I'm studying the biological reduction and oxidation of iron. For this purpose, I have to apply some techniques that isolate iron oxidizing/reducing bacteria, but I'm not sure about the information that I have. Can anyone suggest a few techniques so that I can verify this information?
Dear Francisco, can you tell me what type of samples you are working with? I mean from a particular type of samples you want to isolate iron oxidizers?
However, in general the best method is to inoculate your soil/liquid sample in sterilized 9K medium ((NH4)SO4 3.0 g/L, K2HPO4 0.5 g/L, MgSO4 0.5 g/L, KCl 0.1 g/L, pH 2.0). Add 1-2 % filter sterilized ferrous sulphate after that and incubate for 5-10 days. You will get enriched growth of your iron oxidizing bacteria. However it will not be a pure culture rather a consortium. If you want to get pure culture than you will have to go for either serial dilution method or agarose gel based dual layer solid medium method.
for isolating IOmicrobes....plennty media and work has beeen done.....especiallly as pointed by Patel....the 9K media.
but for IRB......its a medium called balch media.....using VitaminC or Resorcinol as reducing agent......,with mineral rich elixir.....
Hi Francisco,
First of all, you need to figure out pH range of your microorganisms-to-isolate. This will determine media. Fe(II) oxidizing bacteria generally use either oxygen or nitrate as the electron acceptor. The medium preparation depends on pH as at low pH Fe(II) is stable in the presence of oxygen and at near neutral pH - not. The majority of Fe(III)-reducing organisms grow at near neutral pH. But there are some that can grow at acidic pH too. Again, media and methods would be different for those.
Hi Francisco, first of all before you even decide what kind of medium or isolation technique you are going to use , make sure you know the right techniques (and have supplies and perhaps equipment) for growing anaerobic bacteria in general. Especially when trying to enrich and isolate Fe(III)-reducing bacteria, you do not want oxygen in your media (either short or long term) and this can be tricky to achieve. From your question it is not obvious if you are aware of this or not or if you need some references for that?
Hello!
If you are growing microoxic IOB at neutral pH, i suggest using the gradient-tube method (Emerson & Moyer, 1997), which sets up a 'manufactured' redox-interface (oxygen in the headspace, ferrous-iron plug at the bottom).
Anaerobic Fe-oxidizers can use perchlorate and nitrate as an electron donor, there are a lot of published methods out there.
IRB are very easy to grow at neutral-pH using a complexed Fe(III) source, such as Fe-citrate (Kostka, J., Nealson, K.H., 1998).
ron reducing bacteria do in fact tolerate oxygen concentrations, such as Shewenella spp. BUT, if there is molecular oxygen in your media, you would be unlikely to 'isolate' an IRB per se, as you would enrich for bacteria that are using molecular oxygen and ferric iron as a terminal electron acceptor.
Dear Francisco, I wonder if you still need advice in this question... I very much agree with the previous comments, but I would also comment on it
If you intend to isolate Fe-reducing bacteria, you should keep in mind that Fe(III) may be reduced by 2 different ways: non-specifically, by metabolites or via decrease of Eh (i.e. medium becomes more reduced), and specifically, when bacteria use Fe(III) as a terminal electron acceptor (i.e. for respiration).
If you need to isolate strains, which respire with Fe, you need to provide them anoxic conditions because the presence of oxygen will stimulate growth of numerous aerobic bacteria. Also, you should eleminate nitrates, which many bacteria use for respiration, and any media compounds which may be possibly fermented, because this type of metabolism does not need any electron acceptors.
With this approach you will create conditions, which are selective for only Fe-respiring bacteria. You may supply Fe (III) as Fe-citrate complex, which stay in solution or as Fe hydroxide, which is insoluble at pH above cca. 2. Some bacteria (e.g. Shewanella) may respire with Fe(III) precipitate, whereas others probably, so they need its soluble forms..
The composition of medium then depends on your aims - Fe-reducing bacteria may use peptone, molecular H2, acetate etc. as electron donors; try to search papers by D.L. Lovley.
Good luck
Francisco,
a very belated answer - but also, realize that iron oxidizing bacteria can be either autotrophic or heterotrophic. The gradient tube method used by Emerson & Moyer, as indicated above, was designed primarily to isolate autotrophs. Addition of intentional carbon sourcs will enhance isolation of heterotrophic IOB. The latter can also be isolated on various simpler petri dish incubation by adding a suitable Fe(3+) chelator - that will prevent spontaneous Chemical oxidation. Good luck.
Hi, Francisco,
The biological oxidation of iron, usually occurs at acid pH. There are also iron-oxidizing bacteria at neutral pH but more chemically oxidized.
I am work with bacterial iron-oxidation.
these bacteria are difficultuos in isolation for media solid.
A good culture medium for isolating iron-oxidizing bacteria is to Oli Tuovienen: K2HPO4 (0.4 g / L), MgSO4.7H2O (0.4 g / L), (NH4) 2SO4 (0.4 g / L) and FeSO4.7H2O (33.3 g / L). The medium should be at a pH between 1.5 to 3.
You can check these papers:
http://link.springer.com/article/10.1007% 2FBF00425295, http://www.sciencedirect.com/science/article/pii/S0005272812010407
best regards
How much agar we would need in iron oxidizing bacteria isolation medium. Or we need liquid medium? What sample is best for isolation?
In iron-oxidizing bacteria, determine the pH and essential conditions at which the microorganisms are to be isolated. In the case of iron-oxidizing bacteria growing at pH of 5-8, consider the amount of oxygen and iron it needs to thrive.
Reference:
Emerson & Floyd. (2005). Enrichment and Isolation of Iron‐Oxidizing Bacteria at Neutral pH. Retrieved from Article Enrichment and Isolation of Iron‐Oxidizing Bacteria at Neutral pH
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