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Tips on media preparation and how to dissolve starch?

media medium substrates

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#1 tretol

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Posted 18 May 2014 - 03:10 AM

Hello,

 

I tried to prepare the following medium. It is a modified LB agar. I would like to test if my bacteria are facultatively anaerobic. Hence, I have to add sugars since LB does not contain them. Since I am not sure which sugars my bacteria might metabolize I read some papers and decided for a sugar mix.

 

I will give the recipe of my media. Please tell me, if any information is incorrect or incomplete. For example: How can I dissolve the soluble starch I used and what is dipotassium phosphate and magnesium sulfate good for (magnesium sulfate maybe as cofactor for enzymes and the formation of cystein and methionin)?

 

 

nlljgtjy.jpg

 

Thank you very much!



#2 El Crazy Xabi

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Posted 18 May 2014 - 05:41 PM

Pure normal starch is barely soluble even heating. Soluble starch is a modified starch, partially acid-hydrolysed, that is common for starch hydrolysis tests of free iodine detection. Do you use it? 9 g in 180mL should be OK. In the starch-agar (agar 12g/L, soluble starch 10g/L, beef ext. 3 g/L) the aspect of the plates is kind of cloudy if I remember well. It may not give you a clear solution as if it were glucose, but I did it long time ago so better wait for someone with more recent experience on it.

 

The dipotassium phosphate adds extra P and buffer capacity. The magnesium sulfate provides extra Mg, many enzymes need it, and may be used as sulfate source by sulfate reducing bacteria in anaerobic conditions



#3 Phil Geis

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Posted 19 May 2014 - 02:31 AM

Sucrose is fructose and glucose and starch a polymer of glucose.  The complexity is not justified - use glucose. 



#4 lucilius

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Posted 19 May 2014 - 12:24 PM

Hello,

 

I tried to prepare the following medium. It is a modified LB agar. I would like to test if my bacteria are facultatively anaerobic. Hence, I have to add sugars since LB does not contain them. Since I am not sure which sugars my bacteria might metabolize I read some papers and decided for a sugar mix.

 

I will give the recipe of my media. Please tell me, if any information is incorrect or incomplete. For example: How can I dissolve the soluble starch I used and what is dipotassium phosphate and magnesium sulfate good for (magnesium sulfate maybe as cofactor for enzymes and the formation of cystein and methionin)?

 

 

nlljgtjy.jpg

 

Thank you very much!

 

Can anyone explain this:

 

I would like to test if my bacteria are facultatively anaerobic. Hence, I have to add sugars since LB does not contain them.

 

I do not understand your logic. Why would you need the sugars because they are facultative anaerobic bacteria? Would anaerobic bacteria not grow on this medium without the added sugars?

Aerob ones do,  so why would anearobic ones not grow?

Can they not use the amino acids as carbon source?
 



#5 Phil Geis

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Posted 19 May 2014 - 02:30 PM

Good point - I'd assumed both aerobic and anaerobic conditions of incubation but as you say, don;t see this specified.



#6 tretol

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Posted 21 May 2014 - 10:32 PM

Hello,

 

you are right, I can indeed grow the bacteria aerobically. The aim is to develop a media suitable for 1)  testing the oxygen requirements of the bacteria I already isolated and 2) a media rich eough to culture future isolates.



#7 pito

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Posted 23 May 2014 - 09:45 AM

2 very different goals in the end...

 

A lot depends on the bacteria... LB is not really suited for what you are trying to do to be honest.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#8 tretol

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Posted 25 May 2014 - 02:47 AM

why do you think so?



#9 pito

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Posted 25 May 2014 - 02:59 AM

If you add the sugars its ok (but normally LB is made without sugars). If you add sugars, just add glucose (no need to try other, harder to metabolise the sugars) to test.

 

I dont know what kind of bacteria you want to test, but there is a reason why there are so many specific media .....

 

Testing the oxygen requirments, I dont see how you are going to do this using LB.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#10 tretol

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Posted 25 May 2014 - 04:02 AM

okay, then just glucose. I will incubate the inoculated plates in the absence of oxygen. This is the reason why I need sugars. If I do not provide a sugar source, how should the bacteria survive? They will not have components for fermentation. I am isolating bacteria from envorinment on different types of media (LB, Muller Hinton, Columbia Sheep blood) and for some bacteria I had good growth on my modiefied LB and only tiny colonies (or no growth) on the other media.

 

Additionally, I will test the oxygen requirements using thiglycolate broth. Thanks, I am always grateful for criticism.



#11 pito

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Posted 25 May 2014 - 04:32 AM

okay, then just glucose. I will incubate the inoculated plates in the absence of oxygen. This is the reason why I need sugars. If I do not provide a sugar source, how should the bacteria survive? They will not have components for fermentation. I am isolating bacteria from envorinment on different types of media (LB, Muller Hinton, Columbia Sheep blood) and for some bacteria I had good growth on my modiefied LB and only tiny colonies (or no growth) on the other media.

 

Additionally, I will test the oxygen requirements using thiglycolate broth. Thanks, I am always grateful for criticism.

 

I am not sure (as someone else also noted) why you are stating that you need sugars because they are (you think) anaerobic. Even anaerobic bacteria can use amino acids as carbon source.

 

LB is a good way to start, but not sure its the best..

I also wonder: how do you know that what you grow is the bug you are looking for?

 

Is your bacteria already isolated or ? I find it hard to understand what your goal is at the moment.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#12 tretol

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Posted 25 May 2014 - 10:15 AM

Hello,

 

okay, let me explain what I am doing:

 

I isolated bacteria from environmental samples and I am trying to characterize them. I do not think that the bacteria are anaerobic. I would like to test if they can survive under anerobic conditions.

 

I will get more environmental samples from different localities soon and I would like to isolate bacteria from these samples as well (all kinds of bacteria).

The samples are lipophilic and that's why I added Tween. But I am trying to further enrich LB. I also ordered meat extract but I think that yeast extract and meat extract is nearly the same (provide amino acids).

As mentioned before I also use other media.

 

The bacteria I am planning to isolate have never been isolated before. It might be that the samples I will receive are bacteria-free.

Hope this was clear biggrin.png



#13 pito

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Posted 25 May 2014 - 10:56 AM

I see.

 

Its possible there are anaerobic bacteria in the samples too.. (I dont know where they come from, is it from the soil? water? ..)

 

You can use different media but its hard to culture most bacteria so dont expect to find a lot , especially new ones...

You also might try other ways to find novel bacteria.

 

Culturing is often a huge problem for new/unknown bacteria.
 

You also have to be very carefull not to end up with mixed cultures... so make sure you are indeed working with 1 specie when you characterise them.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#14 El Crazy Xabi

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Posted 25 May 2014 - 05:45 PM

I also ordered meat extract but I think that yeast extract and meat

Roughly, yes. They are complex amino acid-rich extracts but their analytical composition is different. Check the Difco culture media manual (free on its web). They have the analysis of the hydrolysates and extracts nearly the end of the manual.


 







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