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Author Topic: need help with a growth medium, bacteria wont grow on it?  (Read 6492 times)

Offline chemi

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Hello,

Well I want to grow a certain bacteria in a liquid medium. therefore I found a paper in which they cultured this bacteria and I followed the same recipe. what they do is add all the salts, vitamins etc together in distilled water except for the aminoacid. Then they autoclave it and afterwords add the amino acid (filter sterilized) before culturing.
I followed all the steps but each time the bacteria won't grow. I have been reading something about that vitamins can be heat sensitive so could it be that I should have to add the vitamins and aminoacids filter sterilized and only autoclave mostly the salts?


 

Offline Variola

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need help with a growth medium, bacteria wont grow on it?
« Reply #1 on: 29/07/2010 19:43:45 »
Hello Chemi

What bacterium are you trying to grow and on what?
I would go for autoclaving your salts, then adding your amino acids and vitamins after. There are similar methods I have used before for making up media and most of the salts and MS, agar etc are autoclaved first then the 'goodies' added after.
Also check your reagents too  :)
 

Offline chemi

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need help with a growth medium, bacteria wont grow on it?
« Reply #2 on: 30/07/2010 09:47:03 »
First of all thanks for your reply.

Well I am trying to grow Helio. mobilis. I got a medium in which it grows but the problem is it contains alot of yeast. The reason why I don't want that is because I want to label the Nitrogens with 15N and since yeast already has a nitrogen source I would prefer not to have any yeast in my medium in order to get a high amount labeled.
 

Offline Variola

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need help with a growth medium, bacteria wont grow on it?
« Reply #3 on: 30/07/2010 10:46:43 »
First of all thanks for your reply.

You're welcome, not sure how much help I will be!  :)

Quote
Well I am trying to grow Helio. mobilis. I got a medium in which it grows but the problem is it contains alot of yeast. The reason why I don't want that is because I want to label the Nitrogens with 15N and since yeast already has a nitrogen source I would prefer not to have any yeast in my medium in order to get a high amount labeled.

Ahhh I see your dilemma now. Out of interest what was the recipe they used in the paper? I would go with your thinking, autoclave your stable essentials first, then add vitamins and aminos after. Vitamins are not all that stable and do suffer when exposed to heat, especially at autoclave temps!

Couple of other forums I use that you might find helpful:

http://www.scienceforums.net/forum/21-microbiology-and-immunology/

http://molecularbiology.forums.biotechniques.com/index.php

They can both be quite helpful with a query or for looking up info.

 

Offline chemi

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need help with a growth medium, bacteria wont grow on it?
« Reply #4 on: 30/07/2010 11:33:24 »
The recipe was like this :
(Per 1 L) 2.2 g sodium pyruvate; 1 g (NH4)2SO4; 150 mg L-methionine (filter-sterilized); 0.9 g K2HPO4; 0.6 g KH2PO4;200 mg MgSO4 7 H20; 75 mg CaC12 - 2 H20; 5mg disodium EDTA; 15 ug biotin; 1 ml chelated iron solution (to prepare: dissolve 500 mg FeCl2 4 H20 and 1 g disodium EDTA in 1 L water, and add 1.5 ml concentrated HC1); 1 ml trace element solution [4]; pH adjusted to 6.8.

And according to the paper it should grow quickly, but I havent been able to grow it yet.

 

Offline Variola

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need help with a growth medium, bacteria wont grow on it?
« Reply #5 on: 30/07/2010 11:53:54 »
Has it not grow at all? Or just poor growth? That's the trouble with papers, they never make things clear.


Biotin is fairly stable to heat, but autoclaving is rather more fierce!


You could try adding your vits, amino acids, and also your trace element after autoclaving and immediately before use??
I am not overly familiar with H.Mobilis though, I spend most of my days playing with S.Coelicolor so it may not make much difference to the growth, but it is worth a try.
You could always drop the paper's author an email and ask too.
 

Offline chemi

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need help with a growth medium, bacteria wont grow on it?
« Reply #6 on: 02/08/2010 10:11:27 »
well it's like this. H.mobilis has 2 forms a green one and a brownish one. Helio normally grow in a surrounding with little oxygen and are therefore brown. However under the presence of oxygen and light the bacteria will react with oxygen (which will cause a change at the chlorophylls) and thus convert into green which will stop the growth. The problem is I need to study the brown form  ;D.
Inorder to prevent this conversion we add 0.5g/L sodium ascorbate which should get rid of the oxygen.
However each time I have used this medium so far it was either not growing at all or everything was in green.
Untill now I have tried alot of variations but so far none seem to really work.

But so you are suggesting to add all these filter sterilized:
sodium pyruvate; L-methionine ; biotin; trace element solution

or is it ok to autoclave sodium pyruvate ?

and thanks again for your replies  :)
« Last Edit: 02/08/2010 10:16:25 by chemi »
 

Offline Variola

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need help with a growth medium, bacteria wont grow on it?
« Reply #7 on: 02/08/2010 19:41:58 »
Ah I see your quandry.

I think sodium pyruvate is heat stable, I can't anywhere that says it isn't or is not to be autoclaved  but I stress I am not a chemist!

Yeah try mixing up yours salts etc and autoclave, then add biotin, methionine and trace element solution after. It may not make any difference but that is the protocol I follow for making up certain types of media.

And you are welcome, as I said I don't know how much help I will be!!
 

Offline wolfekeeper

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need help with a growth medium, bacteria wont grow on it?
« Reply #8 on: 03/08/2010 04:40:04 »
Maybe there's a preservative in something.
 

Offline Variola

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need help with a growth medium, bacteria wont grow on it?
« Reply #9 on: 03/08/2010 21:47:12 »
Maybe there's a preservative in something.

Good idea but manufacturers of microbial products ensure their media is uncontaminated and free from preservatives.
 

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need help with a growth medium, bacteria wont grow on it?
« Reply #9 on: 03/08/2010 21:47:12 »

 

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