8 replies to this topic

[nux]

I have to screen ~2000 bacterial strains for their potentail in forming gaseous compounds which inhibit E. coli in growth. Basically it should be something like a dual-culture test without direct contact, though each antagonistic strain should share the gas phase with E. coli. The only restriction is that all bacteria have to be grown on a solid media. Does anyone have a suggestion how to arrange this screening?
I also wondered if there is a staining method for differentiation between living and/or dead bacteria, that is not based on fluorescence? For example if I have ~50 bacterial cultures on one piece of blotting paper. The method doesn't have to be exact/quantitative, but a discrimination between normal growing and inhibited cells is necessary.

It's my first post here

Edited by nux, 25 April 2011 - 01:01 PM.

[pito]

one of the main problems is that you can do big tests and thus you need to work with small amounts of gas being formed.. this also means small effect most likely.

+ 2000 strains... thats a lot of work..
I have some ideas to use, but maybe you allready tried some of them. What have you tried before?

[nux]

You are right, the biggest challenge is the amount of strains I have to deal with. I worked with two compartment plates and had good results, but it allows testing of only one strain per plate. My idea for a faster screening assay was the usage of deep well plates covered with blotting paper. The wells conatain antagonistic strains while the blotting paper is covered with E. coli. I think that this method may work, but the visualization of individual spots is hard when fluorescent dye is used.
Do you have any good suggestion?

[pito]

A problem is: how do you know the gas that is created by your bacterium is the reason e.coli dies... It can be because of not enough oxigen, not enough food..

So the blotting paper part: sounds weird to me.
How are you going to put e.coli on that paper and keep it alive? You are going to try to put each colony above a well then? or?

[nux]

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how do you know the gas that is created by your bacterium is the reason e.coli dies

By using negative controls, one without any second bacteria and one with a non-antagonistic bacteria.

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How are you going to put e.coli on that paper and keep it alive?

From a liquid culture with a known OD600. It will stay alive for the time the experiment lasts.

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You are going to try to put each colony above a well then?

Yes, one blotting paper for each multi-well plate and an exact amount of E. coli culture above each well.

I think that everything would work fine, except of a quick live/dead analysis. Maybe a wipe test would work after the incubation time...
I still couldn't find any reagent for an easier determination of living/dead cells.

[pito]

nux, on 01 May 2011 - 08:37 AM, said:

Quote

how do you know the gas that is created by your bacterium is the reason e.coli dies

By using negative controls, one without any second bacteria and one with a non-antagonistic bacteria.

Quote

How are you going to put e.coli on that paper and keep it alive?

From a liquid culture with a known OD600. It will stay alive for the time the experiment lasts.

Quote

You are going to try to put each colony above a well then?

Yes, one blotting paper for each multi-well plate and an exact amount of E. coli culture above each well.

I think that everything would work fine, except of a quick live/dead analysis. Maybe a wipe test would work after the incubation time...
I still couldn't find any reagent for an easier determination of living/dead cells.

Yeah, well that last phrase summs it up: I see what you want to do, but how are you going to test it (dead vs alive)?
Putting the bacteria on a piece of paper... its not an easy method.
An idea or solution, could be to plate every "colony" from the blotting paper on a new petri dish to see if its still alive or not....(I think this is what you mean with the wipe test?)
But however you turn it, you well have lots and lots of work.

BTW: did you test it ? Did E.coli grow good enough on the paper? Did it really enter exponential growth on the paper? If it doesnt: then you can test for it to be alive or not, but the information you get out of that doesnt mean a lot. (well it depends on the negative strain).
+the negative control: how long are you going to run the experiment?

Anyway: I think you can try this, but in the end you will need to do more accurate tests with the strains that seem to kill E.coli.

My idea would have been a bit different and a lot more work, so I guess yours is better altough, the idea of using paper with bacteria on it... eum... I also wonder: how many samples are you going to take for each strain? You cant just blot one.. So how many did you think on doing?


If I was you: I would now start with the negative control! Check what you get if you put e.coli on a piece of paper above a multiwell filled with medium.
See of the medium has growth : e.coli falling in it?
See of the e.coli on the paper is still alive (plate it out)
If you start with this, then you allready know if this is an option or not.
Also: check the growth of e.coli at different time-intervals.

[nux]

I performed some tests with the blotting paper and it doesn't seem to be a reproducible method. The colony density varies too much in the negative controls. I think that I have to replace the blotting paper with some kind of foil that allows better growth. It would be great if E. coli could enter the exponential growth phase, but that's impossible without adequate nutrient supply.

[pito]

nux, on 05 May 2011 - 02:32 AM, said:

I performed some tests with the blotting paper and it doesn't seem to be a reproducible method. The colony density varies too much in the negative controls. I think that I have to replace the blotting paper with some kind of foil that allows better growth. It would be great if E. coli could enter the exponential growth phase, but that's impossible without adequate nutrient supply.

Well thats what I feared...

I dont think there will be any other option then use a medium itself.. you need to put e.coli in medium.

[Phil Geis]

Why 2000 isolates and what is your demonstration of concept?