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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Help review my process
#21689876 - 05/17/15 09:32 AM (8 years, 8 months ago) |
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So I'm doing bags and wanna run through my process for flaws if you please.
Grain prep is done as usual and is dry enough going in the bags that I don't have any bacteria problems, I've only had mold issues.
I load 4 qts per bag and insert a sleeve (does it matter the size of the bag and sleeve?) Fold accordian style 3 times and place in PC.
I PC my bags for 3 1/2 hours and it seems sufficient for bacteria and mold is easier to kill. I am now letting my PC and bags cool totally on their own, before I would take them out hot in front of my hood.
After PC I place my PC beside my hood, open, and take out a bag. I wipe down what I can with iso, my gloved and sleeved hands included and pull the sleeve slowly out of the bag and discard.
Then I wipe down what I couldn't with the sleeve in, flame sterilize my scalpel and iso my hands. Then I proceed to open my petri and cut a wedge and place quickly into bag and seal with impulse sealer that's in front of my hood as well.
I have an issue however that I run out of room with a bag, petri, and impulse sealer so I have my torch right beside my hood and have to heat my scalpel there and bring back into sterile air. I always thought this was a non issue since that's pretty much how its done in a sab but I'm trying to find what little thing I'm missing that's still causing me mold.
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Help review my process [Re: Ghatti]
#21689883 - 05/17/15 09:36 AM (8 years, 8 months ago) |
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If I can't get this resolved then if I still wanna try to grow a mushroom my only other option it seems is build a small positive pressure room to have clean air and have my hood in there but that's super extreme and although I do love this hobby I highly doubt I'll spend the extra 300 to do it lol
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Re: Help review my process [Re: Ghatti]
#21689905 - 05/17/15 09:47 AM (8 years, 8 months ago) |
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When are you noticing mold?
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Help review my process [Re: bodhisatta]
#21689934 - 05/17/15 09:56 AM (8 years, 8 months ago) |
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Usually as the first flush comes in or right before. Its usually 50/50 if I can squeeze a flush out first. With that time frame I'm assuming its getting in during inoculation but I was just informed of the apparent importance of using sleeves so maybe they were sucking in ambient mold spores as they cooled?
The bags always look fine but if I leave them to sit for 2 weeks ish after full colonization I can see the mold colonies start to show invitro
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Help review my process [Re: Ghatti]
#21689967 - 05/17/15 10:05 AM (8 years, 8 months ago) |
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I wish I could trust myself to make a clean lc so I can just seal bags immediately and inject. But with not being able to make 100% clean spawn its not an option obviously.
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tripdawg420
low life with no life



Registered: 02/02/09
Posts: 7,071
Loc: illinois
Last seen: 9 hours, 29 minutes
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Re: Help review my process [Re: Ghatti]
#21689997 - 05/17/15 10:15 AM (8 years, 8 months ago) |
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innoc a qt of spawn with the wedge and then use the spawn to innoc the grain bag
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mushpunx
Fungus Punk



Registered: 04/20/14
Posts: 13,394
Last seen: 11 days, 9 hours
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Re: Help review my process [Re: Ghatti]
#21689999 - 05/17/15 10:15 AM (8 years, 8 months ago) |
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I inoculate a pint or quart jar with my wedge first (I've come to prefer the pint jars) and then G2G into spawn bag.
Colonization is pretty lighting fast this way man, especially if you are innoculating 4qt bags I would knock up a master jar first!
Also man I dunno about letting the bags sit for 2 weeks, thats a long time to leave colonized spawn sitting around man. If I have suspicions, I massage up the bag and watch how it recovers but usually I don't do this... only if I'm worried.
I definitely have my share of mold dude haha. What has been helping me (at the sugguestion of some awesome memebers) was switching to pints for masters and squirting ISO up under the rim as I wipe them down, and just washing everything better with ISO in general
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 Amateur Mycologists United AMU Q&A
Edited by mushpunx (05/17/15 10:16 AM)
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tripdawg420
low life with no life



Registered: 02/02/09
Posts: 7,071
Loc: illinois
Last seen: 9 hours, 29 minutes
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Re: Help review my process [Re: mushpunx]
#21690006 - 05/17/15 10:17 AM (8 years, 8 months ago) |
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ive use pints for master so many times they work great in small sab
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mushpunx
Fungus Punk



Registered: 04/20/14
Posts: 13,394
Last seen: 11 days, 9 hours
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You beat me to it dude hahah!
Yea pint masters are awesome they colonize faster and the spawn bags only take a couple extra days usually. I fill them with 4.5qts so with the pint it makes 5.
The pint jars are easier to handle too during G2G I think.
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 Amateur Mycologists United AMU Q&A
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Help review my process [Re: mushpunx]
#21690016 - 05/17/15 10:20 AM (8 years, 8 months ago) |
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I let the bags sit to confirm it was indeed the spawn and not something in tub prep. If you recall I was having issues with jars as well.
Those too I was taking out hot and I believe the slightly imperfect seal was sucking stuff in.
I will do a control bag today, well 2. One I will open for a sec and then seal, the other I will inoculate with a blank wedge. This should tell me what part of the process is flawed.
I'm really just hoping that letting them cool totally in the PC and using sleeves will solve the issue.
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Help review my process [Re: Ghatti]
#21690024 - 05/17/15 10:23 AM (8 years, 8 months ago) |
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Obviously my ambient spore load is through the roof with so many failures. I will make a few pint jars though and use my lids gasket down as I was using them gasket up and tested a few by filling with water and shaking and I could get so me liquid to seep out. If water could get out I would imagine mold could get sucked in.
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Help review my process [Re: Ghatti]
#21690042 - 05/17/15 10:33 AM (8 years, 8 months ago) |
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I really do think/hope that this will solve the issue. My agar work is almost always perfectly clean so that don't think its my sterile technique.
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mushpunx
Fungus Punk



Registered: 04/20/14
Posts: 13,394
Last seen: 11 days, 9 hours
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Re: Help review my process [Re: Ghatti]
#21690127 - 05/17/15 11:00 AM (8 years, 8 months ago) |
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Quote:
Ghatti said: I really do think/hope that this will solve the issue. My agar work is almost always perfectly clean so that don't think its my sterile technique.
Yea dude I get ya here, my agar work is pristine but then I get mold before first flush. I think mine gets in during G2G most of the time
I just started using spawn bags and had to re sterilize a few times because of that My AA is usually still warm the next day after a cycle the night before! With jars I can take out as soon as the PC can be opened but with bags I lose my seal unless I let the PC sit overnight. So maybe youre right about the seal being imperfect
--------------------
 Amateur Mycologists United AMU Q&A
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Help review my process [Re: mushpunx]
#21690131 - 05/17/15 11:02 AM (8 years, 8 months ago) |
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Ionno. I figured if I let em cool in front of my hood that it'd be fine but apparently not
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hamloaf
Loaf of Fam.


Registered: 12/23/09
Posts: 20,192
Loc: Oklahoma.
Last seen: 1 year, 8 months
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Re: Help review my process [Re: Ghatti]
#21690295 - 05/17/15 11:57 AM (8 years, 8 months ago) |
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When bags are folded accordion style with tyvek wrist sleeve for sterilization, before the substrates removal from the PC, it's a requirement that the substrates are allowed to cool to room temps on their own, so that it's ensured that the seal will fully form between the gussets of the spawn bag, and the tyvek wrist sleeve. Inoculating spawn bags with agar wedge is an awkward practice. Done it before, just to spite the fact that I read that inoculating spawn bags with agar wedges is awkward. Was indeed discovered that it is easiest to inoculate bags with fully colonized jars of grain spawn, LC, or the transferring of colonized grains from one bag to another (bag2bag g2g).
In this lab, the scalpel is flame sterilized just outside of the direct downstream of the flowhood. Mold and bacteria are kept from being cross contaminated via the scalpel handle by, wiping scalpel handle & blade thoroughly with an alcohol soaked paper towel IMMEDIATELY before flame sterilization. Once scalpel is flame sterilized, scalpel is brought directly into downstream of flow hood with right hand. Left hand reapplies another dose of rubbing alcohol into the left hand, left hand is then brought into direct downstream, and hands are re-lathered with alcohol before sterile cultures in a petri dish are even thought about being uncapped.
It is also IMPERATIVE that all surfaces of the work area are wiped thoroughly with alcohol before & between tasks, as well as, all media vessels, and tools that are needed for task are wiped thoroughly with an alcohol soaked paper towel before being placed into the direct down stream of the flowhood. It's also important that the flowhood be allowed to run for 1-2 hours in the lab area to scrub the air of airborne contaminates.
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Ghatti
Totally not a Federal Agent

Registered: 06/23/14
Posts: 1,733
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Re: Help review my process [Re: hamloaf]
#21690378 - 05/17/15 12:26 PM (8 years, 8 months ago) |
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I don't run my hood for 2 hours but I do turn it on, clean it,and let it run 30 mins or so. I was unaware that the bags would suck air in and that's why we use tyvek. I always thought the inside of a PC stayed sterile untile opened but apparently I was misinformed.
Agar to bags is awkward but it is the safest method for me atm since I am having issues making spawn.
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