Hi all. One of my media recipes calls for "clarified rumen fluid." From what I found, that means through cheesecloth. The problem is that cheesecloth is very porous and I am trying to filter out more than that. I have gotten it as far as 3um but cannot get it past that. My project manager would like it to get to at least 1.0um or smaller pore size. I cannot get it to this point. Has anyone worked with this or something similar and been successful? I have tried a syringe and also tried a vacuum. Nothing is working. Any tips would be greatly appreciated.
Hi Jorji Siegmundt
Did you think about centrifuging your rumen fluid at 4 C at about 5000 rpm for about 30 minutes? .Then the supernatant can be taken and you can filter it again later if you want using a syringe filter
Hi Sandhya,
We centrifuged it at 6000 x G for 10 minutes, then pulled off supernatant.
I spun the supernatant again at 6000 x g for 15 minutes, then pulled off that supernatant and tried to filter that.
At that point, it will go through a 3um filter but I cannot get it through a 1um. I also tried going through a 3um, then spinning that for 6000 x g for 15 minutes, then tried to filter that supernatant but that won't go through a 1um either.
I also need a lot of it; it makes up 300mL of every 1000mL of media. The syringe method is currently what I am using but it also takes a really long time, and I have to change the filter out quite frequently, even at 3um.
Thanks so much for your input! Any other ideas?
Hi Jorji Siegmundt
Try keeping your centrifuged supernatant in the refrigerator for few hours. Then the particles start settling down. You can remove the upper liquid layer and filter through your sequential syringe filters. Or else try increasing the centrifugal force or rpm up to about 10000 or more
centrifuge it at 6000 x G for 10 minutes, then pulled off supernatant.
Thank you all for your suggestions! I ended up having to centrifuge the rumen fluid at a higher speed (17,000 x g) for a longer period of time. If you centrifuge it more slowly or for a shorter period of time, the pellet stays pretty loose. After I centrifuged it at this high speed, I was able to filter the supernatant through a 0.45um and then 0.2um filter. It was actually still difficult, but much better than it had been.
Jorji
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