10 replies to this topic

[rickyvets]

hello everybody!
I have a problem, stock C.perfringens cultures growth different anaerobe or facultative anaerobic bacteria since one month, I have used to TSC (with D-cycloserine) agar as a media...

Does anybody have any suggestion supplement or antibiotic etc. for killing the contamination flora,
thank you


P.S. I sent the image of TSC with contaminated.

[pDNA]

why not make a fresh stock from a pure colony from your fresh streaked plate?

Regards,
p

rickyvets, on 10 February 2011 - 01:13 AM, said:

hello everybody!
I have a problem, stock C.perfringens cultures growth different anaerobe or facultative anaerobic bacteria since one month, I have used to TSC (with D-cycloserine) agar as a media...

Does anybody have any suggestion supplement or antibiotic etc. for killing the contamination flora,
thank you
100_3440.jpg

P.S. I sent the image of TSC with contaminated.

[rickyvets]

Hi, I tried it but the result didnt change:(( I stained the white colony and the colonies appear gram negative rod at microscopy...

[pito]

rickyvets, on 11 February 2011 - 12:47 AM, said:

Hi, I tried it but the result didnt change:(( I stained the white colony and the colonies appear gram negative rod at microscopy...

Then your stock culture is contaminated. Do you have a stock culture that hasnt been opened?

You could try to make a "new" stock culture, subculering your bacterium untill you get a "clean" not contaminated plate..

[rickyvets]

pito, on 11 February 2011 - 03:28 AM, said:

rickyvets, on 11 February 2011 - 12:47 AM, said:

Hi, I tried it but the result didnt change:(( I stained the white colony and the colonies appear gram negative rod at microscopy...

Then your stock culture is contaminated. Do you have a stock culture that hasnt been opened?

You could try to make a "new" stock culture, subculering your bacterium untill you get a "clean" not contaminated plate..

Unfortanetly I dont have another clean stock culture:(( there was only one culture...  C. perfringens is my PhD thesis but I dont know what to do anymore...

[pito]

rickyvets, on 11 February 2011 - 03:44 AM, said:

pito, on 11 February 2011 - 03:28 AM, said:

rickyvets, on 11 February 2011 - 12:47 AM, said:

Hi, I tried it but the result didnt change:(( I stained the white colony and the colonies appear gram negative rod at microscopy...

Then your stock culture is contaminated. Do you have a stock culture that hasnt been opened?

You could try to make a "new" stock culture, subculering your bacterium untill you get a "clean" not contaminated plate..

Unfortanetly I dont have another clean stock culture:(( there was only one culture...  C. perfringens is my PhD thesis but I dont know what to do anymore...

Well try subcultering it.. Take colonies and subculture them... If you have a plate with your bacterium and contaminats, pick your bacterium, inoculate a fresh plate.. and keep doing this till you get what you want.
Or have you tried this before?

[HomeBrew]

There are two types of colonies on the plate -- white and black.  Which is the correct phenotype for C. perfringens on the media you used?

There are well-isolated colonies of each phenotype.  Pick one of each type and streak it to a fresh plate.  Do they each produce a single phenotype, or do they produce both black and white colonies?

[pDNA]

yes, black should be the right choice! Take a black colony and make a new stock culture!

or does these colonies appear after streaking a single colonie on a fresh plate? ...in this case i would check if your media is contaminated and do the corresponding controls!

Regards,
p

HomeBrew, on 11 February 2011 - 04:25 AM, said:

There are two types of colonies on the plate -- white and black.  Which is the correct phenotype for C. perfringens on the media you used?

There are well-isolated colonies of each phenotype.  Pick one of each type and streak it to a fresh plate.  Do they each produce a single phenotype, or do they produce both black and white colonies?

[rickyvets]

I tried all of your suggestions but the result didnt change,  C. perfringens growth on TSC as a black colony.
For purification; I tried to take middle of typical colonies via loop with needle so the colonies surronded with white zone.  results was same I couldnt get rid of the contamination flora again...  
Certainly there is a solution but What? presently, I dont know:(

I will wait your suggestion...

[pito]

rickyvets, on 11 February 2011 - 05:50 AM, said:

I tried all of your suggestions but the result didnt change,  C. perfringens growth on TSC as a black colony.
For purification; I tried to take middle of typical colonies via loop with needle so the colonies surronded with white zone.  results was same I couldnt get rid of the contamination flora again...  
Certainly there is a solution but What? presently, I dont know:(

I will wait your suggestion...

Well then there must be something wrong with your technique... or your incubator is contaminated or the LAF is...

Did you try to pick a black colony with a thootpick? and then subcultivate it?
(just touch a black colony very very gently at the top and place it on another fresh plate)
BTW: have you tried to incubate fresh plates? (with nothing on it, negative control)?

And you are 100% sure its a different bacterium? Because 2 colors doenst always mean its another bacterium.
(but since you said its gram - and C. perfringens is gram + this seems not possible)

[gebirgsziege]

this problem combined with the discussion on your previous problem sounds like you are not very experienced working in a microbiology lab? But please correct me if I am wrong.
Can you ask your supervisor or somebody in your lab to help you with your technique? People know that you need help when you start a thesis and experiments using methods you have not used before and are prepared to help; because everybody wants you to get proper data, and the experiment running. If you do not ask, it will be the other way round: a lot of time is lost, the experiment is not running and usually this includes you sitting in the lab at weekends doing the experiments (again).
Please do not get me wrong now, all of us here are happy to help you, but as your problem seems to persist for quite some time now, I really think that you should talk to an experienced microbiologist in your lab (or a nearby microbiology lab) and ask for help and advice. Some problems - especially when it comes down to lab techniques - can be understood and explained best directly from (wo)man to (wo)man. Finding somebody at your lab who can help you will save you a lot of (unnecessary) trouble for your PhD.

Edited by gebirgsziege, 11 February 2011 - 07:31 AM.