14 replies to this topic

[Tom]

Hi folks,
Could anyone tell me what speed (rpm/g) i need to obtain on a centrifuge to separate bacterial cells from molecules. I`m trying to obtain a cell free supernatant of a bacterial culture without the use of filters. I know there is a standard value of g which will separate larger cells from smaller sub cellular fractions. Could anyone help me? Thanks

[AquaPlasmid]

Tom, on Feb 3 2009, 09:12 AM, said:

Hi folks,
Could anyone tell me what speed (rpm/g) i need to obtain on a centrifuge to separate bacterial cells from molecules. I`m trying to obtain a cell free supernatant of a bacterial culture without the use of filters. I know there is a standard value of g which will separate larger cells from smaller sub cellular fractions. Could anyone help me? Thanks

What "molecules" are in your mind, salts or cell-free DNA/RNA? Spin at ~12000xg for 2 min will pellet intact bacterial cells but not virus (phages) and bacterial debris. If you add PEG6000-8000 to 3% final concentration, it will pull down phages and cell debris at ~12000xg for 5 min and leave free DNA/RNA in the supernatant.

[scolix]

A lot depends on what you need after the centrifugation.

If you want only the cells, spin at 10,000x g, you should get all bacteria pelleted. There may be a few subcellular organelles which can come down with higher g's. IF you use a ultracentrifuge and spin at higher g's, you could get nearly all subcellular part down and only the molecules remaining int he supernatant.

[Tom]

Thanks AquaPlasmid and scolix for your replies. I`m not sure of what molecules I`d want remaining in the supernatant. I`m trying to isolate the supernatant with any molecules the bacteria would have secreted during its growth in the medium. Thus I want to separate just the bacterial cells from the supernatant. The bacteria are also known to secrete cellulose into the medium and thats something I`d like to retain. Looking forward to ur replies. Thanks

[GeorgeWolff]

Bacterial cellulose is not soluble - usually associated with the cell.  Simple centrifugation prob won't be useful.

In any case - you ought to pl;an out your experiment with an actual objective.

[Tom]

GeorgeWolff, on Feb 6 2009, 12:52 PM, said:

Bacterial cellulose is not soluble - usually associated with the cell.  Simple centrifugation prob won't be useful.

An overnight culture of my bacterial strain gets a bit viscous and hard to filter through a 0.2um filter. Would that be because of secretion into the medium or could the cell associated cellulose also cause such an effect?

GeorgeWolff, on Feb 6 2009, 12:52 PM, said:

In any case - you ought to pl;an out your experiment with an actual objective.

I do have an objective and that is to separate the bacterial cell from its secreted substances (supernatant)  before I can use the supernatant for my assay. I`ve seen an activity that has been narrowed down whatever it is in its supernatant. Filtering doesnt seem to work as the results are ambiguous, probably because the filter material adsorbs the active molecules. I dont know what exactly I`m looking for and I need to try a few more assays with just the supernatant to narrow it down and hence my query. If I knew exactly what I was looking for . my project would be complete.

[gebirgsziege]

maybe you should try to spin the cells down to get rid of most of the cells and then filter it. If you think your active substance is absorbed by the filter, try filters made of different materials.....

And are you sure your active substance is in the broth or can it be associated to the material secreted by the bacteria (e.g. like the mucilage surrunding the bacteria)....then you have to have a plan to seperate the bacteria from this substances befor you can get rid of them.....

[Tom]

gebirgsziege, on Feb 6 2009, 02:43 PM, said:

maybe you should try to spin the cells down to get rid of most of the cells and then filter it. If you think your active substance is absorbed by the filter, try filters made of different materials.....

And are you sure your active substance is in the broth or can it be associated to the material secreted by the bacteria (e.g. like the mucilage surrunding the bacteria)....then you have to have a plan to seperate the bacteria from this substances befor you can get rid of them.....

Heres what I get: I find that when cocultured bacteria A inhibits the metabolism of bacteria B as seen through my assay. If I separate the two by a 0.2 semipermeable membrane which prevents the bacteria from interacting, but allows the medium and therefore their secreted substances to pass, there is very less inhibition. I`ve a larger pore size (0.45) of a different material and found even less inhibition. The logical thing to do would be to separate the cells from the supernatant through a non filter based method. I`m not sure if the substance is in the broth or cell bound, but doing the assay with just the broth through such a separation technique should give me a clear idea.

[gebirgsziege]

Maybe you should look for some kind of centrifugation-technique with a selective barrier....

but have you ever thought about this: bac A is producing the substance that inhibits bac B only if directly challenged by it?? So some trigger substance of e.g. Bac Bs membrane is needed to let bac A produce the substance?

[GeorgeWolff]

Tell us what yu are measuring.

How do you know there is free exchange across this membrane?

[Tom]

gebirgsziege, on Feb 6 2009, 05:43 PM, said:

Maybe you should look for some kind of centrifugation-technique with a selective barrier....

but have you ever thought about this: bac A is producing the substance that inhibits bac B only if directly challenged by it?? So some trigger substance of e.g. Bac Bs membrane is needed to let bac A produce the substance?

Yes, I`ve thought of that, however bac A also inhibits other bacteria of different species which suggests its not a species specific trigger.

[HomeBrew]

Tom, on Feb 6 2009, 12:24 PM, said:

If I separate the two by a 0.2 semipermeable membrane which prevents the bacteria from interacting, but allows the medium and therefore their secreted substances to pass, there is very less inhibition. I've a larger pore size (0.45) of a different material and found even less inhibition.

Does this make sense?  Why would using a larger pore size (= more stuff through) result in a decrease in inhibition ("larger pore size" = "even less inhibition") if the cause of the inhibition is a secreted substance?

[GeorgeWolff]

agree homebrew, and i doubt the concept has legs.  Wonder what data validates presumption that it allows free movement.

[Tom]

HomeBrew, on Feb 7 2009, 01:13 PM, said:

Tom, on Feb 6 2009, 12:24 PM, said:

If I separate the two by a 0.2 semipermeable membrane which prevents the bacteria from interacting, but allows the medium and therefore their secreted substances to pass, there is very less inhibition. I've a larger pore size (0.45) of a different material and found even less inhibition.

Does this make sense?  Why would using a larger pore size (= more stuff through) result in a decrease in inhibition ("larger pore size" = "even less inhibition") if the cause of the inhibition is a secreted substance?

one instance where this can happen if there was the presence of a immune factor of a larger size that would pass through 0.45 but not 0.2. Thus filtering through a 0.2 stops the immune factor from passing but allows the inhibitory factor to pass. This is seen in several bacteria  such as in the case of lantibiotics.
O.K , all i asked for was if anyone could help with the centrifugal speed in separating bacteria from molecules. Now its turning into an open defense of my thesis If you cant help with the original question lets just be say we dont know and leave it at that eh?!

[GeorgeWolff]

Your call, Tom.  But you might consider the "defense" a favor.  These are questions that will be asked and it's easier to hear them now and answer them while your doing the research than at the end.

good luck