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Offlineneonero
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Can you help me evaluating these Agar dishes?
    #27048334 - 11/20/20 06:07 AM (3 months, 12 days ago)

Hi,

I have one failed try going from spores to minitub. This is my second try, and I am trying to find the source of contamination. So this time, I was hoping to get some trained eyes to look at some parts of the process.

I have now started all over with spores to agar transfer. It is a premade Agar/Malt powder that I have mixed with water and sterilized in PC. Pour is in SAB. I have been so dedicated to detail as possible, not to contaminate the dishes.

How do you think they look?






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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27048404 - 11/20/20 07:56 AM (3 months, 12 days ago)

Im thinking you might want to try no pour tek..those plates look contaminated mate.Lets hope a TC chimes in for ya.


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Re: Can you help me evaluating these Agar dishes? [Re: Sunny Skies]
    #27048439 - 11/20/20 08:45 AM (3 months, 12 days ago)

I think I can see bacterial colonies on the agar, but it might just be bubbles from the pour.  I would at least try to take a couple transfers from the flatter/denser growth from colonies in the pic 1/2 dishes and see how they do on a new plate. Not sure I would trust pic 3 dish with that odd growth ring showing up on most of the mycelium colonies.


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Re: Can you help me evaluating these Agar dishes? [Re: PBJ710]
    #27048446 - 11/20/20 08:56 AM (3 months, 12 days ago)

I wouldn't even open them in my house let alone a SAB..are you using a syringe cause it looks like you sneezed on your plates.


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Re: Can you help me evaluating these Agar dishes? [Re: Sunny Skies]
    #27048452 - 11/20/20 09:09 AM (3 months, 12 days ago)

Quote:

Sunny Skies said:
I wouldn't even open them in my house let alone a SAB..are you using a syringe cause it looks like you sneezed on your plates.




I really don´t understand it. Using SAB and all techniques to keep it and my gloved hands clean. Used an inoculation loop and flamed it.


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27048456 - 11/20/20 09:15 AM (3 months, 12 days ago)

how long are you PCing your agar? maybe the issue is there?


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Re: Can you help me evaluating these Agar dishes? [Re: PBJ710]
    #27048460 - 11/20/20 09:19 AM (3 months, 12 days ago)

Quote:

PBJ710 said:
I think I can see bacterial colonies on the agar, but it might just be bubbles from the pour.  I would at least try to take a couple transfers from the flatter/denser growth from colonies in the pic 1/2 dishes and see how they do on a new plate. Not sure I would trust pic 3 dish with that odd growth ring showing up on most of the mycelium colonies.




Thank´s a lot for chiming in.

I am so confused now, that I even don´t know if I am growing mushroom spores or some contamination. Could you confirm that it is cubensis myc and not some air borne fungus?
I have studied all the pictures I can find on the subject, and to me it looks like myc, but a confirmation would be greatly appriciated:-)

You think I should let it grow some more before making a transfer from the dishes 1/2 that you suggest?


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Re: Can you help me evaluating these Agar dishes? [Re: Sunny Skies]
    #27048462 - 11/20/20 09:24 AM (3 months, 12 days ago)

Quote:

Sunny Skies said:
how long are you PCing your agar? maybe the issue is there?




Maybe. PC it for 1/2 hour right after filling the media bottle.


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27048478 - 11/20/20 09:33 AM (3 months, 12 days ago)

do you leave the bottle top a little loose and cover with foil?? Are you venting your PC prior to pressure build up?


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Re: Can you help me evaluating these Agar dishes? [Re: Sunny Skies]
    #27048843 - 11/20/20 01:54 PM (3 months, 12 days ago)

It looks like mycelium to me, but not sure if it's healthy.  Make a few transfers and see how it responds...it may take a few transfers to get good clean growth.

IMO, you want to take tiny transfer from a spore plate ASAP after growth shows so you can identify a healthy/clean colony to transfer from.  If they grow on top of each other, you won't be able to tell which colonies are desirable to keep as easily.  After it's on a new plate let it grow and see what shows up without all the competition going on.

Would you mind sharing your agar recipe and how you got to this point?  Sunny might be onto something with the details of the process being part of the issue.


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Re: Can you help me evaluating these Agar dishes? [Re: Sunny Skies]
    #27048930 - 11/20/20 03:11 PM (3 months, 12 days ago)

Quote:

Sunny Skies said:
do you leave the bottle top a little loose and cover with foil?? Are you venting your PC prior to pressure build up?




I left the bottle top a little open with foil over. When the PC was finishd, I let it cool a little bit, let out the steam and tightend the bottle top only touching the outside of the foil.

I did not vent the PC prior to pressure build up.


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27048974 - 11/20/20 03:34 PM (3 months, 12 days ago)

Quote:

neonero said:
Quote:

Sunny Skies said:
do you leave the bottle top a little loose and cover with foil?? Are you venting your PC prior to pressure build up?




I left the bottle top a little open with foil over. When the PC was finishd, I let it cool a little bit, let out the steam and tightend the bottle top only touching the outside of the foil.

I did not vent the PC prior to pressure build up.




Don't let out the steam. Let the PC drop to 0 PSI naturally then remove the bottles.


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Re: Can you help me evaluating these Agar dishes? [Re: PBJ710]
    #27049013 - 11/20/20 03:57 PM (3 months, 12 days ago)

Quote:

PBJ710 said:
It looks like mycelium to me, but not sure if it's healthy.  Make a few transfers and see how it responds...it may take a few transfers to get good clean growth.

IMO, you want to take tiny transfer from a spore plate ASAP after growth shows so you can identify a healthy/clean colony to transfer from.  If they grow on top of each other, you won't be able to tell which colonies are desirable to keep as easily.  After it's on a new plate let it grow and see what shows up without all the competition going on.

Would you mind sharing your agar recipe and how you got to this point?  Sunny might be onto something with the details of the process being part of the issue.




OK. I will do a transfer tomorrow.

The Agar is a premade Agar/Malt powder mix I got from at scientific store. It is made for mushroom cultivation.

How I got to this point:

Mixed Agar/Malt + Tap water. I live in a place with very clean water with no chlorine.
Boiled it. Filled media bottle. Little loose cap with foil over.
PC 30min. Cooled down a little bit. Let out rest of pressure. Thightend cap touching only foil and bottom of bottle.
Stored it.

Next day. Put Media Bottle in a water boiler that can keep the temp steady on 200*F until the agar has melted. I was a little quick on this one. There were som lumps in the Agar I had not detected.
Made SAB ready. First I wipe table with alcohol, place damp clean cloth on it, SAB on the cloth. I have an oven grate inside the SAB to get some space to the bottom. Also wiped down with alc.
SAB is cleaned inside with alc. Placed on table. Misted inside with water. Holes covered with cling film.
Let it rest a little bit.
I put on washed lab coat, nitril gloves over arm selves, a buff on my head, surgical face mask. OK. Before that, I have put all the things I need on the table.
I sit down. Wipe the stuff and my hands down with alc. Put unopend sterile dishes, media bottle, small clingfilm wrap and a drying paper soaked in alc in SAB.
Put in both hands. Open the dis wrap with sterile scalpel.
The pour is like this: https://www.shroomery.org/forums/showflat.php/Number/24336286#24336286
I then wrap edges with 2 inch wide clingfilm. Take the dishes out. Store them insde lunch zip lock bags in fridge.

When I inoculate, I have taken of the wrap on a dish. Washed it down with alc. Take out one hand, flame inoculation loop, put it back in the SAB, cool it in the agar of the receiving dish, take it back out, open my spore print wwhich is in foil, put the loop inside. Take it back inside the SAB. Inoculate in a Z pattern. Wrap dish with clingfilm. Put it in zip lock bag.

I think that´s about it.


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Re: Can you help me evaluating these Agar dishes? [Re: GrinchGrower]
    #27049017 - 11/20/20 03:58 PM (3 months, 12 days ago)

Quote:

GrinchGrower said:
Quote:

neonero said:
Quote:

Sunny Skies said:
do you leave the bottle top a little loose and cover with foil?? Are you venting your PC prior to pressure build up?




I left the bottle top a little open with foil over. When the PC was finishd, I let it cool a little bit, let out the steam and tightend the bottle top only touching the outside of the foil.

I did not vent the PC prior to pressure build up.




Don't let out the steam. Let the PC drop to 0 PSI naturally then remove the bottles.




Thank´s I will do. Also vent the PC before pressure build up.


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27049129 - 11/20/20 05:23 PM (3 months, 12 days ago)

You can keep your spore print in SAB.  Your agar is definitely clumpy which is from bad pour temp.  I like to get the agar in the PC and then go set up my SAB.  Remove Media Bottle(s) tighten lid and let them cool in SAB.  I've tried reheating it, which I know works for some people, the plates looked clumpy like yours; probably cause I rushed it.  I think you've got myc and will start to see it thin out after a transfer or two IMO 3rd plate looks best to me.


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Re: Can you help me evaluating these Agar dishes? [Re: neonero] * 1
    #27049272 - 11/20/20 06:52 PM (3 months, 12 days ago)

Hopefully you'll be pleasantly surprised after a couple transfers.

For the most part, things sound right.  I would suggest boiling the tap water before adding the agar mix so that it mixes more evenly.  I've seen this cause sediment and clumpy agar when I forgot to do it with BRF based agar (may not apply as much to ME agar though).

Storing the dishes in the fridge may not be necessary and can cause some condensation issues.  I leave mine out at room temp so if anything (contam) does make it to the plate, I'll be able to easily see it before using the dish instead of having a dormant hidden contam waiting to sabotage me.

I've always just tightened my media bottle caps down until it was snug (not tight) and never used any foil, but I don't think you're having any issues from that or you would see obvious contaminations.

I try to prepare my agar a few hours ahead of when I'm going to use it so I don't have to reheat it.  I pour in multiples of 20 even if I only need a few.  The unused ones can wait in a ziploc bag until I need them.


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Re: Can you help me evaluating these Agar dishes? [Re: PBJ710]
    #27052099 - 11/22/20 02:30 PM (3 months, 10 days ago)

I made some new Agar and poured right away after PC`ing it.
The pour came out very nice.

Today I made several transfers, and in a couple of days I will post pics of them. Hope you can help me along so I get a clean growth of mycelium in the end.

Best

NeoNero


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27055489 - 11/24/20 02:48 PM (3 months, 8 days ago)

It seems like you've gotten plenty of help so far from people who know a lot more than I do. But, do yourself a favor and give this link a thorough read. It taught me a lot about the use of a pressure cooker that I otherwise wouldn't have known. There's even a sub-link in there about the importance of venting and what it means/does. Cheers, mate!

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

Bod also has a really nice TEK about doing agar work


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Re: Can you help me evaluating these Agar dishes? [Re: bigfootscreepyuncl]
    #27056569 - 11/25/20 06:38 AM (3 months, 7 days ago)

Quote:

bigfootscreepyuncl said:
It seems like you've gotten plenty of help so far from people who know a lot more than I do. But, do yourself a favor and give this link a thorough read. It taught me a lot about the use of a pressure cooker that I otherwise wouldn't have known. There's even a sub-link in there about the importance of venting and what it means/does. Cheers, mate!

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

Bod also has a really nice TEK about doing agar work




Thank´s a lot for the link. I will look at it this evening. Most of what I have learned so far is from BOD.


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27056576 - 11/25/20 06:53 AM (3 months, 7 days ago)

Hi,

So now the transfers have grown a couple of days on the new Agar pour. The new Agar is PC the right way with venting of the PC.

The transfers came from plates with Agar PC without venting.

As you can see, some of the transfers have dark spots that looks like mold.

To me it looks like dish #6 is the cleanest, and that I should use that one for a transfer.

If you look close, also on #6, there is a narrow opaque looking ring around the white myc. Is this normal, or could it be bacteria?

So, what do you think I should do? Make a new transfer now? Wait until it grows some more, or make some new dishes from spores?









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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27056610 - 11/25/20 07:56 AM (3 months, 7 days ago)

A few plates look like they're having some issues, but 1 and 6 are looking the best so far (maybe 4 as well, but can't tell from pic).  I would give it another day or two so the culture can better show what you're working with.


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Re: Can you help me evaluating these Agar dishes? [Re: PBJ710]
    #27057018 - 11/25/20 01:45 PM (3 months, 7 days ago)

IMO give it a 3-4 days and take a transfer a cm or so on the inside edge.  I think what you are seeing are metabolites usually from the myc fighting a bacteria.


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Re: Can you help me evaluating these Agar dishes? [Re: IdiotCircusBoy]
    #27057933 - 11/26/20 01:00 AM (3 months, 7 days ago)

@PBJ710 @IdiotCircusBoy Thank´s a lot for the advice. I will let it go for a few days and take a transfer.
Right now, it looks like it is #6 that is the only one without mold coming out from the transferred lump of Agar.


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27063982 - 11/30/20 07:00 AM (3 months, 2 days ago)

Hi again,

The dishes have evolved, and it is clear that I am transferring contamination from the gen. 1 dishes (from spores) to gen. 2 dishes.
If you remember, the Agar on the gen. 1 dishes was PC incorrect.

I post pics of all the dishes again, just in the interest of science :-)

Do you think #6 will be useful for transfer?

What do you think is the source of contamination? Gen. 1 Agar, Spores, or airborne in SAB?









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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27064176 - 11/30/20 10:42 AM (3 months, 2 days ago)

Looks like 6 is the only candidate at this point.  I would take a couple tiny transfers from the leading edge and hopefully they will be clean(er).  It's entirely possible that the mold is still riding along with the mycelium at this point.  Personally I would transfer any good looking transfers 1 more time (assuming growth is orderly and not just a bunch of random fuzz) before expanding them, but it could probably be done directly from those T2 plates if they look really nice.


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Re: Can you help me evaluating these Agar dishes? [Re: PBJ710]
    #27064241 - 11/30/20 11:36 AM (3 months, 2 days ago)

Quote:

PBJ710 said:
Looks like 6 is the only candidate at this point.  I would take a couple tiny transfers from the leading edge and hopefully they will be clean(er).  It's entirely possible that the mold is still riding along with the mycelium at this point.  Personally I would transfer any good looking transfers 1 more time (assuming growth is orderly and not just a bunch of random fuzz) before expanding them, but it could probably be done directly from those T2 plates if they look really nice.




Thank´s a lot:-)

I will do so as soon as I get some spare time.  I´ll be back with the results when they have colonized a bit.

Best regards

N


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27064411 - 11/30/20 01:29 PM (3 months, 2 days ago)

I'm def no contamination expert but I think green is typically Trichoderma and, as I understand it, you can't transfer your way out of Trich.  If 6 isn't green then you may have a contender.


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Re: Can you help me evaluating these Agar dishes? [Re: IdiotCircusBoy]
    #27064536 - 11/30/20 02:33 PM (3 months, 2 days ago)

Quote:

IdiotCircusBoy said:
I'm def no contamination expert but I think green is typically Trichoderma and, as I understand it, you can't transfer your way out of Trich.  If 6 isn't green then you may have a contender.




Hmm. #6 is also a little green I think. I can try one transfer and see.


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27064964 - 11/30/20 06:14 PM (3 months, 2 days ago)

I was trying to be optimistic and hoping that it is just bruising.  We should be able to tell from the T2 plates how it's doing in regard to contams.  If they show bad stuff, I would start some fresh plates instead of fighting this out trying to clean it up.


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Re: Can you help me evaluating these Agar dishes? [Re: PBJ710]
    #27065615 - 12/01/20 01:12 AM (3 months, 2 days ago)

Quote:

PBJ710 said:
I was trying to be optimistic and hoping that it is just bruising.  We should be able to tell from the T2 plates how it's doing in regard to contams.  If they show bad stuff, I would start some fresh plates instead of fighting this out trying to clean it up.




:thumbup: :smile:


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27071517 - 12/04/20 12:03 PM (2 months, 29 days ago)

Hi,

It turned out that #6 also developed green mold before I could transfer, so I have made up new dishes and inoculated with spores.

I´ll post pics when they start to grow.

Best

Neo


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27071714 - 12/04/20 02:01 PM (2 months, 29 days ago)

Because I'm learning agar right now, could you please tell me how you think on your 1st attempt you got so many mycelium spots everywhere?

When you used the loop did you drag it all over your entire dish?  I ask because the amount of isolated(?) mycelium clumps from edge to edge of your dish confuse me.  I'm wondering if this is what others were reacting to at first.  I thought you only put a transfer in the middle, or drag your loop a few times.  Does this make sense?  thanks


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Re: Can you help me evaluating these Agar dishes? [Re: rashomon]
    #27073089 - 12/05/20 10:09 AM (2 months, 28 days ago)

Quote:

rashomon said:
Because I'm learning agar right now, could you please tell me how you think on your 1st attempt you got so many mycelium spots everywhere?

When you used the loop did you drag it all over your entire dish?  I ask because the amount of isolated(?) mycelium clumps from edge to edge of your dish confuse me.  I'm wondering if this is what others were reacting to at first.  I thought you only put a transfer in the middle, or drag your loop a few times.  Does this make sense?  thanks




Hi,

I used this streaking technique https://www.shroomery.org/forums/showflat.php/Number/24336286#24336286


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27073544 - 12/05/20 03:08 PM (2 months, 28 days ago)

Quote:

neonero said:


Hi,

I used this streaking technique https://www.shroomery.org/forums/showflat.php/Number/24336286#24336286




I had no idea this was a thing!!  Thanks for explaining!


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Re: Can you help me evaluating these Agar dishes? [Re: rashomon]
    #27082061 - 12/10/20 02:07 PM (2 months, 23 days ago)

Hi again,

OK, so I started over with spores. This time, the making of Agar and the pour was fine. I took the alu foil with the print inside the SAB after washing it with alcohol. Scraped a little bit loose, and holding the foil over the dish, shaking a little bit on the agar plate.

The pics were taken after 2-3 days. It really does not look promising? What do you think?





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OfflinePBJ710
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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27082237 - 12/10/20 03:46 PM (2 months, 23 days ago)

Looks like a little tuft of mycelium on #3 that you should be able to use if that's not bacteria behind it - it may take a few days for them to show more colonies.


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InvisibleIdiotCircusBoy
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Re: Can you help me evaluating these Agar dishes? [Re: PBJ710]
    #27082409 - 12/10/20 05:06 PM (2 months, 23 days ago)

#3 Maybe but its surrounded by something not kosher.  Did alcohol get on the spores or the plate?  I've not heard of the sprinkle on technique so am a little unsure about it.  The trick with a SAB is to not let stuff get above what you are working on.  So using a sterilized inoculation loop to swab the spores and swipe the plate accomplishes this because you're coming in from the side.  Your also using a streak technique so that spores are spread out on the plate.

Check out Bod's video on Spore Print to Agar.
https://www.shroomery.org/forums/showflat.php/Number/24337011#24337011


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Edited by IdiotCircusBoy (12/10/20 05:08 PM)


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Re: Can you help me evaluating these Agar dishes? [Re: IdiotCircusBoy]
    #27083214 - 12/11/20 01:10 AM (2 months, 23 days ago)

Quote:

IdiotCircusBoy said:
#3 Maybe but its surrounded by something not kosher.  Did alcohol get on the spores or the plate?  I've not heard of the sprinkle on technique so am a little unsure about it.  The trick with a SAB is to not let stuff get above what you are working on.  So using a sterilized inoculation loop to swab the spores and swipe the plate accomplishes this because you're coming in from the side.  Your also using a streak technique so that spores are spread out on the plate.

Check out Bod's video on Spore Print to Agar.
https://www.shroomery.org/forums/showflat.php/Number/24337011#24337011



Thank´s for the advice. I have used Bod´s tek in the past, but this time I did the sprinkle. I found it on youtube, but what I did not think of, was that it was used with a flow hood. I will make a couple of new ones with streaking this weekend.


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27083310 - 12/11/20 02:39 AM (2 months, 23 days ago)

All the growth underneath the black specks looks like bacteria.

The print may just be really dirty...

Someone suggested hot pouring a plate to get the myc racing away for a cleaner transfer. Might be worth a shot on #3.


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Re: Can you help me evaluating these Agar dishes? [Re: neonero]
    #27084101 - 12/11/20 04:49 PM (2 months, 22 days ago)

Did you use alcohol on the spore print?

Quote:


I took the alu foil with the print inside the SAB after washing it with alcohol.





No alcohol on the spores or plate.


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