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OfflineAlsetAlokin
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27137250 - 01/09/21 09:50 AM (3 years, 1 month ago)

Quote:

Uzimyco said:
Cubes grow in warm places. Florida Brazil, Hawaii, Thailand. These are all zone 10 and higher.




Before mushroom cultivation, they also only grew outdoors (and not in the quantity we're able to reproduce on here). No need to mimic their natural environment, just give them what they want.

Put your previous experience on hold for just a second and learn how others do it on here, THEN use your expertise to adjust and adapt.

People won't be able to help you diagnose your strange facilities because no one here uses them.

Making things simpler, consistent, and more efficient are the main goals in cultivation.


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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: jcm4620]
    #27137777 - 01/09/21 02:30 PM (3 years, 1 month ago)

Well wait then. Isn’t a glove box synonymous with still air box?  How would one work with anything inside of a closed box ?

Admittedly I’m the first to say that I realize the powerful limitations of any box set up. I hate it. As mentioned I’m patiently awaiting my filter so I can complete my flow hood, which I assume will be a major game changer. Hell it has to be.

I do think though that perhaps once reasonably perfected, agar is somewhat predictable but I would say there are far too many threads on the internet that talk about agar being simple and anyone can do it. I respectfully disagree.

I think agar is very unforgiving likely and requires outstanding levels of sterility. Why do I say that? Because I’ve done this with cakes many times, never had contamination issues and quite frankly wasn’t using the almost psychotic level of care I am now.

Yet every agar attempt has yielded some unforeseen problem. Again don’t misunderstand what I am saying.... I think agar is without question- the gold standard.  I just don’t think it’s quite the way some videos and threads portray. Again I’m saying this from some experience. Pf tek type stuff. Success success success and agar trouble trouble trouble. I know light years more than I did when I made the first cakes and those things blew up huge. Repeated many times.

Agar is challenging or at least so in my opinion without a flow hood. Since I’ve used hoods before in mericloning, meristem tissue extraction and fern spore plating im well aware of what the difference is. With a hood as long as it’s functioning properly and you follow the protocols- you’re basically good to go. With a box or otherwise and agar - it still appears to be a crapshoot.

My agar was pc at 15psi - 45 mins. I use high end lab individually wrapped sterile disposable innoc loops, syringes and uvc with an American lab dosimeter that ensures radioactive destruction of pathogens mechanically (there is no way to be tolerant to it. ). These are the same in research facilities and hospitals with CARF certification around the USA.  Yet still incur trouble?  I’m inclined to believe it’s a combination of agar and the box which no matter what you do at some point requires the entry or removal of something putting your work at risk whereas with a hood you have vastly more flexibility to work with things and ensure they remain in a sterile environment from a-z.  I’m not mycology pro but I’m no novice to science and sterility. 

Trust me. I’m VERY frustrated at myself about it. My experience should be helping to mitigate much of my failures here.  You’re looking at a person who did their first tissue culture plating likely well before most of the members of this forum were born. And believe me, there are numerous fern species growing in botanical gardens around the globe as the result of my spore work. Plants and fungi are not the same of course. Sterility often is.... and so I continue. I know it will get there. Hey, the bright side is , if it was super easy, everyone could do it. :thumbup:


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Thankfully the US Constitution ensures that my rights don't end where your feelings begin



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Offlinejcm4620
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27137800 - 01/09/21 02:42 PM (3 years, 1 month ago)

well then u should know the difference between a glove box and a still air box and understand how a vaccum created airflow would fuckin defeat the purpose of a still air box. hence the term STILL AIR🤷🏼maybe before tryin to impress everyone here with how vast ur scientific background is and writing a long ass post you should take a lil time and use the search engine on here in the upper right. it will tell you all u need to kno. you can basicly ask it a question. but that last long post  i didnt even bother to really read after the part about the glovebox not bein different i just kinda skimmed thru it as im certain thats causing ur problem and then ur placing it in the E Z bake contamination oven and making it even worse.

i bet u eliminate those 2 factors ur gnna be much better off👍


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PANAEOLUS FRUITING MADE SIMPLE



Edited by jcm4620 (01/09/21 02:44 PM)


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Offlinejcm4620
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Re: Anyone have link or pics of early stage agar inoculation? [Re: jcm4620]
    #27137812 - 01/09/21 02:49 PM (3 years, 1 month ago)

heres the difference




the attached gloves are a huuuuge fuckin no no. they create a push/pull vaccum effect with every single slightest movement stirring up the internal air just enuff to shit all over your work.


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PANAEOLUS FRUITING MADE SIMPLE



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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27137820 - 01/09/21 02:53 PM (3 years, 1 month ago)

These are some close ups of the growth in the original plates now. Maybe slightly better pics.

If anyone has thoughts that they are potentially good I’d want to do a transfer soon naturally. I’m not loving the new plates I did with dry spore. Different from the first set but I’m not clicking my heels.

Per my book above there may just be challenges with my box, or the spore prints or something else but I’d love to save something if possible. Please take a look and share your thoughts:




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InvisibleDoctor Mario
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Re: Anyone have link or pics of early stage agar inoculation? [Re: jcm4620]
    #27137836 - 01/09/21 03:00 PM (3 years, 1 month ago)

Check out the links that are in my signature. They're some of my favorite teks and I've ordered them in a way that is sort of from beginning to end as far as process goes. (Except for Bod's Teks) He has a big ass list of of teks he's made that work well for most people.

If you want to be successful, look at what everybody is doing or has done with great results. When you get a better understanding of what we're all doing and why, then you can make adaptions and other's will thank you for it. In the end, we're all after the same result. We want mushrooms and we want to see eachother succeed. This place and the people here can seem harsh at times but it really is for your own good.

Reddit, Discord and Youtube have some good things going on but I'd say about 95% of it is all unnessecary or misinformation. Stay away from those places. The search engine here should be your bible. If you have a question, someone has already asked it and it's documented there. Get familiar with it.

Those plates look good. That's what you're looking for.


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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27137848 - 01/09/21 03:08 PM (3 years, 1 month ago)

Thank you. I appreciate the distinction between the two.

Not entirely sure how using gloved hands in the box with open airspace or the action of inserting ones hands wouldn’t risk carrying pathogens into the box, but I do understand what you’re describing.

I’m absolutely not trying to offend you in the least and certainly not trying to make you angry. Due to my confusion on the issue I did some quick searching around and my misunderstanding is evidently very common as I found piles of threads on this and other forums where the semantics are routinely held synonymous.  Wish I had known. The concept of a still air box appears significantly easier to construct.

I will say however that my glove box is fully stripped so as to eliminate air pressure from moving from outside to within.  Glove boxes are routinely used in hazmat situations where the individual is to be protected from the contents. It seems counterintuitive that such a design would allow for free flow in and out as the person would risk exposure but again the distinction is genuinely appreciated.


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Thankfully the US Constitution ensures that my rights don't end where your feelings begin



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Offlinejcm4620
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27137852 - 01/09/21 03:10 PM (3 years, 1 month ago)

first off its not may be an issue with ur box. if u have gloves on it there IS an issue with ur box. a big one that will surely ruin all kinds of shit. so get a knife n chop that shit off completely.

as for ur plates that first one looks narly and i wouldnt ever touch anything close to an edge of a plate like that. now the second pic if u want u can take tfansfers from about the 2:00-5:00 positions and see what happens but dont place ao close to the edges. also u need to be using spore prints not syringes. streak in a zig zag pattern. plenty of good info in the search to belp ya along


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PANAEOLUS FRUITING MADE SIMPLE



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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Doctor Mario]
    #27137862 - 01/09/21 03:17 PM (3 years, 1 month ago)

Thank you Mario. I have looked at the links in your signature. Had done so before actually.

Not sure why everyone seems to think I’m looking to reinvent the wheel here. It’s simply not the case. I’m really not looking to do anything outside of what’s been described.

I did read your entire thread some time ago on the differences in mycelium vs a pathogenic mold. It’s very hard to tell or maybe my eyes are having a hard time making the distinctions. I know you notated the differences in the puffier fluffy look vs the coarser crunchy look. I am having a hard time
Telling the difference. I did post up some new pics of the spots in question with hopefully some better clarity. Hoping you all might be able to identify it?


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Thankfully the US Constitution ensures that my rights don't end where your feelings begin



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Offlinejcm4620
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Re: Anyone have link or pics of early stage agar inoculation? [Re: jcm4620]
    #27137865 - 01/09/21 03:17 PM (3 years, 1 month ago)

i think u care more about playin with fancy lookin garbage than u do about getting good results


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PANAEOLUS FRUITING MADE SIMPLE



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Offlinejcm4620
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Re: Anyone have link or pics of early stage agar inoculation? [Re: jcm4620]
    #27137869 - 01/09/21 03:19 PM (3 years, 1 month ago)

ur not as smart as u think u are here man ive already explained it. its called a vaccum im really tryin to help u here man ditch that thing


and u may very well have it sealed perfect but guess what theres fuckin air inside of it. or do u suck all that out with another mr wizard unit while ur working??


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PANAEOLUS FRUITING MADE SIMPLE



Edited by jcm4620 (01/09/21 03:31 PM)


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InvisibleDoctor Mario
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27137878 - 01/09/21 03:26 PM (3 years, 1 month ago)

Quote:

Uzimyco said:
Thank you Mario. I have looked at the links in your signature. Had done so before actually.

Not sure why everyone seems to think I’m looking to reinvent the wheel here. It’s simply not the case. I’m really not looking to do anything outside of what’s been described.

I did read your entire thread some time ago on the differences in mycelium vs a pathogenic mold. It’s very hard to tell or maybe my eyes are having a hard time making the distinctions. I know you notated the differences in the puffier fluffy look vs the coarser crunchy look. I am having a hard time
Telling the difference.




It'll come with experience. You'll grow mold and eventually grow mycelium while you're still learning and then it will all make sense and you'll be able to tell the difference between the two when they're still barely visible colonies just beginning to form. If you have doubts, take a transfer and let it grow.

What JCM is explaining to you about the glove box being a bad idea is that when you make a proper glove box the air becomes perfectly still. Mold spores and other microscopic shit settles on the floor of the box or anything that it comes in contact with. When you put your hands into the gloves and move your arms, it creates a positive (or negative) air pressure which is enough force to pull anything off the ground and stir it around into the air allowing it to fall into your plates or anything else. With a still air box, the concept is similar except when everything settles on the floor, it stays there when you use slow movements. Because the box isn't sealed the pressure remains at 0.

I've seen all kinds of box ideas. Fans running with filters attached, bowls of water that supposedly draw the contaminants in because of static electricity... :smbfacepalm:

There's a reason nobody is using any of that stuff.


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Edited by Doctor Mario (01/09/21 03:29 PM)


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Offlinejcm4620
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Doctor Mario]
    #27137883 - 01/09/21 03:28 PM (3 years, 1 month ago)

you want a still air box not a glove box

bingo

all of which could have been found via the search in 30 seconds


Edited by jcm4620 (01/09/21 03:30 PM)


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InvisibleDoctor Mario
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Re: Anyone have link or pics of early stage agar inoculation? [Re: jcm4620]
    #27137899 - 01/09/21 03:35 PM (3 years, 1 month ago)

I feel it's also worth mentioning that my sterile tech is complete shit. I'm staring at 28 contaminated plates as we speak. They never even got to see spores before they were laid to rest.
:futuramamario:

There's a reason JCM has the TC tag and not me. If you're gonna listen to anyone here, listen to the TC's.


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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: jcm4620]
    #27137907 - 01/09/21 03:38 PM (3 years, 1 month ago)

Thanks jcm.  That is what I will do. Sadly they are the same plate at different locations but based on your descriptions and looking at the pics I see what you’re saying.

The first picture shows no threadlike growth around it so it likely is not very good.

2 o’clock and 5 o’clock on the other show clean thread like growth whereas say, 9 o’clock looks muddled and fuzzy.

That is amazingly helpful as I think I have an idea what I’m looking at based on what you’ve advised. Am I interpreting what you’ve said correctly?


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Thankfully the US Constitution ensures that my rights don't end where your feelings begin



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InvisibleDoctor Mario
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27137915 - 01/09/21 03:40 PM (3 years, 1 month ago)

Quote:

Uzimyco said:
2 o’clock and 5 o’clock




:thumbsup:


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Offlinejcm4620
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Doctor Mario]
    #27137927 - 01/09/21 03:44 PM (3 years, 1 month ago)

2 thru 5 take several and see what ya get. but plz dont do it in a gloved box ul really increase chances of failure.


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Offlinejcm4620
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Re: Anyone have link or pics of early stage agar inoculation? [Re: jcm4620]
    #27137934 - 01/09/21 03:48 PM (3 years, 1 month ago)

u should be streakin plates in a zig zag pattern like so.  (just note this is pan myc)



or c10 has another very good streak method but i always just streak like my pic above.


heres a cube tissue clone



just cube myc


this plate was in the fridge for a long time which is why it looks like it does but if i was to want to take transfers off it id take from the leading edgeds of the sectored areas



then this is why u should stay away from syringes and why u should never go spores go grain




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Edited by jcm4620 (01/09/21 03:54 PM)


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OfflineUzimyco
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Doctor Mario]
    #27137958 - 01/09/21 03:57 PM (3 years, 1 month ago)

It was a quick and painless surgery. :grin:

Haha thank you!




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Offlinejcm4620
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Re: Anyone have link or pics of early stage agar inoculation? [Re: Uzimyco]
    #27137964 - 01/09/21 03:58 PM (3 years, 1 month ago)

good deal make sure u got all the sleeve parts too. u dont want nothin that can move


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