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Registered: 01/10/05
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Contaminated jar; thoughts, please? (pics)
    #4226824 - 05/27/05 10:45 PM (17 years, 8 months ago)

These are pictures of a jar of WBS, inoculated on 5/19 with a multi-spore solution of Palenque.

If you cannot see it, everything is green mold, from top to bottom right along the inoculant path. If you saw this in your first jar from this particular syringe, what would your thoughts be?

Cleanliness at time of inoculation?

Pressure cook for longer?

Where did all this contamination come from?



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Re: Contaminated jar; thoughts, please? (pics) [Re: Leimana]
    #4226861 - 05/27/05 10:55 PM (17 years, 8 months ago)

It could be so many things you have to tell us step by step what u did.



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Re: Contaminated jar; thoughts, please? (pics) [Re: Leimana]
    #4226905 - 05/27/05 11:06 PM (17 years, 8 months ago)

1. Contaminated syringe.

2. Contaminated during inoculation.

3. Not filtered properly.

4. Not PC'ed properly.

By the looks of it, syringe was contaminated, or needle was during inoculation. Because contaminate is growing along the line of injection.

But, who knows....contaminates can pop out about anywhere, if they are in the jar, outside the jar, or injected into a jar.


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Re: Contaminated jar; thoughts, please? (pics) [Re: agar]
    #4226922 - 05/27/05 11:12 PM (17 years, 8 months ago)

give us some idea of how long you pc'ed and all your steps otherwise you guess is as good as mine

Check out the official MushroomWiki at http://www.mushroomwiki.com. Stop by and contribute.

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Re: Contaminated jar; thoughts, please? (pics) [Re: Leimana]
    #4227698 - 05/28/05 03:21 AM (17 years, 8 months ago)

From the pics, I'd bet on a contaminated syringe, or heavily contaminated needle. It looks like the green is only growing from the inoculation drizzle, along with mycelium. You don't see green in other parts of the jar that weren't touched by this solution, which implies the sterilization process was OK. With mold, it's rarely incomplete sterilization as they are very easily killed. The inoculation step itself [dirty glove box, bad air, etc] wouldn't produce a continuous line like that if it were to blame. . .usually it's only an errant few mold spores that get in. What you have there looks like a green mold/cube mixed inoc. I'm assuming you just inoculated and didn't shake it up which is what it looks like. That's just what it looks like to me, a couple of negative controls would have helped out---one jar sterilized but never opened, another 'fake inoculated'/treated like the real jars---both incubated with the rest.
If it ends up that the syringe was contaminated, there's really no hope for it. Even agar clean up would be tough. This problem does happen, usually from prints that are slightly moist for too long before syringe-ing.

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Re: Contaminated jar; thoughts, please? (pics) [Re: Suntzu]
    #4229952 - 05/28/05 09:27 PM (17 years, 8 months ago)

Thank you for the response.

I have been dreading the possibility of a bad syringe, as all of my syringes, at present, have been purchased from Shroomery sponsors. The last thing I want to do is approach an established and well respected merchant that some of the syringes he sent were contaminated. I'm hoping that it is something correctable that I did wrong in my culturing technique. Here is the procedure:

The grains were soaked for 72 hours in the refrigerator. They were thoroughly drained and loaded into quart jars and PCed at 15 psi for 90 minutes with the lids slightly loose.

The lids themselves are metal Ball or Kerr lids with 5 holes drilled into them. 4 small ones, evenly spaced around the edge, each one with a blob of silicone over it as a self-sealing injection site, and 1 large one in the middle with tyvek siliconed to the top side and another to the bottom side (double layer).

After 90 minutes at 15 psi in the pressure cooker, the cooker was opened as soon as pressure reached ambient, and the jars were removed, lids tightened, and shaken, then returned to the PC to finish cooling.

The next afternoon, the jars were removed from the PC and place on my work surface. The spore syringes were taken from their packages and shaken. The protective nub-cap that shipped with the syringe barrel was removed, and, holding the syringe with the tip end down, the needle package was opened, the needle removed, and screwed onto the tip of the syringe.

The top of the jar was swabbed with alcohol.

The needle protector was then removed from the needle. The needle was flamed till glowing, cooled with an alcohol swab and the first point was inoculated. The syringe was then shaken vigorously (small droplets of water were flung from the needle. Shaken too hard?), flamed, swabbed and the next point was inoculated. This was repeated for each inoculation point on each jar.

The jars were then place in my incubator, and the results for the 2 Palenque jars are as shown. The two Ecuador jars that were inoculated with them have not shown signs of growth yet (but no contamination either).

So far, 3 syringes out of this last batch have shown contamination like this, and now I'm paranoid and am going to do a HUGE batch of half pints to test every syringe.


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