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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: AlsetAlokin]
    #27131874 - 01/06/21 10:13 PM (3 years, 1 month ago)

Many thanks for this post. The kind of info I’m looking for.

Yes it’s been 3-4 days since inoculation.

I appreciate that input. I maintain my offer if anyone decides to chat with a goofball for 10-15 mins. Understand if no takers and I appreciate the help everyone is giving. I’m a science hobbyist snd I don’t like being unsuccessful. I’m usually not.

This confuses me. My new flow hood will be compelete in the next couple of weeks when the damn filter gets in. Has to be better than the box. Cant stand the SAB


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InvisibleJosex
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Re: Anyone have link or pics of early stage agar inoculation? [Re: AlsetAlokin]
    #27131877 - 01/06/21 10:16 PM (3 years, 1 month ago)

People usually work in a still air box or in front of a laminar flow hood. I've heard some professional hoods come with a bulb like that but you won't ever really need that for any sterile procedure in mycology.

That bacteria is located on the areas you streaked, which makes me think the inoculum (spores) was dirty, it's either that or the disposable loops, but I admit those look legit. The weird thing here is that you said you used a print and even sloppily made prints won't give ya that much bacteria unless they weren't allowed to dry properly. Are both prints from the same source? How old are they?

also;
Quote:

AlsetAlokin said:
After innoc from spores nothing happens on days 1-3. That's probably why ya can't find anything.

Germination usually happens between day 5-14 IME.

You got some bacterial blotches there, give it some time. Myc might show up in a week.




:whathesaid:


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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Josex]
    #27131882 - 01/06/21 10:17 PM (3 years, 1 month ago)

Yes both prints from the same vendor.

Dated 11/20


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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27131887 - 01/06/21 10:21 PM (3 years, 1 month ago)

Only thing I can think is I used some distilled water. Boiled and put into beaker. First placed spores into cooled water and let sit covered in SAB (and uvcd as well).

Maybe should have just used dry spore???


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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27131892 - 01/06/21 10:23 PM (3 years, 1 month ago)

At any rate I think you have some myc in the third pic you posted. Mario circled it for you. You could transfer that to a new plate.

Quote:

Uzimyco said:
Only thing I can think is I used some distilled water. Boiled and put into beaker. First placed spores into cooled water and let sit covered in SAB (and uvcd as well).

Maybe should have just used dry spore???




Ok problem solved lol

Yea man, you seriously fucked up there.

Use dry spores, of course.


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OfflineAlsetAlokin
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27131898 - 01/06/21 10:26 PM (3 years, 1 month ago)

BINGO.

We found our winner, I was also puzzled at how wet those streaks looked.

DRY spores work fine!

If they're old and stubborn you can hydrate them using a swab maybe, but that shouldn't be necessary.

There's still hope though, granted you didn't use a liter of water to one small print there's probably some spores mating in those dishes as we speak.

The bacterial blotches are likely caused by your use of water. Try again with a dry print.


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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: AlsetAlokin]
    #27131911 - 01/06/21 10:31 PM (3 years, 1 month ago)

Ok got it.

I saved spores just in case so I’ll redo with dry.

Still don’t understand however how the water is the vector.  Boiled the shit out of it snd it went into a lab quality test tube. Sealed. Moved to SAB then poured into a sterile beaker. Covered beaker with sterile foil that was cleaned with iso and physan first?  Just sat there for about 6 hrs but can’t imagine how boiled distilled could be the culprit but don’t get me wrong. I’ll redo it tomorrow dry. Just confused about the water??


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OfflineAlsetAlokin
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Re: Anyone have link or pics of early stage agar inoculation? [Re: AlsetAlokin]
    #27131913 - 01/06/21 10:32 PM (3 years, 1 month ago)

Spores to agar

Here's some videos if you're more of a visual learner. None with dry spores to agar but you can reference the spore swab video in replacement of dry spores and an inoculation loop.


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Re: Anyone have link or pics of early stage agar inoculation? [Re: AlsetAlokin]
    #27131916 - 01/06/21 10:34 PM (3 years, 1 month ago)

I was like how the hell did he get that much bacteria from a damn print?? Fuck that's even too much even for dirty spore solution lol
Even if you were to fold the print still wet I doubt it would give ya so much bacteria on agar, so it was obvious something else was up. Glad we found the last piece of the puzzle.


Edited by Josex (01/06/21 11:02 PM)


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OfflineAlsetAlokin
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27131920 - 01/06/21 10:37 PM (3 years, 1 month ago)

Quote:

Uzimyco said:
Ok got it.

I saved spores just in case so I’ll redo with dry.

Still don’t understand however how the water is the vector.  Boiled the shit out of it snd it went into a lab quality test tube. Sealed. Moved to SAB then poured into a sterile beaker. Covered beaker with sterile foil that was cleaned with iso and physan first?  Just sat there for about 6 hrs but can’t imagine how boiled distilled could be the culprit but don’t get me wrong. I’ll redo it tomorrow dry. Just confused about the water??




Waters a vector, the pot it was in was a vector, the transfer of water to the tube is a vector, the iso is a vector, the foil is a vector.

Sometimes with this, the simplest way is the most sterile way and there's no need to complicate it,

The methods described in the TEKs here have been tested for years by hundreds of members, follow them and then you can deviate if you found a better way.


Edit: benefit of the doubt you could have all this extravagant equipment that could help you, but that print probably had some bacteria in it and you gave it a media and environment to multiply in. If you go dry spores to agar that myc will likely out compete the bacteria to a point where you can make a decent transfer.


Edited by AlsetAlokin (01/06/21 10:42 PM)


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Re: Anyone have link or pics of early stage agar inoculation? [Re: Josex]
    #27131922 - 01/06/21 10:39 PM (3 years, 1 month ago)

UVC is def unnecessary. I've used loops like that at a clinical lab I used to work at. Its probably better to just use cottonswabs sterilized in foil in the PC. If you feel good about the print and your technique you can save that swab instead of going back to the print next time. But all that bacteria around the inoculation site means something's up. Usually that the inoculant is bacteria laden. Could come from condensation and mishandling of the plate, but have to keep your mind on many other factors in applied microbiology.

Edit: yeah, the water's unnecessary and not done correctly. Just boiling won't sterilize, have to pressure cook (similar to autoclaving). Just use a sterile swab and swab it on some firm agar.


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Re: Anyone have link or pics of early stage agar inoculation? [Re: AlsetAlokin]
    #27131924 - 01/06/21 10:40 PM (3 years, 1 month ago)

Yea you did too many unneccessary steps, all of them huge vectors.


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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Josex]
    #27131949 - 01/06/21 11:07 PM (3 years, 1 month ago)

Understood

Thanks to all. I got thrown off by people saying “just make  a spore syringe snd drop a drop in the middle. Figured ok. But this way I could easily streak it.

Well - fuck up #10, #11?  Christ. Just frustrating.

Again much thanks to you all for your insight. I seem to have to learn this from endless mistakes. Just so much contradictory info out there. Hard to parse out correct and incorrect.

Thank you again genuinely. Tomorrow I’ll try again. Wonder if I should pressure clean my refrigerated plates first ?  Couldn’t hurt right?

-update-  agar was pc before poured. But why not re pressure cook while already in the dishes?  Not a bad idea right?


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Edited by Uzimyco (01/06/21 11:09 PM)


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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27131957 - 01/06/21 11:16 PM (3 years, 1 month ago)

You can't sterilize petris, they'll melt unless they're borosilicate petris and even then, the agar would boil over and they'll come out empty after the cycle. You can use those that are in the fridge no problems.

Incidentally, not the best idea to store blank plates in the fridge. If there's bacteria present due to an insufficent cycle or an user error when you poured them, you want it to grow so you know not to use those plates.


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OfflineAlsetAlokin
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27131959 - 01/06/21 11:17 PM (3 years, 1 month ago)

Quote:

Uzimyco said:
Thank you again genuinely. Tomorrow I’ll try again. Wonder if I should pressure clean my refrigerated plates first ?  Couldn’t hurt right?

-update-  agar was pc before poured. But why not re pressure cook while already in the dishes?  Not a bad idea right?




If you have glass plates I've seen people sterilize them in ovens, most people here use pre-sterilized single use 100mm plates.

Use the "search posts" button in the banner up top and narrow your results accordingly. Try to use up to date information, if it's old there's a good chance we found a better way to do it.

Key words, search: glass petri, no pour, holy grail plates, agar - click the "trusted cultivators" box for sound advice, and be skeptical of most other (including myself) a lot of us are new here or only skilled at a few things.


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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: AlsetAlokin]
    #27131983 - 01/06/21 11:48 PM (3 years, 1 month ago)

Many many thanks again. 

My dishes are all borosilicate glass but the over boiling of agar makes it a no go.


Illl try some refrig. Hate to make new agar 🧫 with 20 parafilmed plates.

Otherwise guess. It’s yet another do over.

I’m thankful for the experienced voices and advice. Many thanks to you all!


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Edited by Uzimyco (01/06/21 11:50 PM)


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InvisibleDoctor Mario
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27132311 - 01/07/21 07:14 AM (3 years, 1 month ago)

You're asking the right questions and maintaining a good attitude. I hope to see your future progress. Let's get you a rating.


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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Doctor Mario]
    #27134079 - 01/07/21 09:04 PM (3 years, 1 month ago)

Thank you very much!  Again the advice is so genuinely appreciated!! :thumbup:

I admit, I’m not a fan of the internet for more reasons than I can list.. The forums of the internet, such as shroomery are one of the very few exceptions in my opinion, Information is shared purely out of the desire to teach and assist. I think there are few things on the internet that offer such value.

*I did plate 6 dishes tonight with dry spore as instructed and I took extra precaution to ensure I didn’t miss anything that I could disinfect.

Ironically as I went to empty the incubator, I noticed one plate that looked like it might be trying to out run something. Pic is below. I left it and 1 other in with the new plates as they looked like maybe something positive was brewing.  High hopes for this new batch.

Here is the one that looked like maybe it was something. If not, maybe something bad but I thought it was worth a bit if a grow out. With no real frame of reference it’s certainly a challenge to know what it is (the pics I posted yesterday were taken within minutes of posting so the one day certainly made some difference. This pic was from 30 mins ago). It’s not in great focus, there is glare from the lights and some overlapped parafilm. I don’t know if I could have possibly added any more detracting features to the photo if I tried.




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InvisibleDoctor Mario
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27134162 - 01/07/21 10:10 PM (3 years, 1 month ago)

It's definitely hard to tell because of the low quality photo. It looks like myc but I've encountered mold that looks like that too.


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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Doctor Mario]
    #27134707 - 01/08/21 08:27 AM (3 years, 1 month ago)

Agreed. No way to tell and certainly not by my naive eye.

I’ll keep you all updated and perhaps you can tell more in some time. The 6 additional plates I did with dry spore yesterday are a bit ridiculous. So much spore it looks like a race track on the agar, but I went with the “more is better” concept. Just figured at least if something grows out I can take a
Section and move on but I was definitely “heavy handed” to say the least.

Always thankful and appreciative for your experience snd assistance!!


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