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OfflineJagermeister1
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Registered: 08/09/06
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Are my sterilization temps too low?
    #6006727 - 08/28/06 10:45 PM (17 years, 5 months ago)

I keep getting contamination in my jars after just a week or two. I'm using BRF/vermiculite just as perscribed by the PF tek. I don't have a pressure cooker, so I've been just steaming them for a couple of hours. I've kept everything very clean and sterile to the best of my ability, so I'm starting to wonder if the temperature isn't getting high enough. Does anyone have a suggestion? Because at this point, I'm getting tired of wasting my spores. Do I need to invest in a pressure cooker?


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OfflineDharmaBum
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Re: Are my sterilization temps too low? [Re: Jagermeister1]
    #6006745 - 08/28/06 10:51 PM (17 years, 5 months ago)

I may be able to help. When sterilizing, are you jar lids closed tight? Do they have holes in the lids? Fill me in on those two points. I use steam, and only leave pint jars in there for one hour after water is boiling. I never have any problems. Give us a run through on how you go about your process, and we may be able to help better. Also after you sterilize, what is your inoculation process... Hope to be of some help. Talk to you soon.


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OfflineJagermeister1
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Re: Are my sterilization temps too low? [Re: DharmaBum]
    #6006788 - 08/28/06 11:02 PM (17 years, 5 months ago)

No, the jar lids are not closed tight. They have 4 holes in each lid, covered with heavy duty masking tape added before the steam bath. I have steamed them for about 1 and a half to 2 hours each time just to be on the safe side. When inoculating, I sterilize my needle every 2 or 3 jars to avoid cross-contamination, using an alcohol lamp. I use anti-bacterial cleaning products on all the surfaces that my equipment comes into contact with, and I wear a surgical mask during inoculation as well. After the steam bath, I let them cool overnight, and then begin the inoculation first thing in the morning. The only other idea that I have is that my air quality in my place isn't the best, but contact with the air is as minimal as I can make it. If there's any other detail I've left out, please let me know. I appreciate all your help. By the way, all my jars have become contaminated, so I don't think there's a singular screw-up that I made.


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OfflineMacBluntski
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Re: Are my sterilization temps too low? [Re: Jagermeister1]
    #6006797 - 08/28/06 11:06 PM (17 years, 5 months ago)

come to my house and we'll pressure cook your jars and hang out and play scrabble. peace
-alan


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OfflineRogerRabbitM
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Re: Are my sterilization temps too low? [Re: MacBluntski]
    #6006830 - 08/28/06 11:17 PM (17 years, 5 months ago)

It's important to know where the contamination is starting. Take a look at the picture in this thread http://www.shroomery.org/forums/showflat.php/Number/5999103#Post5999103 The contamination started at the top of the jar and worked its way down. You can see healthy mushroom mycelium growth at the inoculation points, but it's being overtaken by the contamination that started from above. That's an example of improper jar technique, most likely a damp/wet vermiculite barrier.

If your contamination starts at the inoculation points, check your syringe making or inoculation procedure.

If your contamination starts at random places in the jar away from both the top verm barrier AND inoculation points, it's in the sterilization procedure, which I doubt.
RR


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InvisibleAnand
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Re: Are my sterilization temps too low? [Re: RogerRabbit]
    #6007300 - 08/29/06 06:11 AM (17 years, 5 months ago)

It might be handy to make blank jars with each batch, its costs a bit more but helps to find the problem.

Blank jars are jars with substrate but not inoculated, keep them with the other jars all the time, if they contam you will know its not they syringe but more likely the sterilization tek.


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OfflineJagermeister1
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Re: Are my sterilization temps too low? [Re: Anand]
    #6013192 - 08/30/06 07:54 PM (17 years, 5 months ago)

Okay, first off, thanks for all the help so far. I think we're starting to get somewhere. The contamination doesn't start at the top of the jars. It definitely starts at the inoculation points, and not at the top or at random places. So does this tell me that the sterilization temperatures are definitely high enough and that the contamination is coming from somewhere in the inoculation procedure? If so, judging by the routine I detailed in my second post of this thread, could anyone give a suggestion or two about how I might eliminate this little problem of mine? For example, is there something I should do differently with the masking tape or tightening fo the jar lids?


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Invisiblecreamcorn
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Re: Are my sterilization temps too low? [Re: Jagermeister1]
    #6013716 - 08/30/06 09:48 PM (17 years, 5 months ago)

if the water is boiling, the temp is high enough.  water boils at the same temp for everyone. :smile:  (wellll... of course there's variance depending how high above sea level you are if you wanna get technical, unless you live up in the mountains at a really high elevation its not a concern.)  the only place you could really go wrong there that nobody's mentioned is you should be steaming them with a tight fitting lid on the pot.  start counting time when the water reaches a boil, turn the heat down so its high enough to sustain a boil but not a violent bubbling boil.  count 1 hour from that point and you're good.  (though when i steam sterilize pf cakes, i go slightly longer, 1 hr 15  minutes to be on the safe side.)

leaving the lids loose is fine, then tighten as you take them out of the pc.  i wouldn't bother with the tape at all, you have a piece of foil over them (i hope) to prevent water from dripping in.  when its time to inoculate, lift the foil, inoculate through the holes, and just loosely crumple the foil back on top (so as not to completely seal it so they can breathe, but to protect the holes from the "outside world"... remember your verm layer is a contaminate barrier, you don't need to also tape the holes... you DO need to let them breathe.)

to reiterate what RR said since you're still not clear:

if contams start near the top and work their way down, you have a problem with your verm barrier.

if contams start in totally random spots, you didn't sterilize well enough.

if contams start right in the areas your needle reached into, they either travelled in on the needle itself, or were inside the syringe to begin with.

control jars like anand mentioned are a helpful troubleshooting tool if you have repeated failure.  eg, if a jar that you never stuck a needle into but otherwise sterilized and incubated still contaminates - well then you can rule out the incoulation. :smile:


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OfflineRogerRabbitM
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Re: Are my sterilization temps too low? [Re: creamcorn]
    #6014264 - 08/31/06 12:40 AM (17 years, 5 months ago)

If the contamination starts at the inoculation points, then either your syringe needle, or the contents of the syringe where contaminated.
RR


--------------------
Download Let's Grow Mushrooms



semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison


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OfflineJagermeister1
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Re: Are my sterilization temps too low? [Re: RogerRabbit]
    #6015969 - 08/31/06 05:01 PM (17 years, 5 months ago)

At this point I doubt that it was the needle itself. I heated it to red several times throughout the process, and still all the jars have ended up the same way, partly overrun by what I believe is green mold.

Given that I bought the spore syringes from one of the more popular websites used among the members of this board, I'm confident that they're legitamite, but isn't it uncommon for the contents of the syringes to be contaminated? Could something have happened during the shipping process? By the way, I've kept them refridgerated, and then the night before inoculation, I've pulled the syringe out that I plan to use the next day to let it warm to room temperature. Is this an acceptable strategy?


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OfflineSinthetic
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Re: Are my sterilization temps too low? [Re: Jagermeister1]
    #6018184 - 09/01/06 11:22 AM (17 years, 5 months ago)

You might want to look into a glovebox.


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OfflineJagermeister1
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Re: Are my sterilization temps too low? [Re: Sinthetic]
    #6032367 - 09/05/06 05:38 PM (17 years, 5 months ago)

Does anyone have a cheap alternative to something like this?

http://www.mycosupply.com/cgi-bin/shopper.cgi?preadd=action&key=GLBX01

I'm not going to drop $350 on something like this. Is there a way to build one of these cheaply?


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OfflineRogerRabbitM
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Re: Are my sterilization temps too low? [Re: Jagermeister1]
    #6032389 - 09/05/06 05:47 PM (17 years, 5 months ago)

Here's mine. It cost less than $25, including a dozen pairs of those tyvek sleeves. I just wear latex surgical gloves and the tyvek sleeves allow me to pull my arms in and out easily, so things like alcohol and the alcohol lamp can remain outside the glovebox.
RR


--------------------
Download Let's Grow Mushrooms



semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison


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OfflineJagermeister1
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Registered: 08/09/06
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Re: Are my sterilization temps too low? [Re: RogerRabbit]
    #6036725 - 09/06/06 09:50 PM (17 years, 5 months ago)

I'm going to give something like that a shot. Thanks to everyone that's been helping me with this little problem of mine.


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Offlinespeeddealer
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Re: Are my sterilization temps too low? [Re: Jagermeister1]
    #6042636 - 09/08/06 09:54 PM (17 years, 5 months ago)

I am VERY new to this whole thing. And I have successfully colonized numerous jars without any contams at all. I have been quite careless in my noobism. I also boil for about 1:30, using washcloth to insulate the bottoms, adding a few cups of boiling water halfway through and being sure that the foil is tightly secured and that water is not sloshing all over the place. The one thing that stands out to me is the tape, tape can be FILTHY...especially since most of it is produced in third world countries (droppings, mold spores, etc) I have never used tape at all and have successfully colonized dozens of jars with no contams (fingers crossed) I just cover my jars with foil nice and tight and I am sure to leave the lid on the pot overnight. My first few attempts (before discovering shroomery.com) were downright reckless...I never sterlized needles at all, blew chunks of verm off the needle, and even wiped one with my finger before innoculating!) Maybe try different sources for your substrate too. Different brands or whatever.

I am not trying to act like I know a whole bunch but maybe just add an insight or two about what I've done that's worked...Hope you can solve the problem and hope maybe I've helped just a bit. Good luck.


Edited by speeddealer (09/08/06 09:57 PM)


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