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Offlinegreenladel
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some questions to improve my sterile practice
    #27039875 - 11/15/20 04:51 AM (11 days, 19 hours ago)

i have attempted to colonize two batches of BRF cakes now totaling 16 jars and have had a contamination rate of 100%, so it is clear that something is going very wrong.
after doing more searching, watching videos, etc i have made a list of some things i want to do different next time, but i have some questions regarding some of the points i have noticed.
i think the major flaws are with my dry verm layer. on my first run it slipped down the sides a lot, so the second time i packed the BRF mix down a bit by tapping the jar on the table, but the second time it still slipped a little and it seemed to slow any growth considerably. next time i am going to grind some verm down to a finer size to be used on my dry layer and i am going to only pack the top edge of the cake to try to stop the dry barrier slipping down the sides.

there are a couple of other things i am thinking about too, but i have some questions about them.

1.
the holes in my lids are quite big, allowing for some movement of the syringe when it is in the hole. the holes are not huge, just not as small as they could be. i dont think this is a huge problem in itself as long as the dry verm layer is okay, but there is obviously a slightly higher chance of contamination. is it a waste of time and money for me to buy more lids and make smaller holes?

2.
i was thinking about using micropore tape or masking tape as well as my dry verm layer just to be on the safe side, but i have also read it can slow or even stall growth. if i try taping the holes and it stalls growth will the mycelium continue to grow after i remove the tape again?

3.
i saw a video where this guy stored his jars in an opaque plastic storage tub during colonization. it seemed to work okay for him, but wouldnt this slow or stall growth due to poor air exchange?
if i can do this then i would use a clear box to allow ambient light in and it could help me a lot as i dont have many 'out of harms way' places to store the jars.
i guess this would be pointless to combine with micropore tape?

4.
i am planning to put rubber bands around the foil during sterilization in the PC. is it possible to put this too tight? is the jar likely to break if there is not enough of a gap to allow equalization of pressure?


thank you very much for the help :heart:
i will get there eventually!


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Offlinemad_dingo
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Re: some questions to improve my sterile practice [Re: greenladel]
    #27039903 - 11/15/20 05:34 AM (11 days, 18 hours ago)

Quote:

greenladel said:
i have attempted to colonize two batches of BRF cakes now totaling 16 jars and have had a contamination rate of 100%, so it is clear that something is going very wrong.





If you fail that consistently i would assume that your spore syringe is  either very dirty, or you forget to flame the needle and wipe the lid with iso when inoculating. Also possible your jars my be way to wet, so your dry verm layer isn't dry for long.

Quote:


after doing more searching, watching videos, etc i have made a list of some things i want to do different next time, but i have some questions regarding some of the points i have noticed.
i think the major flaws are with my dry verm layer. on my first run it slipped down the sides a lot, so the second time i packed the BRF mix down a bit by tapping the jar on the table, but the second time it still slipped a little and it seemed to slow any growth considerably. next time i am going to grind some verm down to a finer size to be used on my dry layer and i am going to only pack the top edge of the cake to try to stop the dry barrier slipping down the sides.





Packing the substrate by tapping the jar on a surface is totally fine. In fact pack it a bit more if the dry verm still slips into the wet subtrate. You do fill the jars to the rim with dry verm, right? The lid should press down on it.

Quote:


there are a couple of other things i am thinking about too, but i have some questions about them.

1.
the holes in my lids are quite big, allowing for some movement of the syringe when it is in the hole. the holes are not huge, just not as small as they could be. i dont think this is a huge problem in itself as long as the dry verm layer is okay, but there is obviously a slightly higher chance of contamination. is it a waste of time and money for me to buy more lids and make smaller holes?





Your hole size should not be a problem.

Quote:


2.
i was thinking about using micropore tape or masking tape as well as my dry verm layer just to be on the safe side, but i have also read it can slow or even stall growth. if i try taping the holes and it stalls growth will the mycelium continue to grow after i remove the tape again?





I sometimes used micropore tap over the holes. If you use it, only apply after inoculation. I tried once to run the tape through the PC and found it so hard to remove afterwards that i had to inject through it. Incidently that was the one batch that was contaminated. If your verm layer is intact the tape is redundant anyway.

No masking tape though, your mycel might suffocate. Once it's dead it's dead and not likely to recover.

Quote:


3.
i saw a video where this guy stored his jars in an opaque plastic storage tub during colonization. it seemed to work okay for him, but wouldnt this slow or stall growth due to poor air exchange?
if i can do this then i would use a clear box to allow ambient light in and it could help me a lot as i dont have many 'out of harms way' places to store the jars.





Unless your jars are colonizing in a literal barn, there is no point in putting them in another box.


Quote:


4.
i am planning to put rubber bands around the foil during sterilization in the PC. is it possible to put this too tight? is the jar likely to break if there is not enough of a gap to allow equalization of pressure?





No point of doing that, but should not be a problem either.

Good luck with your grows and consider learning about agar.


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OfflineRoscoeReturns
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Re: some questions to improve my sterile practice [Re: mad_dingo]
    #27040007 - 11/15/20 09:04 AM (11 days, 15 hours ago)

Your dry verm layer will almost always slide down the side to some extent. As the myc grows, the cake shrinks as the water is used up, then the dry verm falls down the side to fill in the gap. Likely none of what you listed is your contam issue. Give us some more details on your procedure. Are you wiping the rim of the jar clean after loading BRF before adding dry verm? How are you loading your PC? What pressure do you run you PC, and for how long? Do you have pics of you contams?


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Offlinegreenladel
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Re: some questions to improve my sterile practice [Re: mad_dingo]
    #27041569 - 11/16/20 06:10 AM (10 days, 18 hours ago)

Quote:

mad_dingo said:
If you fail that consistently i would assume that your spore syringe is  either very dirty, or you forget to flame the needle and wipe the lid with iso when inoculating. Also possible your jars my be way to wet, so your dry verm layer isn't dry for long.





thank you for the reply
i dont think the syringe is the problem. they came from a vendor with a lot of respect around the internet, including here. i guess it could be them, but i think it is highly unlikely.
i do flame sterilize my needle, however i do not wipe the top of the jars with iso. i dont think that step was in the tek i followed, but does that not count as working backwards as far as clean practice goes? i sterilize my jars in the pressure cooker then leave them sealed in there until the next day when i am ready to inoculate, then i place them in my SAB and leave the SAB to settle before removing the foil. shouldnt the lid be sterile at that point?

regardless, i will do that next time. obviously it works fine for you so it is already better than what i am doing :smile:

Quote:


Packing the substrate by tapping the jar on a surface is totally fine. In fact pack it a bit more if the dry verm still slips into the wet subtrate. You do fill the jars to the rim with dry verm, right? The lid should press down on it.





ive got to be honest, as embarrassing as this is, i packed down the BRF cake by tapping the jar, but didnt think to do the same with the dry layer added :facepalm: hahah
as far as filling it goes, i have been pouring the dry layer on top so it creates a mound, then running a straight knife across the top of the jar to level it. when i put the lid on it touches the dry verm, but i wouldnt say it is pressing down on it very hard. next time i will tap the dry verm down too and i will leave a slight mound so the lid presses it down. i was also thinking about grinding some verm down to a finer grade to use as my barrier.

Quote:


I sometimes used micropore tap over the holes. If you use it, only apply after inoculation. I tried once to run the tape through the PC and found it so hard to remove afterwards that i had to inject through it. Incidently that was the one batch that was contaminated. If your verm layer is intact the tape is redundant anyway.

No masking tape though, your mycel might suffocate. Once it's dead it's dead and not likely to recover.





thank you. i will use micropore tape next time just to be sure. i did read somewhere here about somebody who used only micropore tape, no dry verm layer, but i think i will use both for now and once i am happy then i will experiment with removing the tape and keep the verm layer

Quote:


Unless your jars are colonizing in a literal barn, there is no point in putting them in another box.





thanks. i wont bother doing that, particularly if i can use micropore tape, it seems like an unnecessary extra precaution.

Quote:


No point of doing that, but should not be a problem either.





thanks. i will try it. i noticed a couple of the jars across both batches had some droplets of water under the foil when i removed it. i am obviously using too much water. i will use rubber bands for now while i learn my PC (never used one before picking up this hobby).

Quote:


Good luck with your grows and consider learning about agar.








thanks, funny you say about agar. while digging for info on good inoculation practice i stumbled across a video about agar and it definitely grabbed my attention. i think the best thing for me would be to nail the tek i am doing (BRF cakes grated into shoebox) first, then start looking into agar once i am comfortable with what i am doing. in the mean time i will continue to read about it and understand what it is all about.

thank you very much for the help :heart:


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Offlinegreenladel
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Re: some questions to improve my sterile practice [Re: RoscoeReturns]
    #27041580 - 11/16/20 06:28 AM (10 days, 17 hours ago)

Quote:

RoscoeReturns said:
Your dry verm layer will almost always slide down the side to some extent. As the myc grows, the cake shrinks as the water is used up, then the dry verm falls down the side to fill in the gap. Likely none of what you listed is your contam issue. Give us some more details on your procedure. Are you wiping the rim of the jar clean after loading BRF before adding dry verm? How are you loading your PC? What pressure do you run you PC, and for how long? Do you have pics of you contams?




thank you for the reply.
my dry verm layer is falling down during sterilization in the pressure cooker. the second batch was much better because i packed down the BRF cake before adding the verm barrier, but still some of it slid down. hopefully my changes next time (tap the jar down with the dry verm included :facepalm: and use a finer verm) will improve that even more.

i wiped down the rim of most of my jars before adding the dry verm, but i did realise after finishing some of them that i had forgotten to wipe it. this only happened about 4 times though out of the 16 jars, so for the rest i did do that and i did it carefully too.

i am loading the pressure cooker with a home made trivet about an inch high, then filling it with water just past the top of the trivet and loading 4 jars in stood upright.
as i mentioned above, i think i am filling it with too much water as some of my jars had water droplets under the foil. next time i wont even let the water touch the bottom of the jars and i will use a rubber band to be safe.
the PC is a 10psi one. i experimented with times from 90 minutes to 160 minutes. i figured that if i can do it in a pot with a tight fitting lid in 90 mins then that should be good for a PC even as low as 10psi.

here are some pics of my first jars. the contamination was the same in all 12 of them. but the contamination in the last 4 (with a better verm layer) was different. it was much closer to the top of the jar. i will get pics up of them asap.
note: the dry verm layer in these pics looks very thick, but it is just where it slid down. i actually filled it to the lip that is just underneath the lid.




edit:
here is a picture of the contamination in the last 4 jars. only one jar, but the contamination is the same in all 4. thanks




thank you very much for the help :heart:


Edited by greenladel (11/16/20 06:44 AM)


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OfflineRoscoeReturns
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Re: some questions to improve my sterile practice [Re: greenladel]
    #27041593 - 11/16/20 06:49 AM (10 days, 17 hours ago)

The dry verm falling down the sides in your pics is pretty normal. Happens like that to me too. Also I get drops of water under the foil, your rubber bands won’t stop it, nor does it cause contams. In the PC the moisture in the BRF mix turns to steam too. Some will get out the inoculation holes and condense under the foil, totally normal. Have all your jars been nocced up with the same syringe? It doesn’t matter how well respected a vendor is, spores are collected from fruit that form in open air. They are inherently dirty. Some dirtier than others. You can try all those things you listed, but none of them are necessary, and likely won’t solve your problem. Try a new syringe, or streak your syringe to agar and clean up your culture.


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Offlinekarri0n
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Re: some questions to improve my sterile practice [Re: greenladel]
    #27042435 - 11/16/20 05:39 PM (10 days, 6 hours ago)

Quote:

greenladel said:


thanks, funny you say about agar. while digging for info on good inoculation practice i stumbled across a video about agar and it definitely grabbed my attention. i think the best thing for me would be to nail the tek i am doing (BRF cakes grated into shoebox) first, then start looking into agar once i am comfortable with what i am doing. in the mean time i will continue to read about it and understand what it is all about.

thank you very much for the help :heart:




If you have spore solution left, learn agar now while your jars colonize. You've got almost a month.


--------------------
Anything written above is just as likely to be accurate as it is to be corrected by someone smarter than me two posts from now.

My Journal / Grow Log


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Offlinegreenladel
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Re: some questions to improve my sterile practice [Re: RoscoeReturns]
    #27046739 - 11/19/20 06:39 AM (7 days, 17 hours ago)

Quote:

RoscoeReturns said:
The dry verm falling down the sides in your pics is pretty normal. Happens like that to me too. Also I get drops of water under the foil, your rubber bands won’t stop it, nor does it cause contams. In the PC the moisture in the BRF mix turns to steam too. Some will get out the inoculation holes and condense under the foil, totally normal. Have all your jars been nocced up with the same syringe? It doesn’t matter how well respected a vendor is, spores are collected from fruit that form in open air. They are inherently dirty. Some dirtier than others. You can try all those things you listed, but none of them are necessary, and likely won’t solve your problem. Try a new syringe, or streak your syringe to agar and clean up your culture.




the first 12 jars were from one syringe and the last 4 were a completely different syrnge and new, sealed needle. the only similarity was the vendor.
i plan to try again when i get paid, and i will keep trying until ive got some healthy fruits! but i am interested in agar and will start to learn more about it.
what do you mean by "streak your syringe to agar"?

thank you for the help :heart:


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Offlinegreenladel
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Re: some questions to improve my sterile practice [Re: karri0n]
    #27046743 - 11/19/20 06:43 AM (7 days, 17 hours ago)

Quote:

karri0n said:
If you have spore solution left, learn agar now while your jars colonize. You've got almost a month.




i have none of those spores left, but when i get more i will save some.

my only concern is cost. what would i need to get started with agar? i guess some agar and some petri dishes, but is there anything else?

thanks for the help :heart:


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OfflineRoscoeReturns
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Re: some questions to improve my sterile practice [Re: greenladel]
    #27046767 - 11/19/20 07:55 AM (7 days, 16 hours ago)

Quote:

greenladel said:

what do you mean by "streak your syringe to agar"?

thank you for the help :heart:




Basically putting spores from your syringe onto agar, and then transferring clean mycelium to a new plate. It’s called streaking because you would normally use an inoculation loop and rub the spores onto your plate in a zig zag motion. Like this:



That spreads the spores out, which allows you to pick a clean colony. You can just put a small drop of spore solution in the center of your plate and tilt to make it run across. Or some just put a drop in the middle and get a big pile of colonies growing on top of one another. It all can work.

Something you might try to narrow down the source of contamination is to hold back a jar next time you try. Prep all jars as normal, but keep one aside, do not inoculate it. If all your jars go bad, then you’re not sterilizing properly. If all except the one you didn’t inoculate go bad, then the problem is in the syringe or your handling/inoculating.

Also stop wasting cash on expensive spore syringes. If you want to try agar to grain to bulk, and skip all this, I’d be happy to send you a spore print. You can grow for a lifetime off one print.


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Offlinekarri0n
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Re: some questions to improve my sterile practice [Re: greenladel]
    #27047060 - 11/19/20 12:19 PM (7 days, 11 hours ago)

Quote:

greenladel said:
Quote:

karri0n said:
If you have spore solution left, learn agar now while your jars colonize. You've got almost a month.




i have none of those spores left, but when i get more i will save some.

my only concern is cost. what would i need to get started with agar? i guess some agar and some petri dishes, but is there anything else?

thanks for the help :heart:




You could do agar without a pressure cooker if you order pre-made plates. Without a pressure cooker, you can't sterilize grain or make your own agar plates, though. You could buy a bag of pre-sterilized grain if you wanted to try this out and decide to buy a pressure cooker later.

You could also inoculate pf cakes with agar if you really wanted to go the agar route but don't want to get a pressure cooker.

I got started with a 40 dollar pressure cooker. It's not ideal since you can't fit quart jars in it, but it's working for my purposes for now. I will have 23 quart come Christmastime, or if I decide to open it sooner.


--------------------
Anything written above is just as likely to be accurate as it is to be corrected by someone smarter than me two posts from now.

My Journal / Grow Log


Edited by karri0n (11/19/20 12:21 PM)


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Offlineblack strat
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Re: some questions to improve my sterile practice [Re: karri0n]
    #27047573 - 11/19/20 05:36 PM (7 days, 6 hours ago)

Quote:

karri0n said:

I got started with a 40 dollar pressure cooker. It's not ideal since you can't fit quart jars in it, but it's working for my purposes for now. I will have 23 quart come Christmastime, or if I decide to open it sooner.




yo dude, jus read your grow log, you haven't updated in a month, and the last pic was ur tub looking well on its way.... what's happening with it now man?


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Offlinekarri0n
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Re: some questions to improve my sterile practice [Re: black strat]
    #27047945 - 11/19/20 09:19 PM (7 days, 2 hours ago)

Yeah heh, I got a bit paranoid when it started fruiting and stopped taking pics. I have to get a proper update in there soon.

I wouldn't call it wild success but acceptable for a first shot at it with PF tek.

I've pulled probably 12g dry off of the two shoeboxes. Been eating some fresh and drying some, gave some to a buddy, so I don't have a super accurate count.

Nothing to write home about, but they were made with a half pint and two half pints, when a good shoebox tek calls for 1 quart and should give you 30g for one shoebox. So the math still kinda works out.

I wouldn't call any of them "flushes" though. I've been pulling between five and fifteen small fruits off every day for like a couple weeks. It's slowing down quite a bit now though.

I screwed around with it a lot and didn't use a proper substrate height for the shoeboxes. Tons of aborts, and the fruits are small. I don't have a handle on proper moisture levels. There's likely bacteria as well, not sure though.

As of now I've also made a bunch ofn oat grain spawn which is colonized with golden teacher,

I spawned a pint of golden teacher spawn to a tupperware container which should fruit soon,

I've got a fully colonized golden teacher LC ready to go to grain,

I've got three PF redspore BRF cakes colonizing,


I've got Pf redspore spores colonizing agar,


And i've made grain spawn, sawdust spawn, and hoping for successful LC's of Chicken of the Woods and Lion's Mane.


--------------------
Anything written above is just as likely to be accurate as it is to be corrected by someone smarter than me two posts from now.

My Journal / Grow Log


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Offlinegreenladel
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Re: some questions to improve my sterile practice [Re: RoscoeReturns]
    #27048242 - 11/20/20 02:33 AM (6 days, 21 hours ago)

Quote:

RoscoeReturns said:
Quote:

greenladel said:

what do you mean by "streak your syringe to agar"?

thank you for the help :heart:




Basically putting spores from your syringe onto agar, and then transferring clean mycelium to a new plate. It’s called streaking because you would normally use an inoculation loop and rub the spores onto your plate in a zig zag motion. Like this:



That spreads the spores out, which allows you to pick a clean colony. You can just put a small drop of spore solution in the center of your plate and tilt to make it run across. Or some just put a drop in the middle and get a big pile of colonies growing on top of one another. It all can work.

Something you might try to narrow down the source of contamination is to hold back a jar next time you try. Prep all jars as normal, but keep one aside, do not inoculate it. If all your jars go bad, then you’re not sterilizing properly. If all except the one you didn’t inoculate go bad, then the problem is in the syringe or your handling/inoculating.

Also stop wasting cash on expensive spore syringes. If you want to try agar to grain to bulk, and skip all this, I’d be happy to send you a spore print. You can grow for a lifetime off one print.




thank you for the info and the generous offer! :heart:
i feel like i would be letting myself down if i gave up on this now :smile: i do want to learn agar soon, but i want to get this right first.
i already have everything i need to do quite a lot more jars too (apart from spores). i will research agar and buy the things i need in the meantime and hopefully i will have some fruits of inspiration to get me started by the time i have what i need :smile:

maybe this is laughable, but my assumption is that i could also clean the agar on plates then put the good myceluim into a BRF jar to colonize and move forward with my shoebox method as before?
if i could do that it means i could buy agar and plates first and still play with my new toys and learn while i wait to have the money for grains, fruiting chambers etc

thank you for the help :heart:


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Offlinegreenladel
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Re: some questions to improve my sterile practice [Re: karri0n]
    #27048250 - 11/20/20 02:48 AM (6 days, 21 hours ago)

Quote:

karri0n said:
You could do agar without a pressure cooker if you order pre-made plates. Without a pressure cooker, you can't sterilize grain or make your own agar plates, though. You could buy a bag of pre-sterilized grain if you wanted to try this out and decide to buy a pressure cooker later.

You could also inoculate pf cakes with agar if you really wanted to go the agar route but don't want to get a pressure cooker.

I got started with a 40 dollar pressure cooker. It's not ideal since you can't fit quart jars in it, but it's working for my purposes for now. I will have 23 quart come Christmastime, or if I decide to open it sooner.




i already have a pressure cooker, but it is a cheap 10psi one, all i can afford for now. i assume it will do the job with adjusted times for most things though? it will be a long time before i can upgrade that to a good one.

i think you have answered my question to RoscoeReturns regards inoculating BRF cakes with petri dish mycelium.

as for your new pressure cooker. open it now! open it now! open it now! :smile:

thank you for the help :heart:


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Re: some questions to improve my sterile practice [Re: greenladel]
    #27048403 - 11/20/20 07:55 AM (6 days, 16 hours ago)

You can do agar to BRF jars, but you have to leave off the dry verm layer. That means making filtered lids to keep nasties out. At that point it’s cheaper to use grain instead of BRF. You can still spawn grain to coir in shoeboxes.


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Offlinegreenladel
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Re: some questions to improve my sterile practice [Re: RoscoeReturns]
    #27049872 - 11/21/20 07:07 AM (5 days, 17 hours ago)

Quote:

RoscoeReturns said:
You can do agar to BRF jars, but you have to leave off the dry verm layer. That means making filtered lids to keep nasties out. At that point it’s cheaper to use grain instead of BRF. You can still spawn grain to coir in shoeboxes.




thank you
i think i will get another spore syringe for now, but a smaller one just to try to get something while i learn agar and gather the tools i will need.
i will do some control jars along the way and use a small amount of spores until i can get it right.
once i have some agar and plates i will streak some spores from the syringe and try to get something clean stored ready for when i can get grains and start using that. after that i will consider methods of increasing yield if i need to.
i think that might be the best path for me to take to try to actually get some fruits while slowly funding agar. does that sound like a good path to take?

thank you very much for the help


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Offlinepickle jar pete
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Re: some questions to improve my sterile practice [Re: greenladel]
    #27051025 - 11/21/20 09:16 PM (5 days, 3 hours ago)

You can tap your jar for even distribution while filling with substrate. An important step is to take a spoon and lightly press down the top of the substrate creating a smooth even top (this will stop the verm from falling down the side).  Wipe outside and inside of rim with iso and add verm to top of brim (verm top layer should be about 1/2in--3/4inches thick). 

temp is the most important aspect for jars 67f-74f is ideal.


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Offlinegreenladel
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Re: some questions to improve my sterile practice [Re: pickle jar pete]
    #27053317 - 11/23/20 05:30 AM (3 days, 18 hours ago)

Quote:

pickle jar pete said:
You can tap your jar for even distribution while filling with substrate. An important step is to take a spoon and lightly press down the top of the substrate creating a smooth even top (this will stop the verm from falling down the side).  Wipe outside and inside of rim with iso and add verm to top of brim (verm top layer should be about 1/2in--3/4inches thick). 

temp is the most important aspect for jars 67f-74f is ideal.




thank you for the reply

great tip, using a spoon, i will do that. i was also wiping the rim with a clean, dry paper towel but i will use 70% iso next time.

i think temperature was the reason my last 4 jars took so long to show anything. i had the jars in a different place where the temperature was typically slightly below 67°F, only going above that for certain periods of the day.
the first 12 jars (in a room temperature location) did start to show growth before contamination showed up.
i have a better spot for them next time where they will be consistently in that range.

thank you for the help :heart:


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Onlinebigfootscreepyuncl
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Re: some questions to improve my sterile practice [Re: greenladel]
    #27055511 - 11/24/20 03:09 PM (2 days, 9 hours ago)

Since you mentioned being new to using a Pressure Cooker and that you're dealing with contamination issues, read through Bods Pressure Cooker TEK (linked below). Like you I've never used a PC prior to this and there was a lot of good information in there that I would have never thought of/figured out. Additionally, there is a sub-link in the TEK about venting your PC prior to starting the sterilization process. Give it a read, apply it and see if it helps clean up your jars. Cheers, mate!

https://www.shroomery.org/forums/showflat.php/Number/24180711/vc/1#24180711


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