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Filter Sterilization of Hot Gellan Gum Media


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#1 Pippy

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Posted 14 May 2011 - 05:33 PM

I'm looking for a quick way to sterilize gellan gum based solid media containing thermolabile phyto-hormones and organics. I'm especially concerned because the time required to properly autoclave the amount of media we use (12x 1L at a time 50 min) seems to break down a lot of the organics especially the B5 vitamins. I found an article presenting data that about 9-25% of sucrose breaks down under standard autoclaving times. My suspension cultures of various species don't grow as well in autoclaved media when parallel tested against filter sterilized liquid media but thats acceptable for now because our cost/kg of biomass would go through the roof if we filter sterilized all of our media for scale-up and we haven't scaled up enough to where autoclaving is no longer an option.

The reason we autoclave though is because of time and cost but I've decided to use filter sterilized media during growth optimization in order to minimize confounding variables.

I know I can autoclave 1L bottles with just micro and macro B5 salts with gellan gum and aseptically add 4x sucrose, 1000x vitamins and phyto-hormons but that would be a pain because of the amount of sucrose needed and contamination is a real issue at the volumes that we need.

Last week I tried mixing a 2x gellan gum water solution with 2x filter sterilized B5 media but that didn't work. I did this by taking a 1L bottle with 500mL 2x gellan gum, autoclaved it, added a 0.22u filter cap and dumped the 2x B5 media on top, filtering into the 1L bottle while stirring. The first time I did this the thing solidified on me. The second time I heated the 2x B5 to 50 *C and it still solidified on me. The 3rd time I turned the heat on the stir plate and heated the gellan gum just below boiling but as soon as the filtrate touched the gellan gum solution the whole bottle bubbled up and into the vacuum line (and hence no longer sterile).

Can I just take the B5/gellan gum solution heat it until the gellan gum melts (~80 *C) and filter the whole thing? Or would this just clog/damage the PES membrane?

#2 bob1

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Posted 15 May 2011 - 03:33 PM

How about autoclaving the medium, but filter sterilizing the vitamins etc. to get around the whole problem. If they are thermolabile this would be the best option.

You could also autoclave for a shorter period, most sterilization of liquids is done for 30 min at 121 psi.

#3 bob1

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Posted 15 May 2011 - 03:35 PM

How about autoclaving the medium, but filter sterilizing the vitamins etc. to get around the whole problem. If they are thermolabile this would be the best option.

You could also autoclave for a shorter period, most sterilization of liquids is done for 30 min at 121 psi.

#4 phage434

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Posted 15 May 2011 - 04:08 PM

You should autoclave the components that can survive 121C, then cool the result to 55C (or whatever temperature allows your gum to remain liquid). Heat your other components to 55, then filter them into the warm gel. Your problem with foaming was due to superheated agar, which will be solved by cooling it.




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