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InvisibleTheDoobskerS
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Input on these agar plates?
    #26801462 - 07/03/20 01:25 PM (3 years, 7 months ago)

Happy Friday to all you beautiful people

Getting ready for my 2nd grow here with the game-plan being:

KSSS spore syringe -->  BRF cakes --> Spawn to coir in a shoebox  -->
  Clone the best looking and any good looking mutants from each flush  -->  Clean up on agar  -->  Inoculate grain jars with agar wedges --> Spawn grain jars to coir in multiple shoebox grows


I also nocced up some plates from the MS syringe. I'm not completely convinced this is mycelium because it's not even white. I'm also not convinced it's all bacteria because that just doesn't make any sense to me.
I am pretty confident that I made clean agar plates and even left one plate to sit without inoculating; nothing grew. The inoculation was done in a SAB, quickly, flame sterilized, alcohol everywhere, gloves, clean long sleeves, both sprayed heavily with IPA, parafilm wrapped shortly after.
The syringe was provided by XXXXX(trusted vendor), so I'm sure it was fairly clean. Also the brf cakes I inoculated are looking beautiful.





What do you guys think?

Squirted onto first plate straight from the syringe (squirted out a bit too much on accident, maybe 1/4cc max).




Made the transfer to a 2nd plate on June 26th. This is what I have now on July 3






This plate I inoculated by scraping spores off a print I made from my last batch of Nepal Chitwans. Also inoculated on June 26. Upon a few searches, I'm thinking this is clean mycelium but just wanted to get another opinion. (Sorry I know that condensation makes it hard to see)



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Edited by TheDoobsker (07/03/20 02:22 PM)


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OfflineJus
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Re: Input on these agar plates? [Re: TheDoobsker]
    #26801477 - 07/03/20 01:33 PM (3 years, 7 months ago)

The syringe and transfers look like bacteria to me. Slimy looking, flat, irregular outline...

The spore print plate in the second pic looks like it had a shiny outline, possible contamination.

Try scraping the spore print with an inoculation loop or similar tool and scraping in a Z pattern to get some clean cultures towards the bottom of the plate.


Edited by Jus (07/03/20 01:34 PM)


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OfflineTheGreatGig
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Re: Input on these agar plates? [Re: TheDoobsker]
    #26801483 - 07/03/20 01:38 PM (3 years, 7 months ago)

Remove venders name from the post. Pretty sure that's a main rule when posting anything about cultivating. Other than that, looks like you are transferring nothing but bacteria, doesn't look like mycelium


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OfflineGastronomicus
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Re: Input on these agar plates? [Re: TheGreatGig]
    #26801545 - 07/03/20 02:12 PM (3 years, 7 months ago)

What they said. First set of plates and transfers are pure contam. You have myc in the last 2 pictures though. Take some transfers from there


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Make my Funk the P Funk, I wants to get Funked up

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InvisibleRoger Clemency
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Re: Input on these agar plates? [Re: Gastronomicus]
    #26801575 - 07/03/20 02:25 PM (3 years, 7 months ago)

Yuuup. The last two pics of that plate show bacteria thickening up around the germ site. You want to try to grab some myc as soon as you can because it looks now like the bacteria is grown out farther than the myc so you will still be fighting it on the next transfer. but you can DO iit


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Sour grapes, sweet revenge
Heaven starts right where hell ends


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InvisibleTheDoobskerS
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Re: Input on these agar plates? [Re: Jus]
    #26801591 - 07/03/20 02:33 PM (3 years, 7 months ago)

Quote:

Gastronomicus said:
What they said. First set of plates and transfers are pure contam. You have myc in the last 2 pictures though. Take some transfers from there





Bummer, but this answer doesn't surprise me.

Any ideas on what went wrong with the syringe plates? No mycelium growth whatsoever and a whole bunch of bacteria confuses me. I would be less confused if there were also signs of myc growth. I most definitely got lots of spores onto this plate from the syringe. Is this a common problem when trying to go from MS to agar? Maybe just so much bacteria in total that the spores never germinated?


Quote:

Jus said:
The spore print plate in the second pic looks like it had a shiny outline, possible contamination.

Try scraping the spore print with an inoculation loop or similar tool and scraping in a Z pattern to get some clean cultures towards the bottom of the plate.




That's a good idea. I'll probably continue to try cleaning this one up with transfers but will also inoculate a couple plates using the streaking method.


So this is probably a pool of bacteria that my myc is in the middle of. Then where do I even transfer from? Won't I get bacteria no matter where I cut from?


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OfflineElViajero
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Re: Input on these agar plates? [Re: TheDoobsker]
    #26801607 - 07/03/20 02:39 PM (3 years, 7 months ago)

yes youll get bacteria, hopefuly the myc will outgrow the bacteria in the next agar plate, and after that you snatch a piece of the edge into a new clean agar


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OfflineGastronomicus
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Re: Input on these agar plates? [Re: TheDoobsker]
    #26801728 - 07/03/20 03:52 PM (3 years, 7 months ago)

Yeah you pretty much said it yourself. Basically the syringe was contaminated and when you squirted a bunch of liquid onto the plate a bunch of bacteria came with it. The myc lost the race to get to be alive because bacteria is faster.

If you're gonna use a syringe to go to agar (a lot of folks advise against this) try to only use a single drop.


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Make my Funk the P Funk, I wants to get Funked up

LAGM2024


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OfflineJus
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Re: Input on these agar plates? [Re: Gastronomicus]
    #26801802 - 07/03/20 04:41 PM (3 years, 7 months ago)

Sometimes syringes are just not good for agar.
The one syringe I've ordered (from a trusted vendor) gave me plate after plate of contamination with no spores germinating.

I was dipping my inoculation loop in some of the solution, then streaking it.

I still have two plates that never developed contamination just sitting there, in the hope of a Christmas Miracle.


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Offlinejcm4620
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Re: Input on these agar plates? [Re: Jus] * 1
    #26801925 - 07/03/20 05:48 PM (3 years, 7 months ago)

lol this is from a very trusted vendor. prolly the most trusted vendor there is even.  at least i think so but this is a prime example why prints are always better when using agar.



spore syringes no matter where you get them will usually always have some contams of some sort. its just what happens. most spores themselves are dirty before even being put into a syringe. all they can do is try to be as clean as absolutely possible and do the best they can and only get their prints from trusted proven growers who take the time to make their prints as clean as possible to limit the amount of contams. i mean its not like a  vendor here in the us is cranking out completely sterile fruits in a giant lab and taking completely sterile prints and making completely sterile syringes.


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PANAEOLUS FRUITING MADE SIMPLE



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InvisibleTheDoobskerS
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Re: Input on these agar plates? [Re: jcm4620]
    #26802158 - 07/03/20 07:44 PM (3 years, 7 months ago)

Sounds perfect thanks for all the input. You guys definitely clarified this for me.

I had read a lot about syringe --> agar not being a very good practice, but I also saw that some people do have success with it. After trying it though, I don't think I'll try it out again. I think I'll be better off doing a MS grow and then cloning/printing some fruits.

I have some agar in the PC right now so I can do this transfer later tonight. Will also streak a couple plates from the rest of my NC spore print.


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OfflineGan
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Re: Input on these agar plates? [Re: TheDoobsker]
    #26802223 - 07/03/20 08:21 PM (3 years, 7 months ago)

If you keep having issues with the bacteria and myc being so intertwined a couple transfers down the road, you can try a hot pour and/or agar sandwich. I've only ever done it a couple times when I had a print that I stored terribly and it was so contaminated, but I eventually cleaned it up.


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InvisibleTheDoobskerS
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Re: Input on these agar plates? [Re: Gan]
    #26802411 - 07/03/20 10:17 PM (3 years, 7 months ago)

Quote:

Gan said:
If you keep having issues with the bacteria and myc being so intertwined a couple transfers down the road, you can try a hot pour and/or agar sandwich. I've only ever done it a couple times when I had a print that I stored terribly and it was so contaminated, but I eventually cleaned it up.




Cool I'll do some searching on that. Hopefully it will clean up easily, but if not, I'll try it out.


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Offlinejcm4620
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Re: Input on these agar plates? [Re: TheDoobsker]
    #26802471 - 07/03/20 11:23 PM (3 years, 7 months ago)

or do what i do and make up a small babyfood jar of grain and knock it up and pluck kernals to plates to clean up


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PANAEOLUS FRUITING MADE SIMPLE



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InvisibleTheDoobskerS
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Re: Input on these agar plates? [Re: jcm4620]
    #26802496 - 07/03/20 11:57 PM (3 years, 7 months ago)

Quote:

jcm4620 said:
or do what i do and make up a small babyfood jar of grain and knock it up and pluck kernals to plates to clean up




That's an interesting idea. You inoculate from a syringe? Would you say it's a method that works well for you?


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OfflineForevergreen
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Re: Input on these agar plates? [Re: TheDoobsker]
    #26802595 - 07/04/20 01:03 AM (3 years, 7 months ago)

Ive made over 100 plates this month with syringes from 2 different vendors and have had 0 luck with any. Not a single speck of mycelium. Prints are supposedly the way to go so I ordered some, on top of our boy JCM here sending me some too. Keep your head up and keep noc'in up dishes til we get something bruh!.


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InvisibleTheDoobskerS
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Re: Input on these agar plates? [Re: Forevergreen]
    #26809510 - 07/07/20 05:18 PM (3 years, 7 months ago)

Thank you all for the advice.


I made a transfer from the NC plate into 2 new plates. As expected, I think I do see an outline of bacteria, but it's not nearly as obvious. The original had a real shiny ring around. This one appears to have a small ring that isn't white around the myc but it doesn't look slimey.



And here is the other plate I transferred to.



The growth is obviously much better than the original plate which I still have growing and looks like shit




I'm wondering if I should transfer these ASAP and also if I should be cutting a wedge from the outer edge of growth or from the middle.


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Offlinew00tmycelium
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Re: Input on these agar plates? [Re: TheDoobsker]
    #26809529 - 07/07/20 05:27 PM (3 years, 7 months ago)

Quote:

TheDoobsker said:
Thank you all for the advice

I'm wondering if I should transfer these ASAP and also if I should be cutting a wedge from the outer edge of growth or from the middle.




I've seen the same bacteria on plates before, your originals were pretty bad looking and the new ones are looking way better.  A few days ago people here suggested I take a rice-sized chunk out of the outer area.  Can anybody explain why taking cultures from the outside is better than the inside?


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OfflineJus
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Re: Input on these agar plates? [Re: w00tmycelium]
    #26810081 - 07/07/20 11:40 PM (3 years, 7 months ago)

It's easier to see if the outside edge is contaminated, and when trying to select transfer sites you can often see where the mycelium is growing strongest, thus which site may have favourable genetics


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