In order to enrich soil ureolytic bacteria, I poured 1g soil into a solution containing Yeast Extract, urea and some other salts. Then I added 1mL (10^7 cell) of the prepared bacteria solution into another bacteria solution (containing Nutrient Broth) to investigate the interaction between bacteria. But I found that YE is precipitated in the new mixture. Although the amount of precipitation is very low but still undesirable, I want to remove it. I know that I can use other sources like soybean meal, corn steep liquor, etc. instead of yeast extract but we do not have these chemicals in our hand now for the urgent case. I was thinking about two of the following ways and would like to know your suggestion.
1st : Centrifuging ureolytic bacteria solution and washing it with the same solution as the solution containing NB and then using it in mixture solution.
or
2st: Preparation of new culture of ureolytic bacteria by pouring 1mL of the old bacteria solution containing YE into a fresh solution containing NB (or NB+urea; which one do you suggest?) and letting them incubate to obtain the same bacteria concentration (10^7 cell/mL). Then I will use this new bacteria solution for the interaction investigation. Can I keep the same number and type of bacteria in the new solution as previous media?
It is better to centrifuge the ureolytic bacteria solution at low "g" to avoid cell breakage and wash it with PBS and resuspend the pellet with PBS. Inoculate the fresh solution with this bacterial suspension.
Regarding medium for ureolytic bacteria, you can reduce the nitrogen source and add urea which enrich ureolytic bacteria in particular. So formulate your medium with less amount of nitrogen and carbon and add urea as a supplement to the medium which acts as Nitrogen and Carbon source. This acts as selective medium for ureolytic bacteria.
All the best!
Dear Kumaresan
Thanks for your answer.
what is your suggestion for g?
Can I use Normal Saline or Tris Buffer Saline (TBS) or the NB culture solution instead of PBS?
Dear Hamed
1. You can run at 3000 to 5000 g for 5 minutes. If you see more cells in supernatant even after centrifugation, extend centrifugation time to 10 minutes.
2. Instead of PBS you can use physiological saline also. NB will add nutrients which will allow the growth of non-ureolytic bacteria.
3. Formulate your medium with less Nitrogen and Carbon and add urea to supplement your minimal medium.
Hello Hamed,
Firstly if you are talking about keeping the bacterial number and type constant it is better to follow it by serial dilution. You can use selective enrichment medium and follow serial dilutions for multiple tubes where in you can start with 10 times and then 100 times dilution. This will allow screening and isolation of ureolytic bacteria from the mixture of bacterial cultures in soil. While reaching the latter dilutions microscopic examination can help to track single type of bacterial cells.
Once this is done you can proceed for centrifugation and suspending the cells in saline and adding to fresh NB ( Selective media at times do not support injured cells to grow). To keep the cell number constant , cells can be suspended in saline after growing for specified time intervals as 6 hours or 12 hours or 18 hours as required in NB.
Just a comment. Bacteria are present in liquid as a suspension NOT a solution.
My first question... are you sure if the precipitate is indeed yeast extract?
It is possible to get approximate numbers only after certain modifications in the media as suggested by other scientists.
Neeru narula
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