16 replies to this topic

[rickyvets]

hello everybody!
I have a problem, my cell cultures have mold contamination since one month,  checked the media, its ok and i changed the laminar hood, do cleaning as much as, still couldnt get rid of them  

Does anybody have any suggestion for killing mold,

please helping
thank you

P.S. my cultures are C. perfringens...

[pito]

So the media is ok?

Then either your bacterial sample is contaminated or the flow/working proces is not good.

You have a bacterial sample in a tube or?
And the medium, you do make fresh medium?
Do others work in your lab with molds?
Spores are everywhere..

[protolder]

Hola, in cell cultures we use amphotericyn to avoid mold contaminations unless sometimes they appears. I don´t now  clostridium sensitivity, but why don´t you try seed your cultute in a plate in isolation and take a new clean clostridium colony?Buena suerte

[gebirgsziege]

I would not use AmphoB in this case, you do not know anything about the contaminant and too many fungi have a resistance against it.

Is your culture of C perfingens really kept anaerob? Because if it is you should take a closer look at the mould...not many moulds can grow really anaerobically. You should check.

You could try heating your culture to 80/90 °C for some minutes (e.g. 10 min), C perfingens should be able to survive this, but most fungi will be killed. You can also make a time/temperature experiment (temperature resistance test), plate it and at some point the mould should disappear.

[rickyvets]

pito, on 25 January 2011 - 02:04 PM, said:

So the media is ok?

Then either your bacterial sample is contaminated or the flow/working proces is not good.

You have a bacterial sample in a tube or?
And the medium, you do make fresh medium?
Do others work in your lab with molds?
Spores are everywhere..

thank you,
I try all of them but unfortunatly:( the result doent change it:(

there are not only molds in tube but also media:(

[rickyvets]

gebirgsziege, on 26 January 2011 - 12:06 AM, said:

I would not use AmphoB in this case, you do not know anything about the contaminant and too many fungi have a resistance against it.

Is your culture of C perfingens really kept anaerob? Because if it is you should take a closer look at the mould...not many moulds can grow really anaerobically. You should check.

You could try heating your culture to 80/90 °C for some minutes (e.g. 10 min), C perfingens should be able to survive this, but most fungi will be killed. You can also make a time/temperature experiment (temperature resistance test), plate it and at some point the mould should disappear.

thanks gebirgsziege!

I will try heating the culture as a result the vegetative. C. perfringens turn to spore form that can be problem for me because I need veg. perfringens...

[pito]

rickyvets, on 26 January 2011 - 01:39 PM, said:

pito, on 25 January 2011 - 02:04 PM, said:

So the media is ok?

Then either your bacterial sample is contaminated or the flow/working proces is not good.

You have a bacterial sample in a tube or?
And the medium, you do make fresh medium?
Do others work in your lab with molds?
Spores are everywhere..

thank you,
I try all of them but unfortunatly:( the result doent change it:(

there are not only molds in tube but also media:(

you have molds in your media? Even when you make fresh media ?

The heating might be a problem for you, but as allready been said here: yours grow without oxigen.. the molds need (most of them) oxigen...

[rickyvets]

pito, on 26 January 2011 - 02:06 PM, said:

rickyvets, on 26 January 2011 - 01:39 PM, said:

pito, on 25 January 2011 - 02:04 PM, said:

So the media is ok?

Then either your bacterial sample is contaminated or the flow/working proces is not good.

You have a bacterial sample in a tube or?
And the medium, you do make fresh medium?
Do others work in your lab with molds?
Spores are everywhere..

thank you,
I try all of them but unfortunatly:( the result doent change it:(

there are not only molds in tube but also media:(

you have molds in your media? Even when you make fresh media ?

The heating might be a problem for you, but as allready been said here: yours grow without oxigen.. the molds need (most of them) oxigen...

of course you r right how it can grow without oxigen but ı dont understand... ı look the media and the tubes still there are molds a short while ago:(  I have used  anaerocult and anaerobic jar for anaerobic condition on the other hand parafilm and liqued paraffin for tubes...

[gebirgsziege]

rickyvets, on 26 January 2011 - 01:47 PM, said:

gebirgsziege, on 26 January 2011 - 12:06 AM, said:

I would not use AmphoB in this case, you do not know anything about the contaminant and too many fungi have a resistance against it.

Is your culture of C perfingens really kept anaerob? Because if it is you should take a closer look at the mould...not many moulds can grow really anaerobically. You should check.

You could try heating your culture to 80/90 °C for some minutes (e.g. 10 min), C perfingens should be able to survive this, but most fungi will be killed. You can also make a time/temperature experiment (temperature resistance test), plate it and at some point the mould should disappear.

thanks gebirgsziege!

I will try heating the culture as a result the vegetative. C. perfringens turn to spore form that can be problem for me because I need veg. perfringens...

But when you subculture you pure bacteria more than twice the spores should be away.

If even the fresh medium without inoculation is contaminated, you have either a problem with your aseptic technique or your autoclave. You should check this!

[pito]

gebirgsziege, on 27 January 2011 - 12:10 AM, said:

rickyvets, on 26 January 2011 - 01:47 PM, said:

gebirgsziege, on 26 January 2011 - 12:06 AM, said:

I would not use AmphoB in this case, you do not know anything about the contaminant and too many fungi have a resistance against it.

Is your culture of C perfingens really kept anaerob? Because if it is you should take a closer look at the mould...not many moulds can grow really anaerobically. You should check.

You could try heating your culture to 80/90 °C for some minutes (e.g. 10 min), C perfingens should be able to survive this, but most fungi will be killed. You can also make a time/temperature experiment (temperature resistance test), plate it and at some point the mould should disappear.

thanks gebirgsziege!

I will try heating the culture as a result the vegetative. C. perfringens turn to spore form that can be problem for me because I need veg. perfringens...

But when you subculture you pure bacteria more than twice the spores should be away.

If even the fresh medium without inoculation is contaminated, you have either a problem with your aseptic technique or your autoclave. You should check this!

I suppose there is no point in subcultering them if the medium itself is contaminated.
But as you allready said: there is something wrong..

Anyway: did you store the medium (do not add anything) anaerobically? Just try this for once (if you still have some left over without contamination) and see if you still get growth in your medium  (or is all your medium allready been contaminated?)

(its possible the contamination happens during the storage in an aerobic storage room.. on the other hand: it should really happen... but you never know I suppose).

Edited by pito, 27 January 2011 - 02:28 AM.

[rickyvets]

Unfortunately, I havent still get rid of mould contamination:(
yesterday I did gram strain from colony contaminated with mold and saw typical Gram (+)basil, sporeforming on microscopy. but interestingly, TSC agar and Cooked meat medium still has growth like molds a lot.

[pito]

You did a gram stain and got gram + bacteria...  you are working with gram + bacteria, so this is normal..  I dont see the link with the molds?


And you have molds on fresh, sterilised media? (the TSC agar and meat medium).
This is not normal, you are doing something wrong or the autoclave is not working like it should..

Anyway, if there is a problem with your medium (the way you prepare it, or the proces of autoclaving) then there is no point in trying to subcultere the bacteria in order to get rid of the molds...

You need to fix the contaminationproblem.

I really dont understand it how you can get mold in fresh medium.
Have you allready tried to simply inoculate an empty plate ? (empty= just medium)

[lab rat]

Is it really a mold, or a filamentous bacterium?

[gebirgsziege]

First: can you post a photo of your "contamination" and your bacteria? This would help to determine the type of your contamination.

Second: can you describe step by step how you set up your experiment and how you prepare your medium? We might be able to find problems in your aseptical technique, that probably solve your problems.

[rickyvets]

Hi everybody,
thank you for your feedback.

I have two news (good and bad): first , there isnt anymore mold contamination:( second news; there are growth alot of atypical colony together my bacteria on media (TSC with D-cycloserine)

I think my stock culture has mix contamination mold and anaerobic bacteria:(
Now I guess have to pressure to the other anaerobic flora... Do you have idea?