We are happily extracting bacterial DNA using a bead beating protocol. Now we need to start processing swab samples containing fecal etc bacteria. If we put the entire swab into the bead beating, the threads fill the beating tube and I'm afraid it interferes with the beating. So instead we shake the swab in a lysis buffer at 56C for half an hour, followed by a brief incubation at 95C, and then proceed with the supernatant. However, I'm not confident that we can extract all the bacterial cells from swabs this way. Any tried and tested protocols for processing the swabs in this kind of application?
We place our swab heads in sterile PBS, horizontally shake that for 10 mins at full speed, transfer the PBS to a new microcentrifuge tube, centrifuge that for 10 mins at full speed. This way, bacterial cells form a pellet at the bottom of the tube.
Taking care not to disturb the pellet, we transfer the supernatant (PBS) back to the tube containing the swab head and repeat the procedure. After this, the supernatant is discarded and we resuspend the pellet in the first buffer (usually the lysis buffer) of our DNA extraction kit and transfer that to the bead beating tube. We then follow the extraction kit instructions.
This works well in our lab and we've got great results sequencing that on the MiSeq. FYI, we are usually working with plant surface swabs - so it's tiny material that we collect.
Better to use QiAamp stool DNA or Mo-Bio power soil kit, because previously you are extracting with bead beating from a single bacterial system and now you will extract from metagenomes. Therefore I recommend to use above mentioned kits for better and good results for any high throughput or NGS.
We place our swab heads in sterile PBS, horizontally shake that for 10 mins at full speed, transfer the PBS to a new microcentrifuge tube, centrifuge that for 10 mins at full speed. This way, bacterial cells form a pellet at the bottom of the tube.
Taking care not to disturb the pellet, we transfer the supernatant (PBS) back to the tube containing the swab head and repeat the procedure. After this, the supernatant is discarded and we resuspend the pellet in the first buffer (usually the lysis buffer) of our DNA extraction kit and transfer that to the bead beating tube. We then follow the extraction kit instructions.
This works well in our lab and we've got great results sequencing that on the MiSeq. FYI, we are usually working with plant surface swabs - so it's tiny material that we collect.
Thanks Wiebke and Michael!
The serial centrifugation sounds clever! I guess your samples (like ours) are so small that you can't really see the bacterial pellet, so you just need to be careful.
MIchael, I'm not completely sure I understand the point in the filtration. So do you use the filters to concentrate the bacteria or to remove the potential swab threads?
I feel that this is a bit late in the game, but thought I would ask a further question: I am extracting from samples with a high mucus content (fish epidermal mucous) on swabs. Would the serial centrifugation method work well for removing the bacteria in the swabs? Would I use normal PBS for that or do I need to include a bit of detergent (such as tween). I used to do fecal samples and we had to wash them quite a bit. . .
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