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AgarStudent
Noob


Registered: 01/25/15
Posts: 550
Last seen: 2 years, 10 days
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Germinating Scerlotia-producing spores on Agar
#23428624 - 07/10/16 08:08 AM (7 years, 6 months ago) |
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Hi, this has been part of a DIY project that has gone on for a few years. The goal has been to transform spores (of scerlotia producing species) to mycelium from a posted print.
My method has been using a flow hood and sterile syringes, also with pre-pressure cooked and cooled Diggers demineralised water.
Over the flow hood in sterile air I suck a bit of water into a clean syringe and then put a few drops over the print. I use a sterile piece of stainless steel (from a dentist kit) to lift and stir the spores into the applied drops and then proceed to suck it back up into the syringe.
I then place a drop on a plate of malt extract agar and allow to colonise at 20 Degrees C or so. The thing is, I have never ever had any luck with prints, but have had luck with a syringe/vial that was accidentally posted to me.
The spores also appear to stick to the sides of the syringe that I have made from prints, but I have heard that there are still thousands in solution that I can't see that will make it to the agar anyway.
The issue I have got is that so far I have just not had any germination. Does it matter if I turn the AC off at night and it drops to 14C or so? The prints have been shipped internationally, but I still don't know if they could have been cooked on transit due to temperature variations? It has been three days since inoculation, but should I wait a few weeks before ordering again? I think I am on my fifteenth print or more and this is a pretty expensive hobby! Still I won't give up.
Still I had luck with vials (cubes) and they germinated no probs and I don't know why?
Thanks in anticipation of your help.
-------------------- Why reinvent the wheel when someone here has already done the science?
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blindingleaf
blue collar underworld


Registered: 07/19/13
Posts: 22,008
Loc: sub-surface unseen
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Re: Germinating Scerlotia-producing spores on Agar [Re: AgarStudent]
#23428638 - 07/10/16 08:17 AM (7 years, 6 months ago) |
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some prints just go bunk man….but 15 of them in a row (if i read that right?) is weird.
I don't think the temp should matter much, but certainly isn't speeding things up. maybe toss them on top of ur fridge and they will warm up some?
can't u e mail the vendor and say WTF is going on? they replace contaminated ones. I'm sure there is a clever way to word "unviable spores" in an e mail so you don't have to keep paying
u can try scraping some spores onto a plate and then pouring agar on top of it. usually the last 2 plates in a sleeve cause the remaining agar won't be so hot. you'll notice the germination around the edges on the plate usually. the warmth of the liquid kinda softens the spore.
-------------------- A few thoughts on cultivation MICROBIAL HUSBANDRY!!!! The whole is greater than the sum of its parts
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Supalemonhaze
Spore syringe hater.



Registered: 10/02/15
Posts: 6,725
Loc: 12" down Europe's butthole
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Re: Germinating Scerlotia-producing spores on Agar [Re: blindingleaf]
#23428667 - 07/10/16 08:33 AM (7 years, 6 months ago) |
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3 days is too early, only the freshest spores are able to germinate within that timeframe and some take longer no matter how fresh they are. You need to allow 2 weeks to pass before giving up on spores.
If you are using agar, I can't see why you are using spore solution when you have a print. Spore solutions on agar are often troublesome. Use an inoculation loop, flame it, dip it into the agar to cool it and scrape some spores with it to inoculate your plate. I like to do a couple of streaks in a zigzag motion and then stab the agar with the loop so the spores get sandwiched between the agar. This helps with spore hydration and germination.
Are you using soft agar? I've had 2 day germination with soft agar, it works wonders IME. Alternatively, you can do as leaf said, pour some plates and just before they set completelely, use your loop to inoculate the semi-solid agar. Just be careful it's not hot or you will kill the spores. This is just as good as a soft agar recipe.
You can also try a warm pour, this works pretty good for spore solutions. Take a spore inoculated plate which doesn't want to germinate, prep and PC some agar and wait for it to cool enough to start to thicken. It needs to be warm, not hot. Once it has cooled down enough, pour that over your inoculated agar plate. The warm, wet agar will soften the spore wall and aid in germination. It works with spore solutions because it sandwiches the spores between 2 "slabs" of agar as well as hydrates them.
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blindingleaf
blue collar underworld


Registered: 07/19/13
Posts: 22,008
Loc: sub-surface unseen
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Re: Germinating Scerlotia-producing spores on Agar [Re: Supalemonhaze]
#23428701 - 07/10/16 08:47 AM (7 years, 6 months ago) |
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i like syringes better than prints but only because they are quicker. flame it and squirt. no unbagging/unwrapping/unfolding/handling step. that said, I never went out of my way to make one out of my own prints so its rare i use a syringe
after i flame the blade (or syringe tip), i cool it in the dish and make several scrapes/slashes/dents and thats where I swipe (or squirt if using syringe) the spores so they are kinda cradled in this moist, nutritious valley.

-------------------- A few thoughts on cultivation MICROBIAL HUSBANDRY!!!! The whole is greater than the sum of its parts
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Supalemonhaze
Spore syringe hater.



Registered: 10/02/15
Posts: 6,725
Loc: 12" down Europe's butthole
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Re: Germinating Scerlotia-producing spores on Agar [Re: blindingleaf]
#23428723 - 07/10/16 08:56 AM (7 years, 6 months ago) |
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I often notice that the first germination spots are usually where I stab the agar so that definitely makes a difference.
I have little love for spore solutions though, I've had shit tons of problems trying to germinate spores that were in a syringe.
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AgarStudent
Noob


Registered: 01/25/15
Posts: 550
Last seen: 2 years, 10 days
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Re: Germinating Scerlotia-producing spores on Agar [Re: Supalemonhaze]
#23430451 - 07/10/16 08:38 PM (7 years, 6 months ago) |
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Thanks guys,
I have not tried the innoculating loop technique, so this will definitely be worth a shot if all else fails. Also soft agar means that you just mix less agar into the MEA mix right so that it does not completely set at room temperature?
Also I never scraped or dinged the agar surface and previously just squirted it straight on with a drop or two. If I try this there should be more contact surface area for the spores to go, which is something I didn't think of before. Thanks guys
-------------------- Why reinvent the wheel when someone here has already done the science?
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Supalemonhaze
Spore syringe hater.



Registered: 10/02/15
Posts: 6,725
Loc: 12" down Europe's butthole
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Re: Germinating Scerlotia-producing spores on Agar [Re: AgarStudent]
#23430462 - 07/10/16 08:42 PM (7 years, 6 months ago) |
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Soft agar will still set but will look wetter than normal agar. Use 1g agar per 100ml of water for soft agar.
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Chk
Reverser


Registered: 09/06/13
Posts: 405
Loc: France
Last seen: 5 years, 3 months
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Re: Germinating Scerlotia-producing spores on Agar [Re: Supalemonhaze]
#23431128 - 07/11/16 02:40 AM (7 years, 6 months ago) |
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i had one old print taking 1 month to germinate, forgot it on my shelf, then one day, bam, everything is possible i guess
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TheChief
Cube Collector



Registered: 07/09/14
Posts: 1,905
Loc: Executing TEKs...
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Re: Germinating Scerlotia-producing spores on Agar [Re: Chk]
#23431388 - 07/11/16 07:15 AM (7 years, 6 months ago) |
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Chk, that mushroom in your sig is downright gross...

OP, sounds like you have bad prints. Follow the advice of other members already posted.
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jcbowling1985 said: "Additionally I did one Armageddon jar with all of the types together."
Edited by TheChief (07/11/16 07:16 AM)
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AgarStudent
Noob


Registered: 01/25/15
Posts: 550
Last seen: 2 years, 10 days
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Re: Germinating Scerlotia-producing spores on Agar [Re: TheChief]
#23431534 - 07/11/16 08:33 AM (7 years, 6 months ago) |
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Does anyone have a recipe for soft agar?, as I live in a dry climate and I am sure I have had previous issues with spores drying up before they have had a chance to germinate. Do you get the MEA mix and just water it down or do you simply put in the same amount of malt extract and water, but half the agar?
I made it once my mistake and can't remember what my ratios were 
Am I right to just put a few drops into the half-set agar to ensure that there are at least some spores in there considering they are mostly plastered to the sides of the syringe. No backing out with that plan as I already made the syringes up ...
Alternatively is it possible to stick with the regular agar mix, but slash it a bit with a sterile scalpel and allow a drop or two of syringe liquid to penetrate? perhaps that way it won't dry out?
Thanks again for suggestions
-------------------- Why reinvent the wheel when someone here has already done the science?
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AgarStudent
Noob


Registered: 01/25/15
Posts: 550
Last seen: 2 years, 10 days
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Re: Germinating Scerlotia-producing spores on Agar [Re: AgarStudent]
#23431537 - 07/11/16 08:35 AM (7 years, 6 months ago) |
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Oh thanks supalemonhaze. Sorry I missed your recipe above.
Cheers.
-------------------- Why reinvent the wheel when someone here has already done the science?
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