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Phage Contamination of Cultures?


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#1 CyrusMallon

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Posted 07 February 2011 - 07:19 AM

Hi Everyone,

Part of the project I'm working on involves isolating 80 strains from soil. I've been through several rounds of isolations now, and in each round appears the same type of problem. In different types of strains I see white streaks or what looks to be smaller colonies nestled within confluent growth. Is this characteristic of phage contamination? Or would anyone else know what I'm looking at?

Any input is extremely thankful! I've stuggled with this problem, and feeling of working with dirty cultures, for over a year now.

Cheers,

Cyrus

#2 pDNA

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Posted 07 February 2011 - 10:28 AM

colonies with different morphologies would mean that you have no pure isolate and that your sample contains different types of organisms ...is this possible?

Do they differ in morphology or they just differ in their size?

A phage contamination would lead to lysis of cells making small to large plaques on confluent plates.

I would check if the small and "normal" colonies look the same under the microscope.

Are you using selective plates for the growth of your bacteria?

Without seeing the plate it is hard to say ...but the things you describe do not sound like phages are involved ...since most of the time they do lyse your cells.

Maybe you can provide more information and we can help you!

Regards,
p

#3 CyrusMallon

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Posted 07 February 2011 - 10:44 AM

Thanks for the quick reply, Veteran.

This is a bit hard to expalin, I will try to take pictures of some of these strains tomorrow with my camera, but here's what I'm seeing: All colonies display the same morphology. However, if you look at the confluent growth of the first streak there are long erratic lines within the culture. And, in some strains, within single colonies the center is white with a very jagged line to rest of the outer colony.

As far as size, they are generally the same size, nothing unusuall there.

And I don't believe some of them are mixed cultures, as I went through several rounds of plating to make sure they were pure. And this is all done with strains from soil, on TSA media.

I'll take a look under the microscope too see if there's anything ususally.

But do you, or anyone else, know what a phage plaque actually looks like? I've been looking all over for a picture of one on a plate to compare but I can't find anything.

Thanks so much!



colonies with different morphologies would mean that you have no pure isolate and that your sample contains different types of organisms ...is this possible?

Do they differ in morphology or they just differ in their size?

A phage contamination would lead to lysis of cells making small to large plaques on confluent plates.

I would check if the small and "normal" colonies look the same under the microscope.

Are you using selective plates for the growth of your bacteria?

Without seeing the plate it is hard to say ...but the things you describe do not sound like phages are involved ...since most of the time they do lyse your cells.

Maybe you can provide more information and we can help you!

Regards,
p


Edited by CyrusMallon, 07 February 2011 - 10:47 AM.


#4 pDNA

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Posted 07 February 2011 - 10:52 AM

i do know how a plaque looks like ...you will never forget it when you once had a phage infection due to spoiled pipettes (therfore if working with phages use special pipettors that you just use for pipetting of phage suspension and not for cells!) or something else.

You can find a picture with plaques here.

Just take a look if this looks like your plates ...if not try to make some pics and post it here ...this would be extremly helpful!

Good luck!

Regards,
p

Thanks for the quick reply, Veteran.

This is a bit hard to expalin, I will try to take pictures of some of these strains tomorrow with my camera, but here's what I'm seeing: All colonies display the same morphology. However, if you look at the confluent growth of the first streak there are long erratic lines within the culture. And, in some strains, within single colonies the center is white with a very jagged line to rest of the outer colony.

As far as size, they are generally the same size, nothing unusuall there.

And I don't believe some of them are mixed cultures, as I went through several rounds of plating to make sure they were pure. And this is all done with strains from soil, on TSA media.

I'll take a look under the microscope too see if there's anything ususally.

But do you, or anyone else, know what a phage plaque actually looks like? I've been looking all over for a picture of one on a plate to compare but I can't find anything.

Thanks so much!




colonies with different morphologies would mean that you have no pure isolate and that your sample contains different types of organisms ...is this possible?

Do they differ in morphology or they just differ in their size?

A phage contamination would lead to lysis of cells making small to large plaques on confluent plates.

I would check if the small and "normal" colonies look the same under the microscope.

Are you using selective plates for the growth of your bacteria?

Without seeing the plate it is hard to say ...but the things you describe do not sound like phages are involved ...since most of the time they do lyse your cells.

Maybe you can provide more information and we can help you!

Regards,
p






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