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Offlinearg1998
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Registered: 03/10/20
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what is the name of this contaminant? how can i fight it?
    #26789518 - 06/28/20 10:52 AM (3 years, 6 months ago)

hello everyone.
I made some PDA (mere PDA with no additives or nutrients) petri dishes several days ago and yesterday, I transferred some PE and B+ genetics in them. before cloning, all dishes were clean and there was no sign of any contamination on agars surface. however, shortly after the transferring process, all plates began to contaminate rapidly.

I use a glove box that is set under UV lamp for 30 min before any operation. I meticulously clean the glovebox with disinfectants and alcohol and flame sterilize the blade several times during any job. i dont use parafilm and tape dishes with micro-pore tapes. ultimately I stack all plates and put them in a plastic bag upside down.

unfortunately, my glovebox has a design flaw regarding the lid which allows air to flow under its curved caps. in my measurements, the airflow was insignificant but nonetheless, I think it plays an important role in my misery to this day.


these are the pictures of my plates after 1 day. in a single DAY !!


can anyone tell me the name of these intruders? are they fungus? or bacteria?
and how can I fight it? does it make any difference if I supplant PDA agar with something else?

another thing is that the pattern of contamination is not consistent. 3 out of 10 plates contaminations are near the lid that leaves no hesitation of air leak through them.
but others infections are concentrated around the center of the plate (where i drop the agar sample)

Note: I have seen this kind of contamination before but they were far less aggressive.

thanks in advance for any aid. may the shrooms help you in return :-)


Edited by arg1998 (06/28/20 10:56 AM)


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InvisibleStrainsfordaze


Registered: 05/10/18
Posts: 669
Re: what is the name of this contaminant? how can i fight it? [Re: arg1998]
    #26789590 - 06/28/20 11:19 AM (3 years, 6 months ago)

Looks like Cryptococcus sp. which is a yeast.
Seems like it may be getting in when you open them up because of that air gap or because of the micropore tape.
I would use Saran Wrap or parafilm instead.


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OfflineTrichodingus
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Re: what is the name of this contaminant? how can i fight it? [Re: Strainsfordaze]
    #26789664 - 06/28/20 11:55 AM (3 years, 6 months ago)

I agree.
I used to get a similar contaminate on all of my plates until I:

1) started taking sanitary procedures seriously.
2) started using parafilm.

Try doing transfers between two plates with nothing on them. Very useful for honing technique!


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InvisibleRoger Clemency
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Re: what is the name of this contaminant? how can i fight it? [Re: Trichodingus]
    #26789750 - 06/28/20 12:47 PM (3 years, 6 months ago)

Are you using a glove box or a still air box?

How are you sterilizing / pouring your agar? Storing plates upside down seems odd, though I guess that itself shouldn't cause problems.

And how did you inoculate these plates? Did you scrape spores onto the areas where stuff is showing up now or did you use a spore syringe or are these transfers from another plate?


--------------------
Sour grapes, sweet revenge
Heaven starts right where hell ends


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InvisibleStrainsfordaze


Registered: 05/10/18
Posts: 669
Re: what is the name of this contaminant? how can i fight it? [Re: Roger Clemency]
    #26791468 - 06/29/20 06:46 AM (3 years, 6 months ago)

Good catch. I quickly read that and didn’t realized he said glove box and not SAB.
Change to a SAB that should help tremendously.


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Offlinearg1998
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Re: what is the name of this contaminant? how can i fight it? [Re: Roger Clemency]
    #26796980 - 07/01/20 01:13 PM (3 years, 6 months ago)

Quote:

Roger Clemency said:
Are you using a glove box or a still air box?

How are you sterilizing / pouring your agar? Storing plates upside down seems odd, though I guess that itself shouldn't cause problems.

And how did you inoculate these plates? Did you scrape spores onto the areas where stuff is showing up now or did you use a spore syringe or are these transfers from another plate?




short version  => normal procedure with agar to agar transfer using default sterilizing techniques

not so short version => I sterilize my agar bottle in a PC under 15PSI for 100 minutes. then fill the plats inside the same glovebox. (this hasn't caused any problem so far. no contamination has been detected at this stage). and finally, I use another agar plate (which I cloned from a sample) and place a single slice of colonized agar on each plate (current problematic stage).

and sorry for the long-delayed response. I was busy with university


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Offlinearg1998
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Re: what is the name of this contaminant? how can i fight it? [Re: Strainsfordaze]
    #26796988 - 07/01/20 01:16 PM (3 years, 6 months ago)

thank you Stainsfordaze. I guess i have no choice but to change my methods entirely to avert such problems in the future.


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Invisiblekrock
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Registered: 12/18/19
Posts: 154
Re: what is the name of this contaminant? how can i fight it? [Re: arg1998]
    #26797129 - 07/01/20 02:14 PM (3 years, 6 months ago)

Quote:

I sterilize my agar bottle in a PC under 15PSI for 100 minutes



You do not need to PC the agar for more than 20 minutes.

And as the others said, it is a good idea to convert your glove box to a SAB (just remove the gloves). In a glove box there is much more air movement because when you move your hands the gloves push and pull the air/contams inside the box. This does not happen as much in a SAB because you are gliding your hands into the air inside the box (think of how water moves) instead of pumping it around.

Also, for future use; you don't really need to disinfect a SAB. It is effective because it keeps the air still, not because it has a clean interior. Avoid flame sterilizing your tools inside the box; it creates air currents.


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InvisibleStrainsfordaze


Registered: 05/10/18
Posts: 669
Re: what is the name of this contaminant? how can i fight it? [Re: krock]
    #26797404 - 07/01/20 04:30 PM (3 years, 6 months ago)

The max for agar would be 45 mins and that would be with grain soak water.
For potato or malt 20 mins is fine.


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