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GermanShaman
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Having issues with Agar work. Just can't seem to get any Myc!!!
#28168737 - 02/02/23 09:12 AM (1 year, 1 month ago) |
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I am having some major issues with my agar work. I just cant seem to get anything to grow.
Here is my process:
Agar Receipes tried.
250ml Tap Water 4g Agar 7g LME
Sterilised at 15psi for 30 mins
Poured into glass /plastic petri dishes, in front of flow hood. Plastic containers have been cleaned with isoprop, glass have been put in microwave for about 10 mins.
Take spore print and create spore syringe with sterilized distilleld water.
Add several drops from spore syringe to cooled down agar plates. Seal up edges of glass dish with sarange wrap, the plastic ones just have their tops placed on. Leave in room at normal room temp (about 21 Deg C) and wait.
I dont have issues with Trich any more since I built the Flow hood.
I just dont seem to get any growth at all even after a month of waiting. I have had some success with Agar work before but now nothing seems to be growing.
I'm also trying the following Agar recipes to see if that helps.
250ml Potato solution (made by boiling some potato peelings) 4g Agar 3g LME
Also trying:
250ml Potato solution 4g Agar 4g dextrose
Would welcome any advices on the matter.
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SirPsycho
Purple Belt in Google-Fu
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: GermanShaman]
#28168743 - 02/02/23 09:15 AM (1 year, 1 month ago) |
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Can we see some pictures?
-------------------- Ask me about free Ps tampanesis, Ps subtropicalis and Ps cubensis (ESS) prints Balance in life is like running on ice. π
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GermanShaman
Stranger
Registered: 01/05/18
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: SirPsycho]
#28168759 - 02/02/23 09:25 AM (1 year, 1 month ago) |
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Here are some pics. Pics 1 - 7 from left to right.
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SimulatedMatrix33
Inter-dimensional Being
Registered: 04/09/22
Posts: 529
Loc: Planet Kai. Hacker of the Matr...
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: GermanShaman]
#28168767 - 02/02/23 09:28 AM (1 year, 1 month ago) |
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How old are your prints you are trying to germinate.
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GermanShaman
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: SimulatedMatrix33]
#28168771 - 02/02/23 09:29 AM (1 year, 1 month ago) |
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The age of the prints varies from a a few months to about 2-4 years.
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DERRAYLD
Constructus
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: GermanShaman] 1
#28168788 - 02/02/23 09:40 AM (1 year, 1 month ago) |
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Your plates are all contaminated with serious bacterial bloom of some sorts. What made you think those plates were clean?
You also don't need to make a spore syringe to use the prints, just streak them to agar directly.
Not sure why you don't have growth but based on your plates I'm not sure your prints are okay to be honest.
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SirPsycho
Purple Belt in Google-Fu
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: GermanShaman] 2
#28168799 - 02/02/23 09:48 AM (1 year, 1 month ago) |
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That's all contaminated and this is why:
Quote:
GermanShaman said: Plastic containers have been cleaned with isoprop, glass have been put in microwave for about 10 mins.
Your containers are not sterile to start with. Isopropyl is a sanitizer, it doesn't sterilize and empty plates in a microwave also don't sterilize because glass doesn't absorb the energy from microwaves and isn't getting heated up
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GermanShaman
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: DERRAYLD]
#28168802 - 02/02/23 09:51 AM (1 year, 1 month ago) |
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I have thrown these plates away now and made some more.
I have tried to scrap the prints to agar directly, that did not really produce any more success. I went to syringe so that I could dilute the spores down into about 20ml of water and therefore make the spore print go further.
I have also managed to take the spore syringe and just inoculate some 1% honey solution LC and managed to get some growth that way.
I am also trying several different prints from several different sources to see if that is the issue. It does seem to be a bit hit or miss!!!!
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GermanShaman
Stranger
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: GermanShaman]
#28168803 - 02/02/23 09:52 AM (1 year, 1 month ago) |
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The glass does get very hot in the microwave.
In any case, could I sterlise the glass in the oven at 200 Deg C for about 20 mins?
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JHOVA
Post whore
Registered: 02/17/17
Posts: 4,727
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: GermanShaman]
#28168807 - 02/02/23 09:54 AM (1 year, 1 month ago) |
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Quote:
GermanShaman said: The glass does get very hot in the microwave.
In any case, could I sterlise the glass in the oven at 200 Deg C for about 20 mins?
https://www.shroomery.org/forums/showflat.php/Number/28006384
Follow the no pour tek for glass petris.
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DERRAYLD
Constructus
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Loc: South Africa
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: GermanShaman] 1
#28168811 - 02/02/23 09:57 AM (1 year, 1 month ago) |
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Quote:
GermanShaman said: I have thrown these plates away now and made some more.
I have tried to scrap the prints to agar directly, that did not really produce any more success. I went to syringe so that I could dilute the spores down into about 20ml of water and therefore make the spore print go further.
I have also managed to take the spore syringe and just inoculate some 1% honey solution LC and managed to get some growth that way.
I am also trying several different prints from several different sources to see if that is the issue. It does seem to be a bit hit or miss!!!!
You need the point of a pin worth of spores, actually a lot less.
No need to dilute anything to make the print go further.
Follow the no pour tek that JHOVA posted, it's your best bet.
I'm also doubting your flowhood to be honest.
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bakedbeings
orbiter of truth
Registered: 09/01/20
Posts: 4,218
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: SirPsycho] 2
#28168813 - 02/02/23 09:58 AM (1 year, 1 month ago) |
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you are pouring agar into plates coated with bacteria
Quote:
I have also managed to take the spore syringe and just inoculate some 1% honey solution LC and managed to get some growth that way
you are squirting bacteria into a jar of bacteria food
Quote:
I am also trying several different prints from several different sources to see if that is the issue. It does seem to be a bit hit or miss!!!!
its not the prints
slow down, stop experimenting, follow trusted teks. i agree that no pour is a good option
Edited by bakedbeings (02/02/23 09:59 AM)
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DERRAYLD
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: bakedbeings] 1
#28168816 - 02/02/23 09:59 AM (1 year, 1 month ago) |
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That lc will be trash as well unfortunately.
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Hindsight
Mad Scientist
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: GermanShaman]
#28168820 - 02/02/23 10:03 AM (1 year, 1 month ago) |
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I wouldn't go spore to LC. Success rate will be low. Need to get your agar working.
200C for 20 mins is fine for sterilization but you have to remember that you can't just do that, take them out, and walk them over to your hood. You have to wrap them in foil so they don't get contaminated between the oven and your hood.
I'm not sure how you are scraping spores onto your plate, but I don't suggest holding the print over the plate and scraping in such a way to let the dislodged spores fall into the plate. Instead, take a wire loop, sterilize it red hot in front of your hood, let it cool in the hood airflow, then rub it on your print to pick up some spores onto it then streak it across your agar plate. Put the lid on. Wrap it with parafilm or cling wrap.
The issue with making spore syringes is that you are FAR more likely to pick up bacteria from the print with the water, and then it can multiply in the syringe, ensuring that any drop or squirt of liquid from that syringe ends up creating a big blotch of bacteria on your plate. Not to say spore syringes are bad, but if you are having bacterial contam issues now, stay away from syringe use.
To reiterate what SirPsycho said, ISO doesn't sterilize anything. It kills most bacteria, but not all, and it does absolutely nothing for mold spores other than spread them around. Anything you pour into must be completely sterile.
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GermanShaman
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: Hindsight]
#28168975 - 02/02/23 11:38 AM (1 year, 1 month ago) |
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I've used a scalpel that is flame sterilized to scrape the spores into a small glass jar that I then squirt some sterilized distilled water into.
All this is done in front of the flow hood. The syringes and needs have all been sterilized in the PC at 15psi.
Maybe I should give the innoc loop a try.
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johnukguy
Learning
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: GermanShaman]
#28177710 - 02/08/23 01:34 PM (1 year, 1 month ago) |
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Can you post a pic of your flowhood and give us the specs please?
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LadysKnight
Hello Ladies
Registered: 10/09/15
Posts: 1,852
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: johnukguy]
#28177799 - 02/08/23 02:46 PM (1 year, 1 month ago) |
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While all the comments above are correct, do keep your eyes open. I see potential myc on half those plates.
This is from the 4th one.
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ruawakeyet
Registered: 05/03/21
Posts: 1,862
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: LadysKnight] 1
#28177810 - 02/08/23 02:57 PM (1 year, 1 month ago) |
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You could also try BRF/Verm pucks, just make sure you get them sterilized beforehand. I've personally seen a BRF puck germinate spores that refused to germinate on agar.
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bakedbeings
orbiter of truth
Registered: 09/01/20
Posts: 4,218
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: ruawakeyet] 2
#28177869 - 02/08/23 03:37 PM (1 year, 1 month ago) |
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Quote:
ruawakeyet said: You could also try BRF/Verm pucks, just make sure you get them sterilized beforehand. I've personally seen a BRF puck germinate spores that refused to germinate on agar.
TEAM PUCK!!!!
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fiddle_head
Registered: 08/05/08
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Re: Having issues with Agar work. Just can't seem to get any Myc!!! [Re: johnukguy] 1
#28178138 - 02/08/23 06:42 PM (1 year, 1 month ago) |
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Quote:
johnukguy said: Can you post a pic of your flowhood and give us the specs please?
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