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Question on indicator/tracing of E.Coli or other bacteria

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Question on indicator/tracing of E.Coli or other bacteria

Postby ChristianFe » Tue Jun 21, 2005 8:37 am

Hello everyone,

I have a problem with an experiment for which I have to come up with a working procedure.

We are trying to observe chemotaxis of e.coli and maybe other bacteria.

What we are trying to find out is if the velocity of such a directed movement towards an attractant is effected by the kind of attractant.
This lab is to be part of a group projekt together with both chemistry, physics and biology students, to experience working together with those students that study different sciences.

My idea was to set up an agar petri dish and divide it up into lets say three parts. One side would contain the attractant and the opposite side would contain the bacteria.

However the problem that I am ending up is how to actually "see" how far the bacteria have moved.
Is there a possibility to use some indicator in the agar dish that turns color when the bacteria have reached it's region, due to some metabolic vaste product emitted by the e.coli bacteria ?

I would be very grateful if someone could maybe post some links to a possible setup of such a chemotaxis observation with e.coli or some other bacteria, that is maybe easier traced ?

Thanks a lot in advance.

Christian
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Postby canalon » Tue Jun 21, 2005 4:11 pm

Hello,

First if you want to see swimming bacteria you will probably have to use "soft agar" plates where agar is diluted (50% normal or 7.5g/l IIRC) where it is easier for bacteria to swim/crawl. The speed of diffusion of the attractant will have to be carefully monitored, because bacteria will be attracted only when in contact with it...
And last but not least, any dye that will be added to your plate will diffuse too making it hard to follow bacteria. But in fact even on TSAgar or LB agar you can see the crawling colonies with naked eyes.

If you want soething more precise you may have to think using microscopy. puuting bacteria on a gradient of your atractant and measuring their speed from one side of the field towar the other. It would be easier if you can make a movie of the bacteria>

HTH

Patrick
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Postby ChristianFe » Tue Jun 21, 2005 4:22 pm

First of all thanks for your response.

You mentioned soft agar, that is something I as well already found as a necessary prerequisite for any movement to occur.

The problem that I see in what you are saying is the concentration of the attractant. How would I control the rate of diffusion of different substances through the agar ?

I hope I would be able to avoid staining and hoped for results that could be makroscopically observed as you mentioned.

The microscope solution sound very interesting, however I won't have much time doing it and there is probably no possibility of making videos of microscope images here.

The main problem that I have takle probably is how to add the attractant on the soft agar plate to allow it to diffuse and how to at least make sure the bacteria gets in contact with it.

I guess I have to spend another night in the library / online to do some more research on the different kinds of soft agar and the attractand diffusion etc.
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Postby canalon » Tue Jun 21, 2005 5:08 pm

Hello Christian,

The problem is diffusion through agar is something quite complicated. It depends on temperature, agar concentration, molecule size, etc.. I Have to deal with it when it comes to antibiotic diffusion, and I have to admit that I am just using published figures without even trying to calculate diffusion rates.

If you can work with colored attractants, it could be interesting, but mixing a dye to a non colored attractant is not going to work since they will diffuse independantly :(

To put the attractant, a simple solution would be to load a sterile paper disc with your attractant and put it the surface of the agar. The molecule wil then diffuse through agar. If you put a drop of bacterial solution on the other end of the plate you will see bacteria swimming in all direction first (growth will appear as a circle) and then the circle is going to be deformed when the first molecules of attractnat are going to be seen by bacteria. The problem is: bacteria from a given strain would diffuse at the same speed (approx.) on the same medium, but not attractants. Hence the deformation will be the product of both its strength and its diffusion speed. I don't know if it is possible to deduce bacterial speed from such data.

For the microscope it would also be quite difficult because you will probably need micromanipulation in order to inoculate both attractant and bacteria at the 2 ends of a field

Good luck with your project, and tell us if you come up with something that worked

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request

Postby cloudnine » Sat Aug 06, 2005 5:34 am

it is a very interesting discussion to me.
i am also on my work with e coli, but not a high-level one.
i have isolated e coli from public water distribution system and now i want to test some of their activities and behaviour.
i am very interested in your work on chemotaxis and i also want to test it.
but my lab is not a well-facilitated and equipped one.
but anyway can you two give me some suggestion and advice?
how to make this experiment on chemotaxis on petridishes, and whether it is possible or not? what is to be used as an attractant?

i am now trying to extract plasmids from these bacteria and so i need some information on wild type plasmids of e coli.
i have tried pub-med that Canalon suggested but i couldn't find what i want. maybe i don't know how to find.
so if you have time, please help me.
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Re: request

Postby iri_black » Sat Aug 06, 2005 6:40 am

cloudnine wrote:i am now trying to extract plasmids from these bacteria and so i need some information on wild type plasmids of e coli.
i have tried pub-med that Canalon suggested but i couldn't find what i want. maybe i don't know how to find.
so if you have time, please help me.


Try this site http://www.fermentas.com/techinfo/appen ... rmsyst.htm

Hope it helps :)
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Postby iri_black » Sat Aug 06, 2005 6:58 am

Also, we now have the site that Opuntia recomended
http://www.textbookofbacteriology.net/
Try that one too :)
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Postby opuntia » Sat Aug 06, 2005 6:59 am

And here is the link to E.Coli :wink:
"The roots of education is bitter, but the fruit is sweet" Aristotle
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Postby cloudnine » Mon Aug 08, 2005 9:49 am

thank you all for your suggestion.
but i am still confused how to test chemotaxis on agar plates.
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