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OfflineChez man
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Registered: 09/03/10
Posts: 741
Loc: Midwest
Last seen: 4 years, 6 months
Where is my contamination originating from?
    #14315366 - 04/19/11 01:34 AM (12 years, 9 months ago)

Hello Shroomerites! Here's my situation:

I have a total of 24 BRF cakes that are all contaminated.

They were all inoculated with 4% Karo LC from baby bottle (with self-sealing injection points created by filling inverted nipple with clear silicone).

The mold began rather quickly. After two days I thought I was seeing normal mycelium growth at each of the injection points, but I now believe it was just trich because around the fourth or fifth day I started noticing a few faint dark spots in a few jars.....and two days after that full blown trich (or blue-green mold?)in every jar.

Because the contamination originated at the injection points in each jar it leads me to think that it came from my lc or my needle or at the very least originated at the inoculation step. It didn't come from improperly sterilized substrate because I pressure cooked (All American unit) 36 total jars that day and I still have a dozen that I never got I chance to inoculate and are free from any contaminates.

My LC looks so healthy though. I'll post a couple pics of it. I wiped everything down with 70% iso alc, except where the micro-pore tape was over each of the four injection holes (I thought it would be bad to get moisture on the tape because it might harbor bacteria....is this wrong should i wipe with alcohol?). I did have foil covering the jars until they were ready to be inoculated though.

Here are some pics:



Both of the jars are contaminated, but a dozen look like the really dark break-out, and the other half have the barely contaminated look.

Maybe it was my glovebox....well it's not really a glove box it's just a tub flipped upside down and placed on the table edge with about a third of it hanging off the table edge so i can get my hands underneath and work off the kitchen table. Come to think of it I was probably creating air drafts when I'd have to pull out my hands each time I flamed sterilized the needle.

The LC was made from an 18 month old syringe purchased from a Shroomery vendor. Maybe too old? The syringe I was using to suck up the LC was an empty vendor spore syringe that I washed and PC'ed along with the jars (it stayed wrapped in foil until ready to use. I hit it with an alcohol soaked pad before I inserted it into the baby bottle. Then I hit up all four holes in the first jar. Flame sterilized on my stove burner, but only about 25% of the needle tip got red hot because I was concerned about melting the plastic part of the syringe. And repeated for each jar. I did a dozen one night and the second half the next night in the same manner.

Can one not tell the difference between mushroom mycelium and trichoderma when they are growing in liquid culture. Is that why RR and so many others do not endorse LC. I've had good results in the past with LC, and although I've had more contaminated jars than I did when using a multi-spore syringe, the super fast colonizing time was a fair trade-off. It also stretched a 10cc syringe into 100 of them!

As I write this I'm PC'ing the contaminated jars and I'll dispose of their contents outside in the morning. Then I'm going to build a proper glove box and inoculate a dozen jars with straight spores from two different, fresh, sponsor purchased syringes. But I'm also thinking about saving a little bit of spores to try a grain LC.

Can anyone tell if my Baby bottle LC looks contaminated or healthy from the pictures above. I shook it a little bit in the second picture. Thanks sorry for long post.:laugh:


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OfflineRogerRabbitM
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Registered: 03/26/03
Posts: 42,214
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Last seen: 11 months, 4 days
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Re: Where is my contamination originating from? [Re: Chez man]
    #14323027 - 04/20/11 01:21 PM (12 years, 9 months ago)

Every day I come in here and rant about how horrible LC is and have explained all the reasons why it shouldn't be used many dozens of times. However, every day some noob advises some other noob to use it and all they end up doing is ruining their project and wasting time. 

I know of absolutely ZERO experienced growers who still use LC.  It's far slower than spores and you don't know what you have until you use it a few weeks later, when you'd already been picking mushrooms if you followed proper procedure.

It's not an oversight that resulted in no LC tek being on "Let's Grow Mushrooms".
RR


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OfflineChez man
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Registered: 09/03/10
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Re: Where is my contamination originating from? [Re: RogerRabbit]
    #14323110 - 04/20/11 01:41 PM (12 years, 9 months ago)

Enough said.

Time for me to move to agar.


Thanks RR.


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OfflineChez man
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Registered: 09/03/10
Posts: 741
Loc: Midwest
Last seen: 4 years, 6 months
Re: Where is my contamination originating from? [Re: Chez man]
    #14349708 - 04/25/11 03:34 PM (12 years, 9 months ago)

Update:

I made a bunch of PF cakes using a few different and fresh, spore syringes. And I also made 3 cakes with the left over spore solution I used to make the above LC in the baby bottles.

Well all the cakes are fine and showing small spots of healthy mycelium growth.....all except the 3 from the suspect syringe. Those cakes are already showing tiny spots of green. This leads me to conclude that the contamination originated from the multispore syringe itself.

My whole point in posting this is that I thought the LC looked perfectly healthy in those baby bottles, but the whole time they were growing pure contamination. So I guess contaminates (trich or whatever it is) looks EXACTLY like healthy mycelium to the naked eye.

I know that trich won't sporulate while suspended in liquid, but I still thought I'd be able to tell the two apart by texture, or color, or something, but I guess not.


--------------------
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OfflineMaverick
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Re: Where is my contamination originating from? [Re: RogerRabbit]
    #14350089 - 04/25/11 04:40 PM (12 years, 9 months ago)

Quote:

RogerRabbit said:
Every day I come in here and rant about how horrible LC is and have explained all the reasons why it shouldn't be used many dozens of times. However, every day some noob advises some other noob to use it and all they end up doing is ruining their project and wasting time. 

I know of absolutely ZERO experienced growers who still use LC.  It's far slower than spores and you don't know what you have until you use it a few weeks later, when you'd already been picking mushrooms if you followed proper procedure.

It's not an oversight that resulted in no LC tek being on "Let's Grow Mushrooms".
RR





Though I certainly agree a syringe to LC is the worst idea ever, I have do Disagree on one thing with you:  If you go agar->LC and use proper technique, your contam ratio should be equal to that of agar->agar transfers, and you shouldn't see contamination.  This is how I make LCs, and I haven't had contamination from LC using this method.  However any other method is a shot in the dark.


I even think syringe->jar is a bad idea compared to print->agar->grain or LC->grain


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InvisibleGretchenmeister
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Registered: 07/23/05
Posts: 1,032
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Re: Where is my contamination originating from? [Re: Chez man]
    #14352912 - 04/26/11 12:18 AM (12 years, 9 months ago)

Quote:

Chez man said:
Hello Shroomerites! Here's my situation:

I have a total of 24 BRF cakes that are all contaminated.

They were all inoculated with 4% Karo LC from baby bottle (with self-sealing injection points created by filling inverted nipple with clear silicone).

The mold began rather quickly. After two days I thought I was seeing normal mycelium growth at each of the injection points, but I now believe it was just trich because around the fourth or fifth day I started noticing a few faint dark spots in a few jars.....and two days after that full blown trich (or blue-green mold?)in every jar.

Because the contamination originated at the injection points in each jar it leads me to think that it came from my lc or my needle or at the very least originated at the inoculation step. It didn't come from improperly sterilized substrate because I pressure cooked (All American unit) 36 total jars that day and I still have a dozen that I never got I chance to inoculate and are free from any contaminates.

My LC looks so healthy though. I'll post a couple pics of it. I wiped everything down with 70% iso alc, except where the micro-pore tape was over each of the four injection holes (I thought it would be bad to get moisture on the tape because it might harbor bacteria....is this wrong should i wipe with alcohol?). I did have foil covering the jars until they were ready to be inoculated though.

Here are some pics:



Both of the jars are contaminated, but a dozen look like the really dark break-out, and the other half have the barely contaminated look.

Maybe it was my glovebox....well it's not really a glove box it's just a tub flipped upside down and placed on the table edge with about a third of it hanging off the table edge so i can get my hands underneath and work off the kitchen table. Come to think of it I was probably creating air drafts when I'd have to pull out my hands each time I flamed sterilized the needle.

The LC was made from an 18 month old syringe purchased from a Shroomery vendor. Maybe too old? The syringe I was using to suck up the LC was an empty vendor spore syringe that I washed and PC'ed along with the jars (it stayed wrapped in foil until ready to use. I hit it with an alcohol soaked pad before I inserted it into the baby bottle. Then I hit up all four holes in the first jar. Flame sterilized on my stove burner, but only about 25% of the needle tip got red hot because I was concerned about melting the plastic part of the syringe. And repeated for each jar. I did a dozen one night and the second half the next night in the same manner.

Can one not tell the difference between mushroom mycelium and trichoderma when they are growing in liquid culture. Is that why RR and so many others do not endorse LC. I've had good results in the past with LC, and although I've had more contaminated jars than I did when using a multi-spore syringe, the super fast colonizing time was a fair trade-off. It also stretched a 10cc syringe into 100 of them!

As I write this I'm PC'ing the contaminated jars and I'll dispose of their contents outside in the morning. Then I'm going to build a proper glove box and inoculate a dozen jars with straight spores from two different, fresh, sponsor purchased syringes. But I'm also thinking about saving a little bit of spores to try a grain LC.

Can anyone tell if my Baby bottle LC looks contaminated or healthy from the pictures above. I shook it a little bit in the second picture. Thanks sorry for long post.:laugh:




1. Maybe its trich in the jars, maybe its some sort of penicillium or bread mold.  Hrm, kitchen....jerry -rigged still air box?

2. Ive never been a fan of baby bottles for anything cultivation related.

3. When you move up to agar, go ahead and learn grain prep.  One hole per jar for innoc is gonna give you far better odds than four innocs per jar.  I love LC, I have a less than 3% contam rate in my last 300 pints of grain, using vendor syringe to lc, and also using grain lc, and also using some isolate/agar. 

If you are relying on vendor syringes alone it would not matter whether you went to lc or substrate jars with it...the contam either came in the syringe, OR, it entered during innoc at some point.

When you pc your jars, be sure to let the pressure cooker depressurize on its own- DO NOT HELP IT TO SPEED UP UNLOADING YOUR JARS- so nasty rogue kitchen biolife isnt sucked into your jars. Try incubating a "blank" jar out of each batch along with your innoced jars, watch for growth in your blank that might indicate failed sterilization.  (Shrug)

Good luck.


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Offlinesolumvita
Q.B.E.
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Registered: 02/12/08
Posts: 2,061
Loc: South Africa
Last seen: 7 months, 20 days
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Re: Where is my contamination originating from? [Re: Gretchenmeister]
    #14359245 - 04/27/11 01:32 AM (12 years, 9 months ago)

LC is a waste of time!  IMO


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