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delcat
Stranger
Registered: 03/13/18
Posts: 126
Last seen: 5 years, 8 days
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Issues with LC And contam - what to do?
#25148255 - 04/18/18 02:05 PM (6 years, 8 months ago) |
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Hi all,
Having a bit of an issue at the moment with LC
I have two spore prints I am trying to turn into a liquid culture. My procedure is as follows:
450ml Glass jars with 2mm hole drilled in lid, and foil over the top of the main lid. 200ml water in each with 2.5ml Honey 2.5ml Maple Syrup PC @ 0.7bar for 40 minutes (A bit long but the first attempt was only 20 mins and at 0.7bar i guess i need longer)
After this, working in a clean area fully wiped with alcohol, I scrape up a a few spores from each print and then open the lid of each jar as little as possible, tap the spores into each jar and close it up again as quickly as possible. I am heating the "scraper" to red hot inbetween spore drops, then wiping it, and my hands with alcohol to get rid of the black residue and cool down the scraper a bit.
Of the two jars I've done so far, after a week and a day one jar looks the same as it did, except with two small blobs of mycelium in it that do not appear to be growing much. The rest of the liquid looks much the same as it did on day 1. The second jar however took off MUCH Faster than the first (Initially I had large myc blobs after 3 days where as jar 1 took 5 days to show even any small strands of myc)
Now the entire liquid in jar 2 has gone very cloudy, to the point that you can barely see the few fairly large blobs of mycelium in it (but the cloudyness clearly isnt the mycelium)
Since both jars were innoced after the same PC with the same tools at the same time in the same area, I Am assuming maybe my spore print for jar 2 has bacteria contam on it.
Is there anything I can do? Is it worth trying to innoc something? PF Cake? Agar and try and separate out the Myc from the contam? What do you think?
I used 1/4 the spore print on this first attempt that doesn't seem to have worked, and another 1/4 on the next attempt (Which I did yesterday)
Anyone have any suggestions? I really want to try and make use of the spore print as its a nice species. At the same time, I am a novice mycologist so I'm unsure whats the best way to go...
Thanks
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wushroow


Registered: 02/03/17
Posts: 369
Last seen: 6 years, 7 months
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Re: Issues with LC And contam - what to do? [Re: delcat]
#25148289 - 04/18/18 02:20 PM (6 years, 8 months ago) |
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I wouldn't use it if I were you. Spores to liquid is a no-no, just learned that the difficult way. If I were you, I'd use a very minute amount of your print (no need to go ham with the spores!) on several agar plates. Agar should be the first step for anyone in my humble opinion. But doing this will allow you to pick some good candidates right from the get-go; you can see the strongest colonies and transfer them to a new plate. This will eliminate contamination, narrow down the genetics, and allow you to make clean grain/LC almost every time. I'll give you some threads. agar: https://www.shroomery.org/forums/showflat.php/Number/19208976 https://www.shroomery.org/forums/showflat.php/Number/18430998 LC: https://www.shroomery.org/forums/showflat.php/Number/24740168 This LC tek has been making a stir. Planning to try it out myself once I get some good agar
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xnirvanax
Frank's biological son

Registered: 05/16/13
Posts: 135
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Re: Issues with LC And contam - what to do? [Re: delcat]
#25148443 - 04/18/18 03:37 PM (6 years, 8 months ago) |
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If agar is possible for you, then do that.
Just scrape some spores on to a dish and let it grow out, cut out the clean growth and transfer.
The cloudy LC is a sign of contamination. I don't think you'll get very far with spores -> LC.
Quote:
After this, working in a clean area fully wiped with alcohol, I scrape up a a few spores from each print and then open the lid of each jar as little as possible, tap the spores into each jar and close it up again as quickly as possible. I am heating the "scraper" to red hot inbetween spore drops, then wiping it, and my hands with alcohol to get rid of the black residue and cool down the scraper a bit.
Are you inoculating this open air or with a still air box?
-------------------- Inspired by FrankHorrigan
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Dr. Freeman
An agent of Order



Registered: 03/27/18
Posts: 351
Loc: Midgard
Last seen: 1 year, 6 months
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Re: Issues with LC And contam - what to do? [Re: wushroow]
#25148454 - 04/18/18 03:45 PM (6 years, 8 months ago) |
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I really love making LC to expand my genetics or make spores last sooooo muuuch looonger. The problem is shooting spores to LC, it simply isn't a good idea. Do agar! From a clean agar sample, make a clean LC.
Make some agar.These are some good teks:
Then Isolate healthy mycelium on your agar. Josex's biopsy method.
And finally, make LC out of agar: Pastywhyte's EzLC tek
Post edit: You could also check my LC tek for the incredibly lazy on my sig, maybe it's for you.
Edited by Dr. Freeman (04/18/18 03:47 PM)
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delcat
Stranger
Registered: 03/13/18
Posts: 126
Last seen: 5 years, 8 days
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Re: Issues with LC And contam - what to do? [Re: wushroow]
#25148530 - 04/18/18 04:11 PM (6 years, 8 months ago) |
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Thats really great information, thank you. It has all helped a lot...
My plan was to do spores to LC To aid in inoculation of 1 pint jars (All I can get a hold of)
I wanted to do LC ao I could pump them full of innoculant in the hope of getting a full colonisation as I know 1 pint jars do struggle. I was following this guide, but using a PC instead, so I skipped the step where he boils the jars on their own:
https://www.shroomery.org/forums/showflat.php/Number/5238137/fpart/1/vc/1
EDIT-UPON FURTHER READING, THE ABOVE TEK IS 12 YEARS OLD AND HAS MANY NEGATIVE COMMENTS MADE ON IT. SEEMS STARTING OFF WITH IT IS A BAD IDEA REALLY...
I think though I'm working with a contaminated spore print so the best thing for me to do at the moment is to isolate it on agar and use the absoloutely awesome biopsy method you mentioned. It looks as if this method could actaully also be used to isolate the contams off of my dirty spore print (doing agar stabs and squirting onto more agar intead of into LC until I Get something clean)
It's more work than I originally wanted to do but if it works then its much better than re-trying the same thing over and over, right!
once I have that confirmed clean LC I Can then look at pouring into grain jars or innoculating BRF Pint jars with it
Until then I Think I may just work with regular online bought shitake spore syringes direct into smaller BRF jars. The speed of LC Colonisation and what I perceive will be the extra fruits of using a pint jar attracted me to LC In the first place, but as I say I think I have a lot of work to do in order to do it properly.
Thanks so much for the great links, I'm not sure how to +1 your post but once I figure it out I'll be sure to do so!
Edited by delcat (04/18/18 04:41 PM)
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delcat
Stranger
Registered: 03/13/18
Posts: 126
Last seen: 5 years, 8 days
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Re: Issues with LC And contam - what to do? [Re: delcat]
#25148557 - 04/18/18 04:18 PM (6 years, 8 months ago) |
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I am not using a still air box presently. I Work in a small room with all the doors and windows closed, being careful basically to not move too much for 30 minutes or so before I do anything.
Obviously a SAB is going to be better, but can I expect to work like this with a reasonable degree of success?
Also, I'm using purell or similar to sterilise my hands to my elbows rather than the 99% alcohol I use for everything elae. Is that ok?
Thanks for all the replies and tips guys!
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Dr. Freeman
An agent of Order



Registered: 03/27/18
Posts: 351
Loc: Midgard
Last seen: 1 year, 6 months
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Re: Issues with LC And contam - what to do? [Re: delcat]
#25148613 - 04/18/18 04:41 PM (6 years, 8 months ago) |
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Quote:
delcat said: I am not using a still air box presently. I Work in a small room with all the doors and windows closed, being careful basically to not move too much for 30 minutes or so before I do anything.
Obviously a SAB is going to be better, but can I expect to work like this with a reasonable degree of success?
You might get over 90% failure rate, some might say even 99%. Not worth the try. SAB are cheap and easy to make, can't cut corners when working with agar and spore prints. Perhaps your spores are clean and you are getting contamination from the ambient.
Quote:
(doing agar stabs and squirting onto more agar intead of into LC until I Get something clean)
Don't squirt water into an agar plate, not if it is from another agar plate you want to clean out. Use a dry needle like this one and push the tissue biopsy with a thinner needle or wire.
Quote:
pacmanbreed said:

I found the image on this post.
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delcat
Stranger
Registered: 03/13/18
Posts: 126
Last seen: 5 years, 8 days
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Re: Issues with LC And contam - what to do? [Re: Dr. Freeman]
#25148628 - 04/18/18 04:49 PM (6 years, 8 months ago) |
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wow, I never realised it was going to be that bad of a failure rate.
What about using commercially available spore syringes without a SAB? Is it the LC/Agar aspect that needs the SAB or am I literally likely to fail completely whatever method I try without a SAB?
I think for now I'd like to just grow some shitakes on BRF in 125ml jars that have been pressure cooked. I'll deal with LC and agar and stuff a bit later once I've actaully done a sucessful grow.
I'll ask anyway just to check, but I'm guessing you guys will also recommend not using 1 pint jars for BRF?
Thanks for the tip on the no water squiring, it makes sense. I have some 18g needles that came with a centre wire that I will use to do just as you say. One question about working in a SAB - I take it I flame the needle OUTSIDE of the SAB (Dont want fire in the confined space)
Moving the needle into the SAB is obviously a contamination vector - how do I cool the needle too? Do I literally just wait for 30 seconds and hope its cool enough to not kill the myc?
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Failboat
Free man

Registered: 02/01/18
Posts: 8,736
Last seen: 1 month, 1 day
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Re: Issues with LC And contam - what to do? [Re: delcat]
#25148668 - 04/18/18 05:02 PM (6 years, 8 months ago) |
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My LC ventures have been 90+% failure, focus on agar work and then put the clean culture to grain. Grain to grain works wonders.
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Grimsweeper
don't fear the sweeper


Registered: 01/29/18
Posts: 4,870
Loc: broom closet
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Re: Issues with LC And contam - what to do? [Re: delcat]
#25148755 - 04/18/18 05:33 PM (6 years, 8 months ago) |
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Quote:
delcat said: I am not using a still air box presently. I Work in a small room with all the doors and windows closed, being careful basically to not move too much for 30 minutes or so before I do anything.
Before I made a glove box, and then a SAB, my very 1st project was done using the oven door method. I nocked up 18 BRF jars to fruit with no contams. Not ideal but no tools needed and better than open air.
-------------------- When you clean a vacuum cleaner you are a vacuum cleaner
Build yourself a Flow Hood in these 99 simple steps
 
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mushboy
modboy



Registered: 04/24/05
Posts: 35,378
Loc: eating the cats
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Re: Issues with LC And contam - what to do? [Re: Grimsweeper]
#25148761 - 04/18/18 05:35 PM (6 years, 8 months ago) |
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Quote:
Grimsweeper said:
Quote:
delcat said: I am not using a still air box presently. I Work in a small room with all the doors and windows closed, being careful basically to not move too much for 30 minutes or so before I do anything.
Before I made a glove box, and then a SAB, my very 1st project was done using the oven door method. I nocked up 18 BRF jars to fruit with no contams. Not ideal but no tools needed and better than open air.

there is no reason not to use a sab. people have built them out of saran wrap and cardboard boxes. play it safe. wear a figurative rubber.
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Dr. Freeman
An agent of Order



Registered: 03/27/18
Posts: 351
Loc: Midgard
Last seen: 1 year, 6 months
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Re: Issues with LC And contam - what to do? [Re: mushboy]
#25149093 - 04/18/18 07:15 PM (6 years, 8 months ago) |
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Don't use one pint for Pf jars! If you use a syringe on a PF cake you are likely to have success. Syringe are never 100% clean, that is Ok for PF, not so OK for LC. Regarding SAB, if you open two gar dishes for 30 seconds one outside the SAB and another inside, one will get horribly contaminated. Yo need to read a lot more
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delcat
Stranger
Registered: 03/13/18
Posts: 126
Last seen: 5 years, 8 days
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Re: Issues with LC And contam - what to do? [Re: Dr. Freeman]
#25149926 - 04/19/18 02:11 AM (6 years, 8 months ago) |
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Thanks guys. I'll make a SAB before I do anything further.
I think the order of operation will be to innoc some 240ml jars with a syringe in a SAB, then start messing around with Agar.
I initially wanted to stay away from it since that LC Tek made LC Sound so easy to get tonnes and tonnes of innoculant, but now I realise its not such a great idea. I read in another thread someone said beginners should:
1.Do straight BRF with spore syringe 2.Mess around with Agar to do strain isolation 3.Worry about LC and G2G later
I was trying to do 3, then 1 then 2. I think I'll stick to the order above.
Thanks again everyone for the valuable advice.
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Grimsweeper
don't fear the sweeper


Registered: 01/29/18
Posts: 4,870
Loc: broom closet
Last seen: 10 days, 16 hours
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Re: Issues with LC And contam - what to do? [Re: delcat]
#25150006 - 04/19/18 04:26 AM (6 years, 8 months ago) |
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-------------------- When you clean a vacuum cleaner you are a vacuum cleaner
Build yourself a Flow Hood in these 99 simple steps
 
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Dr. Freeman
An agent of Order



Registered: 03/27/18
Posts: 351
Loc: Midgard
Last seen: 1 year, 6 months
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Re: Issues with LC And contam - what to do? [Re: Grimsweeper]
#25150016 - 04/19/18 04:41 AM (6 years, 8 months ago) |
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LC is great in my opinion, I really love super thick mycelium syringes, but it has a when and how. LC is not complicated at all, but precautions must be taken or all you'll get is contamination.
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wushroow


Registered: 02/03/17
Posts: 369
Last seen: 6 years, 7 months
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Re: Issues with LC And contam - what to do? [Re: Dr. Freeman]
#25150204 - 04/19/18 08:08 AM (6 years, 8 months ago) |
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If you use a small enough spore scraping, you'll limit contamination. Streak it across the plate to get colonies. Find the agar portal and do agar.
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