Campylobacter swarming

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Campylobacter swarming

Postby naughtoj » Mar 07 2011 6:33 am

Hi, I am trying to do infection assays with C. jejuni and have a problem with the plate counts. The bacteria are swarming across the surface making it impossible to count colonies. Any suggestions would be very useful.
Thanks

Julie
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Re: Campylobacter swarming

Postby relaxin » Mar 07 2011 9:24 am

Can you use higher dilution (less number) of the bacteria?
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Re: Campylobacter swarming

Postby naughtoj » Mar 07 2011 11:53 am

Yep, I tried that. It only seems to happen with internalised bacteria and not those that merely infect the outside of the cells. We're having a good debate here on what is going on but would appreciate any ideas.................
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Re: Campylobacter swarming

Postby relaxin » Mar 07 2011 12:46 pm

I am not familiar with what you are doing. Is it the plate too wet?
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Re: Campylobacter swarming

Postby naughtoj » Mar 08 2011 4:44 am

We're infecting monolayers of mammalian cells with C. jejuni. After infection we isolate total associated bacteria and also internalised bacteria. These are then plated onto agar plates. It's standard plates that we use to culture C. jejuni all the time. The only time we get swarming is when we plate internalised bacteria.
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Re: Campylobacter swarming

Postby relaxin » Mar 08 2011 9:27 am

Some genes have been turned on or off when the bacteria are internalized. This will enable them to move across the surface of agar plate. Perhaps you can mix top agar (as in plating lambda phage) with the dilutions of bacteria before plating. This will immobilise them and you can count the colonies. It is just a thought. It may not work if they stick to each other even in suspension.
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Re: Campylobacter swarming

Postby chiacha » Apr 25 2013 2:50 pm

For some bacteria, the agar concentration affects the swarming ability of bacteria. You might want to consider increasing the agar concentration of your plate. You might need to test different concentrations of agar to find an optimal one.

Good luck!
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Re: Campylobacter swarming

Postby Ilzins » Apr 26 2013 9:46 am

If C. jejuji can resist a few seconds at 40-45ºC you could try the soft agar cover method:

The basic idea behind this method is to limit movility (thus containing colony growth) thru the use of a physical barrier that allows for oxigen diffusion:

1) prepare tubes with 1 or 1.5 mL of soft agar (0.75%) and keep them just above solidifing temp (some 40 - 45ºC coming from liquid phase i.e. cooling ater autoclave).
2) spread your bacterial dilution as usual and wait enough for the surface to be dry (dry as in no visible droplets or wettnes).
3) Empty the soft agar tube onto the center of the plaque with one swift fluid motion, as to allow it to spread over the plaque without buubles forming.
4) make sure he SA covers the whole surface of the plaque, you can use soft movements to ensure this, but no mechanical force.

Alternatively You cuold try the Most Probable Number method (even If it's a lot more work it still gives you an good idea of cell density)

I Think you can find both methods on the book by McFaddin (1976).
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Re: Campylobacter swarming

Postby naughtoj » May 03 2013 3:19 am

That's great, we've grown them at 42 and they're fine so a brief exposure to that temp should be OK. It seems to work most of the time if I just add additional agar to the plates but there is still the odd time it doesn't work.

I'll give the soft agar a try

Cheers

J
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Re: Campylobacter swarming

Postby mchlbrmn » May 03 2013 7:53 am

I've used soft agar overlays before for 'phage, but not swarming bacteria, so I'm just thinking out loud. It seems to me that there's a potential problem that the occasional bacteria will land on, or very near, the surface of the agar and swarm from there. The phage I've used would spread less effectively on the surface. In the case that this is a problem, hopefully it is not, you might try to let the first overlay set for a few minutes, then add a second overlay with no bacteria to submerge the first one. If they swarm more effectively on lower percentage agar, then they'll really enjoy swarming on top of the top agar overlay.
Just a thought.
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