. Simply, but very gently tilt & pour the tube at the opposite opening side of the centrifugal force executed side wall into a new 13 ml tube. Discard remaining solution around 1/3 volume with cell pellet to avoid disturbance of cells precipitated. Transfer the saved cell-free bacteria suspension into 1.5 Eppen tubes and spin down for bacteria. This will save all bacteria live for further use.
Best wishes.
Dear Sang Ho Lee,
thank you very much for this quick and helpful answer. For the centrifugation, into how many "g" would 400-500 RPM at your centrifuge translate?
Thanks a lot.
Best,
Jakob
Hi, Jacob.
Sorry for the rpm. Try 200, and 300 RCF for 6 -10 min. Make a mammalian cell culture dish to let them grow fully (70-80% confluency) in 60mm dish. Typsinize, wash and check them whether you can see cells precipitated at the bottom and walls of the tube from low to high speed in your centrifuge. You will have optimum condition.
Regards.
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