I collected soil and water from the crater lake. Location of the sample I took at Ijen Mountain, Banyuwangi, East Java, Indonesia. The water is extremely acid that the pH is + 0-1. I want to identification the acidophile bacterial by 16S rRNA sequencing but I want to try to isolate the bacterial first.
My method is try to cultivation the sample into Nutrient Broth and/or Nutrient Agar with pH adjustment to 5 to create acid environment on that medium, but no one of them is growth.
Is there any wrong on my method? Or are there better way to identification the bacterial?
Hi
Did you try DAPI staining and see cells under a fluorescence microscope ? I mean that should be the first step to see if there are bacteria in your samples. For water samples I would try to concentrate the bacteria in the fluid by centrifugation and resuspend in a lower volume. Hope that helps for the start.
Do you have any idea of the chemical composition of the natural sample side? Like what are the possible electron donors in your acidic lake ? Is it sulfur compounds for chemotrophs or is there a heterotrophic electron donor source like glucose?
Why did you choose a higher pH (pH=5) for the isolation when in your natural sample the pH is much lower (pH=0-1) ?
I hope these questions can help to develope some ideas!
Hello Florian,
In my laboratory, there is no DAPI staining tool. So, I can't do the DAPI staining. From the research of other people, the chemical composition of the sample there are iron, sulfur, and aluminium on that water.
I try to use the medium with pH adjustment 5, because I think it will be create same acidic environment.
I tried to make the medium use the water from sample, but the medium is ruined. Is it possible if I make the medium using the sample water but the water is diluted?
Thank you :D
Hello Bhoj,
Thank you for your suggest. Previously, I have tried to make agar medium with pH adjustment under 3. But, the medium can't be polymerized so the agar can't be formed. :)
Thank you :D
Well I would use a liquid medium with similar composition and pH of your natural sample ( like Bhoj suggested pH below 3 and soil extract). By the composition of iron, sulfur and aluminium I would choose a medium targeting chemotrophic bacteria. And you will probably need patience as far as I know these bacteria grow slow.
Hello Johan,
You can search information about Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum and Sulfobacillus spp. They are the most commonly isolated from low pH environments.
As mentioned before, enrichment cultures are an alternative to direct plating. This "ensure" that the cells are actively growing before plating.
For liquid media I recommend this:
MgSO4*7H2O 0.4 g/L
(NH4)2SO4 0.2 g/L
K2HPO4 0.1 g/L
As energy source (autotrophic growth); 50 mM FeSO4*7H2O and/ or 5 mM Na2S4O6*2H2O or sulfur 10 g/L
Use H2SO4 to adjust pH. For Iron media pH must be below 2.0 (1.5-1.7). For sulfur and its reduced compounds you can use pH 3.0 (or higher).
I suggest you to use one media for Iron oxidizers and other for the Sulfur oxidizers.
Some acidophiles are mixotrophs, so add 0.05 % yeast extract.
And some others have heterotrophic growth, you can try 0.5 % YE (I do not have experience with those).
For solid media use Gelrite or Phytagel (commercial names) 0.8-1.0 %.
Best regards
Hello Johan,
I would suggest using a liquid medium and dilution to extinction to isolate your bacterium. This can be done in 96 well plates and growth followed by OD.
As for the choice of medium, Mauricio's suggestion is good. For other media options a good place to look for is the DSMZ collection (https://www.dsmz.de/) you can look for known acidophilic bacteria and in their page there will be a link to the medium they were isolated on.
Good luck
Characterize the ecosystem , where your acidophile exist. Try to synthesize a medium which would be very close to the ecosystem and provide the necessary nutrients (I can not say what are these components as I do not know the characteristics of the ecosystem). Keep the pH low as it exist naturally. Now by extinction dilution method, isolate the acidophile.
Hello johan,
I agree with Mauricio Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum and Sulfobacillus spp are the most common for low pH environments. For start you an use enrichment like Mauricio suggested and then you can use the overlay technique to isolate them. You might find helpful the reference below
Okibe, Naoko, Mariekie Gericke, Kevin B Hallberg, and D Barrie Johnson. 2003. “Enumeration and Characterization of Acidophilic Microorganisms Isolated from a Pilot Plant Stirred-Tank Bioleaching Operation
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