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OfflineViolinboy
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Registered: 04/15/20
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MS syringes on agar help
    #26694822 - 05/25/20 11:58 AM (3 years, 8 months ago)

Hello all!

I've been lurking here a few months now trying to get started with mycology and I'm needing some help now.
A few weeks ago I started some multispore syringes (B+, GT, and PE) on agar. I used an inoculation loop in a SAB with proper sterile technique. After about two weeks, the various 'blobs' had spread out a bit, but were still just blobs really, no visible mycelium fuzz or strands. I decided to transfer the whitest cleanest looking blobs at that point to new plates because there were contams scattered throughout the plates that I wanted to avoid getting any more established. Here are photos of the transfers 5 days on now.

B+



PE



GT



How are those looking? Would it be best for me to transfer again right now? Or maybe just transfer the GT (to get away from the mold) and wait a bit on the other two? The PE looks good but I could wait a bit to see what happens? As for the B+, I have no idea what to do with it... it's still looking like a smooth blob, with no visible mycelium (at least not what I'm expecting/looking for).

For a bit more context, here are some photos of a B+ plate I just did up 3 days ago with a loop. This shows the sort of blobs I was talking about on the initial plates. Would you transfer some small white colonies now? Or would you wait until they grow out a bit more?

I'm not necessarily caring about getting to isolates for now... I figured the best thing from this point would be to just get some healthy, clean looking mycelium, spawn that to grain and then to substrate, and grow out an MS grow, and then I can clone from the best fruit from that grow (assuming I get some) for future grows. I see now why so many people suggest spore prints over MS syringes, they can be a bit of PITA!

Thanks so much for any help, I'm looking forward to many more posts and conversations as I really get into this, it's very exciting!


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InvisibleModularMind
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Re: MS syringes on agar help [Re: Violinboy]
    #26694842 - 05/25/20 12:08 PM (3 years, 8 months ago)

The PE may be workable in a few days. The others don’t show anything desirable as far as I can tell.


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Offlinebrikogen
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Re: MS syringes on agar help [Re: Violinboy]
    #26694847 - 05/25/20 12:10 PM (3 years, 8 months ago)

You sure your SAB technique was on point?

Your B+ looks like bacteria. Your PE looks hopeful but too early to tell. Your GT's look like a combination of trich and bacteria.


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OfflineViolinboy
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Re: MS syringes on agar help [Re: brikogen]
    #26694879 - 05/25/20 12:28 PM (3 years, 8 months ago)

Thanks for the help to both of you!
I'll keep an eye on the PE over the next few days.

As for the B+, would you say the fresh plate I did up looks like just bacteria as well? Would it be worth doing some transfers off of that plate? If so, what would you look for in these early stages in terms of promising things to transfer? All the little dots/blobs look about the same... some are white and some are yellow.


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Offlinepoisoned
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Re: MS syringes on agar help [Re: Violinboy]
    #26694910 - 05/25/20 12:38 PM (3 years, 8 months ago)

I think that B+ has a slight chance to grow some myc out. PE looks better. Everything else is pure contam.


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OfflineViolinboy
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Re: MS syringes on agar help [Re: brikogen]
    #26694919 - 05/25/20 12:42 PM (3 years, 8 months ago)

Also, would the bacteria on the B+/GT plates be something I could work around with better technique? Is it likely to have come from my technique, or from the syringe? I'll do a few more plates up being extra extra careful and see what happens, maybe I'll try the multi streak method (where you re-sterilize and streak to a new part of the plate a few times).

On another note, after reading and finding out that many prefer PE to other types of cubes anyway, I might just focus on those for the time being (and keep trying to get some good plates of the others started for the hell of it). No need to complicate the process with more than one variety right now anyway... Besides, I've got gourmets on the go as well, though I've only succeeded in getting my Reishi's to show mycelium, my Shiitake and Phoenix Oysters are super stagnant, is that normal? Sorry if that's getting too off topic for the thread, lots of questions!


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OfflineViolinboy
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Re: MS syringes on agar help [Re: poisoned]
    #26694926 - 05/25/20 12:43 PM (3 years, 8 months ago)

Do you mean the B+ transfer or the fresh plate I streaked up?
The transfer was just one of those blobs from a streak plate (the whitest looking one haha) that had been colonizing for a couple weeks.


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Offlinepoisoned
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Re: MS syringes on agar help [Re: Violinboy]
    #26694950 - 05/25/20 12:54 PM (3 years, 8 months ago)

I can't see any germination plates.

I'd recommend you to try to get one other variety running, PE are supposed to be a bit more tricky than normal cubes. Although, get them running for sure, but I'd take some more transfers from that plate before inoculating any grain.

How did you inoculate your germination plates? If you do a zigzag pattern with an inoculation loop, you can spread your spores and contaminants out to get a cleaner culture from the beginning.


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OfflineViolinboy
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Re: MS syringes on agar help [Re: poisoned]
    #26694958 - 05/25/20 12:58 PM (3 years, 8 months ago)

Sorry my mistake! Totally forgot to attach the germination plate photos :laugh:
Here they are:



This was done with a flame sterilized inoculation loop. This is also very indicative of how the others looked early on as well, I just left them about a week longer before transferring the best looking whitest circle blob thing.


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Offlinepoisoned
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Re: MS syringes on agar help [Re: Violinboy]
    #26694966 - 05/25/20 01:00 PM (3 years, 8 months ago)

No myc there. I'd try inoculating some more plates. Bacteria might be from a syringe, from a needle or from your inoculation loop.


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OfflineViolinboy
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Re: MS syringes on agar help [Re: poisoned]
    #26694980 - 05/25/20 01:07 PM (3 years, 8 months ago)

Alright I'll give four more plates a go today. Would you recommend any changes in technique? Right now I'm setting everything up in the still air box (wiping with iso, spritzing with soapy water, all over a damp towel, gloves wiped with iso too), then flame sterilizing both the needle and the loop, then I squirt enough of the solution on to the loop that I'm confident it's cool enough (3 or 4 drops, all done inside the SAB). Then I take the loop which is holding the drop, and zigzag it across the plate once.

Should I try just zigzag-ing one small portion, rotate (and flame sterilize the loop again), streak to new sector, rinse and repeat? Or is that unnecessary and if I'm not getting any mycelium now then that won't fix the problem... Thanks for the help, I really appreciate it!


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Offlinepoisoned
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Re: MS syringes on agar help [Re: Violinboy]
    #26694995 - 05/25/20 01:17 PM (3 years, 8 months ago)

I do everything the same, except I put a drop on a plate and zigzag it around. Maybe try again with both PE and B+ and see what you get. If you get PE clean again and B+ just bacteria, then your tek is probably ok.


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Offlinebrikogen
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Re: MS syringes on agar help [Re: Violinboy]
    #26695014 - 05/25/20 01:27 PM (3 years, 8 months ago)

Yikes your germination plate looks very bad.

Also If you use a syringe anyway eliminate the inoculation loop, one less point of failure. Flame sterilize the needle and squirt it directly in the plate. Try to spread it around. I never had good luck with a single drop using a MS syringe, I always carpet bomb my petris and start isolating from there.

Here's how my first petris look like:



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Offlinepoisoned
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Re: MS syringes on agar help [Re: brikogen] * 1
    #26695033 - 05/25/20 01:35 PM (3 years, 8 months ago)

Quote:

brikogen said:
Also If you use a syringe anyway eliminate the inoculation loop, one less point of failure. Flame sterilize the needle and squirt it directly in the plate. Try to spread it around. I never had good luck with a single drop using a MS syringe, I always carpet bomb my petris and start isolating from there.





Inoculation loop is fine.


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