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MrPeanutButta
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Registered: 04/29/10
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Last seen: 5 years, 7 months
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Yeast After G2G
#23413511 - 07/05/16 11:46 AM (7 years, 6 months ago) |
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Hello All,
I had two fully colonized rye bags which have been spawned to a manure based compost. They were given one week to consolidate (Maybe not quite enough time) and then placed into fruiting conditions. They seem to be pretty healthy, but I am kind of shocked that they are and this is why...
During the spawn to bulk process I also performed a g2g of some of the rye from the bags into prepped and sterilized WBS quart jars. Admittedly I rushed and did not follow sterile technique like I should have.
I completed the process in a bathroom that had been cleaned up and down, with the AC off, sprayed with Lysol everywhere. Then I would barely open a WBS jar and transfer a bit of the rye over (Used gloved hands and sprayed with Lysol between each one...this is most likely where I messed up and should have used a flamed spoon at minimum) and resealed then a small shake.
Long story short, the trays are looking good, but every single WBS jar has been contaminated with yeast. How does that happen? Does that mean the jars were improperly prepped to begin with? How do I end up with yeast in my WBS, but not in the trays? Is it because the trays are more contam resistent due to more spawn and so it beat it out?
I am just trying to get back up to speed with this stuff, and need to follow everything step by step like I used to and everything will be fine. I have been fine in the past being a bit lazy, but not this time haha.
Edit: Or even worse, do I have yeast in my trays that I just cannot see and they won't end up producing any fruit? Ughh...I need to step my game up. I just used to feel like it all worked without having to worry now I have a problem every step of the way.
Last thing I will add. Someone else prepped the WBS jars. They did not soak the bird seed. They just simmered and then pc'd. The thing is though that the jars sat for 2 weeks without contaminating, so I would think it happened during my g2g due to no flamed spoon.
Edited by MrPeanutButta (07/05/16 11:56 AM)
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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Most yeast comes from cultivator error. In your case it's from doing open air G2G in a bathroom. Use an SAB next time, no spoon, just from the doner jar to receiving jars all in the box.
Open air = fail. I don't give a fuck how much lysol you spray. In fact lysol blows.
Edit, g2g from bag to jar is backward and I would never recommend that unless you were using a large flowhood. You need to start using some proven methods.
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MrPeanutButta
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Registered: 04/29/10
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Last seen: 5 years, 7 months
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Thank you for your feedback. I have used proven methods in the past and have had success. I have also not used those methods and had success as well. I will be using a still air box next time.
As for going from bag to jar, I had to do it that way. I inoculated the bags and then received prepped WBS jars from a friend who didn't want to use them. I had no choice other than to go from bag to jar.
This next round I am going multispore to bags and a few jars which will become grain liquid cultures. That way I won't be doing any transfers that require a jar to be opened. I am also going to inoculate a few WBS jars that could be used for g2g with my SAB as well.
This was just an initial albeit hasty push back into the process. I had near 100% success rates in the past and I got sloppy. Now I am going to tighten it back up because I got burned.
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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You always have a choice. You can do things correctly or you can not do them. It's simple.
Think of it like this, you could simply toss the WBS and not use it. That would be a waste of WBS but you would have saved the bulk material that was then wasted on a failed grow. Could have also used the doner grain assuming it was clean for a successful grow. You would have been out some WBS but, you would have not wasted bulk material, the time to prep it, and still have some fruits in hand.
Sounds like your still on track with risky plans. MS solution to bags and ms to make GLC masters are both pretty big risks to take in order to get around cutting 2 armholes in a plastic box. Your call.
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MrPeanutButta
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Registered: 04/29/10
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Last seen: 5 years, 7 months
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I am going to be using a still air box from here on out. The trays still look good to me, so Im hoping that goes okay.
I didn't mean I didn't have the choice to completely hold off on the WBS. I am saying that due to the order of events that the jars came after the bags.
When you spawn to bulk do you use a still air box? I haven't in the past with zero problems, but now I am going to be super paranoid about contams. I mean I appreciate your feedback, but no need to drive it home...I understand I was cutting corners. I had never had to pay for it before now I am. All that equals is that I will proceed as I should have in the first place.
Edit: Are you saying MS to bags and for GLC masters are risky from a success standpoint? I can see that... Would you suggest MS to jar and then create GLC using sterile water? Then after GLC is established inoculate bags? I'm following you, but i've always read that is how you create a GLC. Or are you saying GLC's are risky/suck period?
Edited by MrPeanutButta (07/05/16 01:17 PM)
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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Spawning can be done in open air with no problems. The grain is already colonized by that point. I personally dislike spores to grain. For me spores go on agar where the culture is confirmed to be clean. Once I am sure it's clean, it can then go to grain. But I don't grow with spores much anyways. Once you get some good clones or isolates on agar you just use those and get predictable results.
Like grain, agar is inoculated and transferred in the SAB always. Only when you spawn to bulk is it safe to expand in open air.
GLC is risky because even well sterilized grain has some latent bacteria in it. Last GLC I made tested bacterial on agar. I still used it and it did okay, but it's bad practice. Bacteria in spawn is a mold vector and sooner or later will catch up with you. If I was to make a GLC I would use several agar wedges to inoculate the grain and colonize it as fast as possible.
MS solution to grain can work. Best way to do that is to just use a few drops to several masters. Then choose the fastest and best looking one and g2g it. Spawn the others. This is not foolproof but will greatly minimize contams. But agar is still king because you can choose not only clean growth, but limit the genetics to faster healthier colonies.
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Spawning to bulk isn't a sterile process so no SAB. Fruiting isn't sterile either.
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MrPeanutButta
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I know....I need to start using agar. It is something I have always resisted because it was an unknown and I didn't have the SAB, but I agree that it would be worth it and minimize mistakes and therefore waste.
For this round I will have to go with GLC to the bags because the bags are already prepped and sealed. I just need to slow down and do this the right way. Most likely I will try to get a clone if these trays fruit and transfer to agar since I will have a SAB.
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MrPeanutButta
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That is how I have always treated it in the past. I guess what scares me is that I have yeast in the jars and attempted the open air g2g at the same time as spawning to bulk.
The bulk is pasteurized manure based compost. Even though spawning to bulk is fine open air I was afraid that somehow a contaminant could have found the manure/compost and caused issues. That isn't how it is looking though...it looks very healthy to me.
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Manure compost gets pasteurization because contaminants will find their way in during spawning and you don't want them to cause problems
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MrPeanutButta
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Last question for you. What would you use in place of Lysol when prepping a SAB?
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Psychedel.EXE
AKA Old Uncle Nutty



Registered: 07/04/16
Posts: 211
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spacechildo
proletarians rise up


Registered: 01/24/13
Posts: 19,243
Loc: Babylon
Last seen: 6 years, 4 months
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ugh, bleach  water with a drop of dish soap for me!
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Quote:
MrPeanutButta said: Last question for you. What would you use in place of Lysol when prepping a SAB?
just water in a spray bottle with a single drop of dish soap. you don't need to sanitize or try to sterilize the inside of a SAB that's now how they work.
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MrPeanutButta
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I am asking about sterilizing foreign objects that you place into the SAB. I know the inside of the SAB doesn't need much. I'm referring to a jar for example that has been incubating and exposed to contams.
Edit: Alright I checked out that tek and did some reading and I feel comfortable with everything except for the concept of flaming outside of the SAB and then going back in to work. It looks like that doesn't cause any issues if everything else is good to go. As far as the objects inside of the box it looks like I just need to add the extra step of wiping them down with iso alcohol or bleach water before they go into the SAB.
Edited by MrPeanutButta (07/05/16 07:18 PM)
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MrPeanutButta
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Thanks for the help everyone!
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spacechildo
proletarians rise up


Registered: 01/24/13
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Loc: Babylon
Last seen: 6 years, 4 months
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Quote:
MrPeanutButta said: I am asking about sterilizing foreign objects that you place into the SAB. I know the inside of the SAB doesn't need much. I'm referring to a jar for example that has been incubating and exposed to contams.
Edit: Alright I checked out that tek and did some reading and I feel comfortable with everything except for the concept of flaming outside of the SAB and then going back in to work. It looks like that doesn't cause any issues if everything else is good to go. As far as the objects inside of the box it looks like I just need to add the extra step of wiping them down with iso alcohol or bleach water before they go into the SAB.
I wipe master jars with 70% ISO, receiving jars usually goes straight from the PC to the SAB. anything that can be flamed red hot, flame it. flaming outside the sab works, the tools are still hot. dont hover hands over open work and move smoothly. pretty much it.
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MrPeanutButta
Stranger

Registered: 04/29/10
Posts: 643
Last seen: 5 years, 7 months
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Quote:
spacechildo said:
Quote:
MrPeanutButta said: I am asking about sterilizing foreign objects that you place into the SAB. I know the inside of the SAB doesn't need much. I'm referring to a jar for example that has been incubating and exposed to contams.
Edit: Alright I checked out that tek and did some reading and I feel comfortable with everything except for the concept of flaming outside of the SAB and then going back in to work. It looks like that doesn't cause any issues if everything else is good to go. As far as the objects inside of the box it looks like I just need to add the extra step of wiping them down with iso alcohol or bleach water before they go into the SAB.
I wipe master jars with 70% ISO, receiving jars usually goes straight from the PC to the SAB. anything that can be flamed red hot, flame it. flaming outside the sab works, the tools are still hot. dont hover hands over open work and move smoothly. pretty much it.
Thanks for the advice. When I think back to my previous successful grows, I must admit that I was quite a bit more thorough. This experience and this thread among a few others have really helped me to snap out of it. I didn't get into this to cut corners...I got into this because I like a challenge and I like to be detail oriented. I don't need to be obsessive, but I at least need to follow sterile tech.
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MrPeanutButta
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Last seen: 5 years, 7 months
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Well, my trays are still looking good but no fruits yet (9 days so far). I did not give them enough time to fully colonize the bulk. Like I said they look good though, so hopefully i'll see something over the next week. I have definitely had mono tubs that took this long a few times.
I have prepared everything I need to push forward including my SAB. I have some spore syringes as well, but I am actually thinking about trying to clone instead if these trays come through. My plan is to use Cloneufc's cloning tek to agar. Then I will complete a few more transfers to further isolate. Next I will transfer to grain (either WBS or Popcorn). I will have a few grain jars for g2g and a glc.
The glc will be used to inoculate my rye bags. I know g2g would be preferable, but the rye bags are already prepped and sealed. My other option would be to start from MS again. I think the main thing I want to figure out is how to keep masters for long periods of time. This is not supposed to be big...it's more experimental and I like variety.
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Supalemonhaze
Spore syringe hater.



Registered: 10/02/15
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You should put cultures inside slants if you want to keep them for a long time. Master grain jars can be kept for a couple of weeks past 100% colonization, probably even more but it's always best to use them ASAP.
You should try LI rather than GLC, it's safer and definitely cleaner. Grains always have some bacterial endospores present after PCing and by the time the mycelium colonizes the jar, they would have already recovered or close to it.
Even with G2Gs, I always notice signs of bacteria and I PC my grains pretty well.
Wedges would be the best option, now that you have decided to start using agar, you should give those a go. They are a bit slow compared to LI, LC or g2g but they make up for that with clean inoculant. If you start inoculating jars with a routine (say, once a week), waiting for wedges to colonize your grains is not a problem since you always have something going.
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