Isolating bacteria from grass roots - (May/28/2009 )
Hi,
I'm trying to isolate a group of bacteria from roots of two type of grasses and I'm looking for the best way to treat the roots. What I've tried before was removing the roots from the plants and grinding 1 gram of root material with a mortar and pestle, performing sets of serial dilution and then plating on selective media.
When I performed this experiment, I did not recieve any growth on any of my plates. I have a few hypothesis of why I didn't get any isolation, but one concern that I'm unsure of is if using a mortar and pestle is the best way to go about grinding the roots. Does anyone have any ideas or better ways for isolating bacteria from roots.
Are you trying to isolate bacteria from on the roots or in the roots? If it's on the roots, try shaking them gently for about 20 minutes in pBS buffer. Then you can either dilute from that or pull it thru a .45 membrane filter to concentrate and put that directly on the plate.
microgirl on May 28 2009, 08:42 PM said:
and if you try inside the roots....did you surface sterilise before grinding? If you did so, maybe the time was too long for the small roots and you killed the bacteria as well.....
But as microgirl said....maybe more details on you experiment will help....
what medium are you using?
microgirl on May 28 2009, 02:42 PM said:
microgirl-I'm acutally wanting to look at both types, bacteria on the roots and in the roots. Do you think vortexing in PBS may work as well?
I'd be worried that vortexing with the roots and everything in there would be stressful on the bacteria so when you try to isolate them they won't grow well, but you could try. I think it's better to gently shake/rock them so that they eventually detach. Also, some environmental bacteria can take a long time to come up on plates if they ever will. You need to put an antifungal into the plates so that you can let them sit for a long time (like a couple weeks).
microgirl-I'm acutally wanting to look at both types, bacteria on the roots and in the roots. Do you think vortexing in PBS may work as well?