Hello,
I am working with a Gram- bacteria that has been isolated form soil, tentatively identified as Enterobacter sp.
I am infecting seedlings and after germination and few days of plant growth, I wash a certain number of root systems and try to collect the bacteria from the washings.
During the last experiment, I collected 1000 root systems and tried to concentrate the bacterial suspension by repetitive centrifugation. At the end I obtained 1 ml of bacterial suspension where I was not able to see any pellet, just some fluffy material floating (most likely platen debris) but CFU counting gave me 10^7 CFU/ml.
From my previous experience, 10^7 cells should form a visible pellet.
I feel I am doing something wrong and maybe loosing my cells with centrifugation... What are the reasons why bacteria would not sediment (centrifugation at max speed for 5 min)? How can I improve the process?
Is there certainly an inconsistency among "optical density" and CFU counting. Any idea why could cause this?
I am really lost with all this, so any advice would be really appreciated.
Cristina
Did you measure the OD of your 1 mL bacterial suspension? And what kind of solution you used? If you are centrifuging the 1 ml solution it could be possible that you can not see the pellet. I would centrifuge for longer time for example 20 minutes instead of 5 minutes and mark a note where the pellet should be formed in the tube if you use a fixed angle centrifuge. If you use swing centrifuge you would expect the pellets at the very bottom surface of the tube. Probably in this case the pellet will not be visible as such. However, as you said it could be cultured, so why not enriching them and centrifuge to have pellet of quite a large population.
What means max speed? Can you estimate approximate "g"? I agreed with Dr. Khan:
possible solution - a lengthening of centrifugation time.
20 min 30min max speed es refrigerada tu centrif?
si no ves el pellet, es poca quantidad (1ml) ,
tratalo como si tuvieras un cultot resupendelo en medio de cultivo y cultivalo!!
en el top del tuvo que tienes? has mirado al microscopio??
besos
Max speed has no meaning without defining the centrifuge and the rotor being used to determine the g-force. There are some centrifuges whose maximum speed is only a few hundred RPM and those might require an hour. A micro centrifuge should pellet cells in 2-3 minutes. So the simplest solution is to centrifuge for longer, 20-30 min might be necessary.
Hi,
I agree with previous comments. In my opinion, you should measure the OD of your suspension first. 107 CFU/mL is not that much. Probably it is normal that you are not able to see any pellet. You can try to spin for longer time, but probably you will not see any pellet again. You should increase the sample, or you can try to preculture your solution, but depends on your final goal. What do you want to do with your bacteria? DNA extraction for microbial community analysis, or something else?
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Microorganisms
- Bacteria