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Offlinedream.culture
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Agar 5th transfer contaminated.
    #26839375 - 07/22/20 09:48 PM (3 years, 6 months ago)

This is the 5th transfer from clean agar plates. I got this contamination in most of the dishes. I suppose it was bad sterile technique. The reason was that I had 70 percent rubbing alcohol that I used to clean everything. But the problem is that the rubbing alcohol was laying around for quite a while and most of it probably evaporated hence making the solution more like 30 % instead of 70. Could there be any other reasons? 10 %Bleach solution usually works fine for me but I don't like using bleach because it smells terrible and it ruins my clothes by bleaching them if I get any on my clothes. What do you use instead ok bleach?





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Invisiblerickyswamps
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Registered: 11/08/18
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Re: Agar 5th transfer contaminated. [Re: dream.culture]
    #26839389 - 07/22/20 09:58 PM (3 years, 6 months ago)

Thats a lot of contams.  The amount that have bacteria in them makes me think your agar wasn't sterilized completely.  What is your process?  I don't usually get any bacterial contamination unless I transferred it.  So you would see it in the center with the growth, not from the edges.  Also, are all our your plates wrapped completely?


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Offlinedream.culture
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Re: Agar 5th transfer contaminated. [Re: rickyswamps]
    #26839830 - 07/23/20 05:55 AM (3 years, 6 months ago)

Maybe that is the case. I pc'd my agar for about 20 minutes. I was blaming the alcohol solution for being not strong enough to kill any contams in the SAB. I never get contams when i transfer. Maybe 1 dish gets contaminated once in a while. Also I did transfer the mother dishes to some grain jars. So if it was the agar the jars will be fine. If it was the rubbing alcohol then the jars should have the same mold/bacteria soon.


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Offlinedream.culture
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Re: Agar 5th transfer contaminated. [Re: dream.culture]
    #26839833 - 07/23/20 05:57 AM (3 years, 6 months ago)

Do you think I can still transfer what's left? Actually I really want to keep this strain and I don't have a backup.


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Invisiblerickyswamps
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Re: Agar 5th transfer contaminated. [Re: dream.culture]
    #26839845 - 07/23/20 06:21 AM (3 years, 6 months ago)

Yes, you can definitely transfer those again.  The sooner the better so it doesn't get harder to do.


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Offlinedream.culture
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Re: Agar 5th transfer contaminated. [Re: rickyswamps]
    #26839852 - 07/23/20 06:27 AM (3 years, 6 months ago)

Thanks a lot for the help. I am on it now.


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OfflineMarxcelium
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Re: Agar 5th transfer contaminated. [Re: dream.culture]
    #26840179 - 07/23/20 09:57 AM (3 years, 6 months ago)

If you're looking for a different disinfectant, I've been using Lysol with great results while Iso has been scarce, but I just picked up some 80% hand sanitizer ethanol from Home Depot in a big gallon jug and diluted it to 70%, and it works great, too.


Edited by Marxcelium (07/23/20 09:57 AM)


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Offlinedream.culture
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Re: Agar 5th transfer contaminated. [Re: Marxcelium]
    #26863738 - 08/05/20 02:21 AM (3 years, 5 months ago)

Ok so here is an update. I checked the agar bottle that I used to pour the agar into dishes. I found mould in the bottle, that means the agar was not properly sterilized in the pressure cooker. I thought I had given it less time (20 minutes) so then I decided to make the transfers again. I pressure cooked the agar for 25 minutes (maybe more). I still found mold growing in the dishes and the bottle a few days later. I am not sure what is going on. I have done several transfers in the past and it hasn't been that bad. Maybe the agar packet was left open for a while. I use bleach and 70% alcohol to disinfect. I transfer everything in a SAB. Do you have any input on this issue? My pressure cooker seems to be working fine...


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Offlinesouthbounpachyderm
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Re: Agar 5th transfer contaminated. [Re: dream.culture]
    #26863745 - 08/05/20 02:39 AM (3 years, 5 months ago)

Quote:

dream.culture said:
Ok so here is an update. I checked the agar bottle that I used to pour the agar into dishes. I found mould in the bottle, that means the agar was not properly sterilized in the pressure cooker. I thought I had given it less time (20 minutes) so then I decided to make the transfers again. I pressure cooked the agar for 25 minutes (maybe more). I still found mold growing in the dishes and the bottle a few days later. I am not sure what is going on. I have done several transfers in the past and it hasn't been that bad. Maybe the agar packet was left open for a while. I use bleach and 70% alcohol to disinfect. I transfer everything in a SAB. Do you have any input on this issue? My pressure cooker seems to be working fine...



The way the contaminates are spreading from the edges out imo indicate an issue with your wrapping technique/sterile technique inside of your SAB. I personally don't like saran wrap and think its a large contaminate vector especially if not stored properly.
Mold in your bottles doesn't necessarily indicate the agar being improperly sterilized. Most mold spores die off between 140-160 degrees. There is no way your media is not getting hotter than that during your sterilization cycle. That would indicate kicking up air currents that draw outside air into the bottle/sab imo.


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Offlinedream.culture
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Re: Agar 5th transfer contaminated. [Re: southbounpachyderm]
    #26864085 - 08/05/20 09:07 AM (3 years, 5 months ago)

You are right it is probably the sterile technique and maybe the saran wrap but nothing has changed in my sterile technique and i used the same cling wrap. Maybe I should go back to using bleach. I have no idea what might be causing this.
Below are the new transfers and the left over agar media in the bottle. Please have a look. Its suprising how the mycelium is trying to take over on top of the mold in one of the plates.

















It's just the agar media bottle contamination that is confusing me.
Saran wrap/cling wrap problem ruled out as there is none used in the agar media bottle.
The culture is clean the new media is not. Better try transferring again but I still want to get to the root cause. I think the PC is fine. Will the spores survive a 20 minute PC cycle? Given that my agar was left open in a closet and maybe the dextrose I used has been also contaminated with spores.


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InvisibleRoger Clemency
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Re: Agar 5th transfer contaminated. [Re: dream.culture]
    #26866043 - 08/06/20 09:09 AM (3 years, 5 months ago)

Mold could be getting into your media bottle from bumping your SAB while working maybe. I’d also try upping your pc time in case it isn’t getting to proper pressure, ensuring you vent for 10 min, let the water in PC get to a boil before putting the lid on to make sure it’s hot af quickly..just for peace of mind.

As far as sanitizing idk. All I do is spray plain water in my SAB. I work half the time with no gloves, I’m just real smooth with movements, don’t bump, and never hover my hands or arms over top of my stuff.

I don’t think I’ve ever cleaned my SAB either, I store jars and stuff in there when not in use and it just sits on my flo.


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OfflineRapjack
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Registered: 05/15/17
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Re: Agar 5th transfer contaminated. [Re: Roger Clemency]
    #26866060 - 08/06/20 09:20 AM (3 years, 5 months ago)

How big are your SAB's holes? I originally had ones with six inch holes but after reading about the piston effect (arms moving creating back pressure / turbulence)  I redid them to eight and contams reduced by about 1/3. You can make the holes as big as you want, too.


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InvisibleJosex
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Re: Agar 5th transfer contaminated. [Re: Rapjack]
    #26866108 - 08/06/20 09:50 AM (3 years, 5 months ago)

Your problem has nothing to do with an insufficient PC cycle. Molds simply don't stand a chance in the pressure cooker, they'll die very quickly.

At any rate, a 20 minute cycle (as you said) is even enough time to properly sterilize agar in most cases, depending on the media used. And the fact that not all your plates show bacteria rules out an insufficient cycle.

Likewise, I don't think the problem has anything to do with sanitizing. Actually, sanitazing is not compulsury. You can get away without it, although it's always recommended you do it.

If nothing really changed and suddenly your contam rate is getting higher, I'd look for a possible mold source in the room or near the room.


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Offlinedream.culture
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Re: Agar 5th transfer contaminated. [Re: Josex]
    #26866529 - 08/06/20 01:43 PM (3 years, 5 months ago)

The same SAB has been used for over a year now. Never had any problems. I wash it before I use it.
I have never had this problem before. I agree with Josex here. Neither the PC nor the sterile technique is to blame here. I am careful usually, gloves, mask and all the precautions are taken even though one of you mentioned its unnecessary.
I did have mold in my media bottle before but K rinsed it with bleach and cleaned it up well enough later. It also went through a PC cycle after that. I am using PDA for agar.
I might not have been careful enough with the movements so I'll try again in a few days and share the results with you all. I think I'll search for a mold source too. Thanks everyone! Really appreciate your input.


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