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OfflineGRETO
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Journey of a rookie - Contam and weak colinization - Any advice are welcome
    #28626004 - 01/18/24 02:40 PM (10 days, 2 hours ago)

Hello Shroomers,

Last year I've launched a mushroom farm in south of France.
I started with ready-to-fruit blocks in a hydroponic tent the time to finish the renovation of the basement which will welcome my activites and got quite good results. As the renovation is finished, I am now doing my own subtrate and inoculating it with mycelium from a well known belgian producer but got lot of struggles.

Here is all my setup and process, maybe some of you will find where my failures come from :

Subtrate :
I use 80% oak pellet and 20% wheat bran + 55% water. I found out that with 60%, my mix was too wet as when I squiz it, lot of droplet would fall. Here is my mixing station



Super Pasteurization :
Then I put 20 to 25 bags in my super pasteurizeur. It take 4h to get to 92°C / 198° F then I let it run for 10 more hours. looks ok, I run test bags every batch that are still free from contam 1 month later.



Lab and inoculation :
Two days later when the bags are about 25°C / 77° F, I unload the bags in my lab. Then inoculate in front of a 2x4 flow hood.  The day before, I spray the whole lab with H2O2 solution and I turn on the fan 1h before inoculation.  I wear gloves, face mask, crocs, lab coat. Everything in front of the flow hood is swiped with 70° alcohol. Surface, tools, mycelium and subtrate bags. I did the lighter test 45° everywhere !




Incubation :
Then, after shaking, blocks go in my incubation space and sit here at a temp of 20-21° C / 68-70°F


Bonus, my fruiting room with some nice Lion's mane growing


My problems :

- I have a high rate of green mold contamination. Always the same thing, bags looks great during 12-14 days then there is an explosion of green mold everywhere in the bag. I never get only 1 or 2 green points, it cover 1/4 of the bag.By being in an old basement, I understand that the moldload would be high but still, half of my bags look like this. 

- Even healthy bags never get totally white like here is a 2 week old and 3 week old bags. Look like the mycelium grow strong hyphe but avoid half of the surface.



SO :smile:

For the contam problem, I do not know how to get it more aseptic. I wash and disinfect everything, I make sure that my hands never get between the filter and the bags. Plus I don't understand how could there be so much contamination point. Looks like there is more than mycelium contact point.

The mycelium I buy looks healthy but is there a way to be sure that it do not come from there?

Any idea about the weak colonization bags? Too low temperature? Bacterial Contam (Oh no :confused: )


Thank you and have a good mushroom day !


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Offlinedeadmandave
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: GRETO]
    #28626083 - 01/18/24 03:53 PM (10 days, 1 hour ago)

try increasing your sterilization time and temp. Use 96C and 20-24 hrs cook time. pull them out same day they're done cooking to let them cool more quickly.

60% moisture should be correct. You might want to test the water content of the materials your using and adjust the numbers from there. I dont squeeze test the substrate anymore, just go by numbers.

what was the substrate used in the pre made blocks?

(In my opinion you will save time and just add the dry ingredients to the bag and then add the proper amount of water per bag, mix after you have added spawn.)


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InvisibleSoutherner
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: deadmandave]
    #28626166 - 01/18/24 04:45 PM (10 days, 10 minutes ago)

Is it specific species/strains or is it all of them? I only ask because the lions mane looks fine.

Lions mane only looking part colonized is normal in my experience. A lot of brown with white spots. It can whiten up quite a bit if left to consolidate. The oyster block looks unusual to me but I haven’t messed with a large amount of strains. Mine are usually full white.

While you’re waiting to see if what deadmandave suggested helps out you could do a couple of tests. Open a sterile Petri dish in front of the hood for a couple of minutes and close it up. See if anything grows in the next week or two. Could also shake up a bag and before inoculating your substrate bags take a little to some dishes and see what the mycelium looks like and make sure there isn’t something else riding along.


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OfflineGRETO
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: deadmandave]
    #28626706 - 01/19/24 03:26 AM (9 days, 13 hours ago)

Next batch, I will give a try with higher temp and time. Two questioning about that :
As my test bags show 0 contaminations after 1 month. This told me that the pasteruisation time was sufficient. Could this be wrong?

The blocks are placed in the pasteurizer in such a way that a large portion touch the metal edges. I'm also going to run a batch with fewer blocks in case the part that touches the edges doesn't rise to full temperature.

Regarding the water, I did not test the water content but based on producer information. The water content is between 8 and 12% which give me between 3 to 5% of the total. So my 55% are not so far I guess (I will test this to be sure)
You are making a point about dry baging technique ! I am thinking about this to save some time but I have bigger problem to solve for now :grin:

Pre made blocks were aok sawdust + mix of agricultural waste bran, rie + gimpsum


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OfflineGRETO
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: GRETO]
    #28626715 - 01/19/24 03:58 AM (9 days, 12 hours ago)

I currently grow blue oyster, white oyster, golden oyster, black pearl and lion's mane.
I knew that lion's mane have a weaker mycelium but so far they're the ones who do best. They're the only ones who have also managed to completely get the upper hand on mold. Literraly eat the green spot.

I also find this really abnormal with oyster mushrooms when I see all this photo of nice white bag and mine always stay with theese brown spots.
I try to eliminate the causes :
Temp : Oscillates between 20 and 21, look ok for me
Humidity : 55% maybe little bit too low from deadmandave com but taking material water content it's about 2-3% so I don't think it come from here.
Material treatment : I use biological wheat bran and additive-free oak pellets
Light : there's a very weak led bar in the room, not enough to bother the mycelium. In addition, the bags at the back in the shade have the same problem.
PH : I did not test that, could be a possibility
Weak mycelium : I do trust my producer about the quality but maybe transportation from belgium to france weakens the mycelium
Contamination : Some of theese weak bag have yellow coloration but it is not squishy and do not smell weird

I will buy some petri dish to do your recommanded test. I also looking in my circle for a particule detector maybe my flow hood is just bad but why? The lighter test is perfect 45° angle and the flame stays sharp
Last batch, I did some "equivalent" test with bag.
One just opened in front of the flow hood and closed.
Second one opened 30 sec then closed
Third one, opened and mimed the action of filling the bag.

Will see in few days if I have contam in them


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InvisibleSoutherner
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: GRETO]
    #28628149 - 01/20/24 06:06 AM (8 days, 10 hours ago)

If it’s in everything it’s got to be insufficient substrate sterilization, the spawn, or the filter/technique. You don’t have an AC vent or something blowing into the room while you work with the hood do you? I’ve forgotten mine open before and lost some Petri dishes to it.


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Offlinedeadmandave
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: GRETO]
    #28628204 - 01/20/24 07:19 AM (8 days, 9 hours ago)

Quote:

GRETO said:
Next batch, I will give a try with higher temp and time. Two questioning about that :
As my test bags show 0 contaminations after 1 month. This told me that the pasteruisation time was sufficient. Could this be wrong?

The blocks are placed in the pasteurizer in such a way that a large portion touch the metal edges. I'm also going to run a batch with fewer blocks in case the part that touches the edges doesn't rise to full temperature.

Regarding the water, I did not test the water content but based on producer information. The water content is between 8 and 12% which give me between 3 to 5% of the total. So my 55% are not so far I guess (I will test this to be sure)
You are making a point about dry baging technique ! I am thinking about this to save some time but I have bigger problem to solve for now :grin:

Pre made blocks were aok sawdust + mix of agricultural waste bran, rie + gimpsum




I don't trust a bag sitting around un-inoculated as evidence that it's 100% clean. There's something that happens when a bag is opened, inoculated and had mycelium running through it.. I might just be crazy myself tho..

Southerner is right, it's either the flow hood, the technique, the spawn or the sterilization .

The insulation is thin on those bubba barrels. Maybe a thicker layer would help temps at the edge stay consistent.


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OfflineGRETO
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: deadmandave]
    #28629809 - 01/21/24 12:52 PM (7 days, 4 hours ago)

The door of my lab is not totally sealed but there is no vent or air moving inside the lab so I do not think it come from that.

It is not a "real" bubba barrel, I made it myself but in fact you are right, I could put more isulation. Now it is isulated with armaflex van isulation. an extra layer of neoprene wouldn't hurt.

Anyway, Thank you both very much for your help !

Next week I will do a range of tests :
- 95° for 20h and less bag so they to not touch edge at all
- Order spawn from another producer
- Open some pretri dish in front of the hood. At the center but also at the height of my bag opening (I suspect some turbulence at the edge of the filter may be to blame)
- I also ordered few Kg of different subtrate. Not for the contam but the weak colonization of the others bags. Maybe that a treatment of my wheat bran, even organic, may be a problem
- Even try to inoculate 2 or 3 bags in my lab with the flowhood switched off. I'm not betting much on it, but if these give better results then my flowhood is the problem.

I will redouble my efforts to ensure that my techniques in front of the hood are perfect. But honestly, when I watch some producers' videos on youtube, I feel like I'm already more careful that what I see.

I really hope that my problems do not come from my flowhood, if it the cause I'll have no choice but to order a ready-made one... $$$


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Offlinecybork
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: GRETO]
    #28630885 - 01/22/24 10:35 AM (6 days, 6 hours ago)

"The mycelium I buy looks healthy but is there a way to be sure that it do not come from there?" + "I will buy some petri dish to do your recommanded test. " makes me think you maybe you are not familiar with QC (Quality Control)?

I don't really understand how one can have such a nice and big and awesome (!) setup, but doesn't have petridishes available for instant use. I always (try) to have about at least 10 petridishes for instant use. In your scenario I would have much, much, much more for ready use.

I get the feeling your organisation grew a bit faster than your experience? Well, speed up the learning curve about petrisdishes :wink: And I'm sure many more challenges will come :thumbup:

Maybe I interpret things wrongly on what you said, could well be. Then ignore the above.


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Offlinecybork
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: cybork]
    #28630948 - 01/22/24 11:29 AM (6 days, 5 hours ago)

Oops. Just saw your last thread better than first. Yeah, confirms a bit what I just stated. OK. Because I care, Let me respond to your last message... (PS: I do not see myself as very experienced yet. So, do with it as you please.).

The door of my lab is not totally sealed but there is no vent or air moving inside the lab so I do not think it come from that.
>>If your flowhood is ok, then it is the only air source flowing in and over your bags (I think you're referring to the alternative airflow stream).

It is not a "real" bubba barrel, I made it myself but in fact you are right, I could put more isulation. Now it is isulated with armaflex van isulation. an extra layer of neoprene wouldn't hurt.
>>nice build.

Anyway, Thank you both very much for your help !

Next week I will do a range of tests :
- 95° for 20h and less bag so they to not touch edge at all
- Order spawn from another producer
>>first do QC on some of your current producer(?) Also changing spawn is changing a constant (if your producer is constant with their mycelium and spawning substrate). Changing it will quite surely also affect your other 'knowns'. PS: if you have also spawn grow in your own control...
- Open some pretri dish in front of the hood. At the center but also at the height of my bag opening (I suspect some turbulence at the edge of the filter may be to blame)
>>place them on strategic places, and keep them open for 5 minutes. I'd do about 10 of them I think (looking at your enormous hood).
- I also ordered few Kg of different subtrate. Not for the contam but the weak colonization of the others bags. Maybe that a treatment of my wheat bran, even organic, may be a problem
>>I haven't got any experience with sawdust. Changing and experimenting with substrate seems smart, but first get your other things working satisfactory (?).
- Even try to inoculate 2 or 3 bags in my lab with the flowhood switched off. I'm not betting much on it, but if these give better results then my flowhood is the problem.
>>Eh. No. I don't think flowhood off could give less contamination. Unless your room is a still air box (which it isn't). On the other hand, if your hood is blowing pollutions like crazy, you have a point. Try it out anyways, but then with petrishes (you will be better to see contam in a petrish). And or with bags. Again with 10 dishes (?).

I will redouble my efforts to ensure that my techniques in front of the hood are perfect. But honestly, when I watch some producers' videos on youtube, I feel like I'm already more careful that what I see.
>>ow, that about youtube is a tricky one to state on this forum. :wink: That said, yeah, can well be. I bet on your flow hood as the culprit then, and/or the not so clean substrate bags after all.

I really hope that my problems do not come from my flowhood, if it the cause I'll have no choice but to order a ready-made one... $$$
>>not sure why you can't make or (re)assemble the materials yourself? And what you have currently: could there be something in the setup that can cause inappropriate working? Did you assemble it yourself? Also I'm not so sure about the lighter method you mentioned earlier, for testing the correct workings.

PS: during scrolling I now also read your subject. Missed that too (that always happens to me, also in my office).


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OfflineGRETO
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: cybork]
    #28631116 - 01/22/24 02:19 PM (6 days, 2 hours ago)

"I get the feeling your organisation grew a bit faster than your experience? Well, speed up the learning curve about petrisdishes :wink: And I'm sure many more challenges will come :thumbup: "
YOU ARE 100% RIGHT :grin:
I recently took a giant step.
My idea about learning was to move backward. As I am now able to get a nice control over my fruiting room and get pretty decent crop, then I moved to the next step : do my substrate and fruiting blocks. And later I would try my hand at making my own spawn.
In my mind, heavy lab work and petri dish would be the last of last step, but it looks like I've got it all wrong...

I understand that by quality control, you are not talking about visual control but you mean put a little piece of spawn on a petri dish to see what happen. As said, my belief being that it was still too complicated/risky at the time. And if my flowhood (or my technique) sucks, result will still be bad.

So everything points to the fact that the first step is to test my flowhood with some petri and if conclusive, I will then be able to test the spawn

"ow, that about youtube is a tricky one to state on this forum. :wink: "

Don't misunderstand what I meant, I have some much respect for all those people who, in addition to being ultra-qualified growers, take the time to make content to help young people like me. No, what I meant was that I feel I'm already taking far too many precaution some of which are probably not even necessary. You should see my hysoprop alcool consumption hahaha


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OfflineGRETO
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: GRETO]
    #28631133 - 01/22/24 02:36 PM (6 days, 2 hours ago)

Oooh just seen some of your comments between the lines regarding my other test.

The most important now is for sure petri dish all over the flowhood and sterilization temp/duration test for now
But it won't take me any longer to try out the others at the same time.

20 bag batch with
5 old spawn / old subtrate
5 old spawn / new sub
5 new spawn / old sub
5 new spawn / new sub

Before you say it, I know it won't do much good if my problem comes from the lab and all end up contaminated, but I still think I can get some knowledge out of it.


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InvisibleSoutherner
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: GRETO]
    #28631814 - 01/23/24 07:53 AM (5 days, 9 hours ago)

Man, been thinking about this issue a bit and this is a tricky one :lol: Although I see the reasoning behind working backwards when shit hits the fan it becomes a lot harder to diagnose. For instance, if you open a Petri dish in front of the hood to test and it contaminates, how do you differentiate between whether it’s the hood or technique?

https://youtube.com/@StipeCap?si=k7zfIj0XTYntgZhR

Here are some really good videos from Stipe n Cap, a TC on the shroomery. Awesome to see they’re back up. He covers pretty much everything with technique on front of a hood.

If you built your hood yourself you may also consider making a post on the psychedelic side of the forum discussing the specs. There are some pretty knowledgeable folks that can make sure everything is correctly spec’d to have sterile laminar flow. Sandman and Stipe n Cap are both really good with that side of things and they both may have some posts you can look over. That shit is way out of my league haha.


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OfflineGRETO
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: Southerner]
    #28632199 - 01/23/24 02:56 PM (5 days, 1 hour ago)

"For instance, if you open a Petri dish in front of the hood to test and it contaminates, how do you differentiate between whether it’s the hood or technique?"
>> So true every of theese test will not tell what is the problem but what is not the problem hahaha :lol: 
At least if this is concluant and I do not get contam on the petri, I will look for others sources. I still really hope that it's just a matter of too low temp and sterilization time.

Thanks for the link, I am currently watching some vid this is great.

Regarding the spec of my hood, I spent days to well understand numbers and I have checked and double ckecked that my filter and my fan are compatible.
I put a regulator and the fan running at half its power to get the requested 1240 m3/h at 150 Pa. This makes me think that for the airflow, the lighter test remains rather conclusive since it's at this wattage only that I get a smooth flame at 45°.


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InvisibleSoutherner
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: GRETO]
    #28632588 - 01/23/24 08:11 PM (4 days, 20 hours ago)

Sounds like you did the work for the flowhood so probably one of the other variables. Keep us posted on how the tests go.


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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: Southerner]
    #28632768 - 01/24/24 02:04 AM (4 days, 14 hours ago)

Can you describe in detail how you prepare your inoculant and how inoculate your spawn?


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OfflineGRETO
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: Floret]
    #28633478 - 01/24/24 03:54 PM (4 days, 1 hour ago)

Sure, here is alllll my lab process from the beginning :grin: (Maybe something will jump out at you even before I use the flow hood).

The day before inoculation day :
- I wash the workbench and different shelves with soap and I mop the floor.
- Last time, I also sprayed H2O2 solution all over the lab, ceiling, walls, floor.
I usually get the bags out of the sterilizer on inoculation day but it could happen that I get them out the night before, they sit on a shelf left to my flow hood.

Inoculation day :
Preparation :
I turn on my flowhood 1 hour before inoculation.
I put some clean clothes, wash my face, hands and arms.
I bring everything I need in the lab, tools and spawn which sit on another shelf to the right of my flowhood. I enter the lab and close the door.
Now I put mask and gloves. Good amount of iso on gloves. I spray a first time my cutter and the entire spawn bag and break the mycelium to make it easier to pour.(on the shelf not in front of the hood). Then I wipe iso all over plexi, hood edges sealer and workbench from top to bottom.

Inoculation :
I take 5 subtrate bags and stack them at the right side (why 5? I make 5 bulk bag with 1 spawn). Give them a nice iso spray.
I take the spawn bag and cutter and spray iso + wipe it down. I cut a small opening about 5-8cm wide at the corner of the spawn bag and let it sit right in the center of the flowhood.
I take a first bag, spray iso all over it and wipe where the opening is. I open it by pulling off one edge, then the other, avoiding running my hands in front of the flow (About that, Stipe and Cap show nice technique in one of his vid, where he breaks the block a little to make opening easier I did not try but I  like that :thumbup:).
I pour 1/5 bag by eye and put the spawn bag right in front of the flow.
When I pour, the subtrate bag is at less than 1cm of the flowhood.
"Pinch/close the bag" to move it to the sealer about 30cm on the left and give it a nice seal. Check if there is any leak, put on the shelf.
Again next bag : iso my gloves, take a bag, iso the bag, open it, pour spawn, pinch, seal, check, put on shelf. 5 times.
Before doing another batch of 5 bags, I wipe the workbench again with iso before taking 5 news bags, etc...

Where I see that my technique could be a problem :
-My surface disinfection sucks...:sad: (honestly I dont think so)
- Stack block is bad? I got the idea that less I leave the workbench, better it could be but even if they are at the edge, they could drag turbulence
- I do something with my hand when I open the bags that bring contam
- When I pour spawn and put it back in place, maybe I get it to high, in the danger zone where the flow is not laminar anymore.
- I recently seen something about iso needs to dry on gloves to be really effective. Do you know anything about that. It is sure that I do not let it dry


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Offlinedeadmandave
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: GRETO]
    #28633747 - 01/24/24 07:49 PM (3 days, 21 hours ago)

Your technique seems pretty solid. I don't know what others do with 24" tall hoods, mine is only 12" tall so I keep my bags on their side and inoculate down near the table. My lab also has carpet, kids, cats and doubles as a bedroom where all kinds of nonsense takes place. I try to vacuum it sometimes.



I like to think of iso alcohol as a solvent, wiping away contams rather than killing them outright. No need to let it sit for 30 seconds even tho that is probably closer to the length of time it takes to kill everything.

Not trying to be pedantic but brushing teeth is also something I have in my lab clean up routine.


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Offlinecybork
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: deadmandave]
    #28633885 - 01/24/24 10:19 PM (3 days, 18 hours ago)

nice write up.

One question that popped up regarding the spawn bag that you use: was that made with the intention of being a spawn bag, or do you use their grow bag as your spawn bag?


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OfflineGRETO
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Re: Journey of a rookie - Contam and weak colinization - Any advice are welcome [Re: cybork]
    #28634023 - 01/25/24 05:31 AM (3 days, 11 hours ago)

I had brush my teeth in the process. Would be funny if it come from that but let's get every chance on my side.

They are grain bag made for spawn use :thumbup:

Yesterday, I did the first batch with longer steam time BUT I wasn't able to get higher temp. The best I can get is 93° C and at this temp, the PID is running full power during the 20h. Not even close to 95° I have to check the calibration of my prob. If it usually give me 92° but in reality it is only 85° maybe the problem come from there. The prob is placed at the higher part of my sterilizer just under the hole where the steam come out.

Spawn from another producer is also on its way.

I found a thread regarding causes of weak colonization and tric. I think it was from Stipe n Cap. I can't find it anymore...
Both can come from a bacterial contam which weaken the mycélium and so give space for others contam to thrive. From what I remember from the thread symptom match very well.


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