Tips on media preparation and how to dissolve starch? - (May/18/2014 )

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)?

 

 

 

Thank you very much!

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

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

tretol on Sun May 18 11:10:50 2014 said:

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)?

 

 

 

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?
 

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

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.

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.

why do you think so?

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.

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.