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OfflineNunyabiz
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Agar...more like Ughar - need expert interpretation
    #25246314 - 06/03/18 01:20 PM (5 years, 7 months ago)

Howdy,

Agar is killing my soul... First grow was successful pftek. All 36 cakes came out as expected, no contams, 3 flushes. Great!

Moved up to monotub attempt. Using mostly Bod's and Pasty's teks. Bought 6 new spore syringes as my first attempts with prints were marginal at best. Agar contams like crazy using SAB and lots of sterile supplies (gowns, gloves, drapes, etc). Green mold everywhere. Work with surgical aseptic technique professionally. After a couple hundred plates, managed to get 5 clean spawn jars (oats) to 100%. Went to the tub and got green stuff a few days in. Son of a....

I live in a very humid climate. So, I thought, build a Flowhood to fight the mold. 24x24 with the dual speed Dayton blower. Tested out with good flow. Poured 60 plates and maybe one in 10 grew anything.

I use the standard 10 agar 10 ME and 500cc distilled. I have tried all kinds of agar recipes. I am using telephone brand agar. I have tried 3 different types of ME (barley, light, and regular) I have also tried Pasty's PDA. I have added yeast as well. I have slightly altered the ratios with less agar and more ME. I have used petris, mini rounds, glass half pints all with the same limited results. I have tried many variations with seemingly crappy results. I have done hundreds of plates and transfers. Yet I currently have only 10 decent jars percolating. I am very frustrated and borderline disheartened. On the verge of going back to PFtek.

The flow hood cut down on the contams greatly (now maybe 10% which is still very high compared to what I have read). However, very little is growing. Since I am using spore syringes, I take a 1cc syringe and aseptically fill it from the 10cc spore syringe. I make a little scratch in the center of the petri and transfer .1cc per petri. I follow the inoculation techniques I found on here. I own and have watched the mushroom video series. I have not used inoculation loops. I have them. I just hadn't seen them used with spore syringes. I had only see them used with prints. I plan on trying that later today.

Any advice would be greatly appreciated. The following pics are 10 days out. 8 out 10 of my plates resemble these. No mycelium. I have six different syringes and they all look similar. No idea what's goin on... Too much condensation? Dilute syringes? Thanks in advance for the expert input.













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Invisibleelasticaltiger
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Re: Agar...more like Ughar - need expert interpretation [Re: Nunyabiz]
    #25246357 - 06/03/18 01:52 PM (5 years, 7 months ago)

So you've tried all your syringes and all you get is bacteria?

How are you sterilizing your agar?


--------------------
First time growing cakes? DON'T make a Shotgun Fruiting Chamber

The Shmuvbox. - The Old TC's Like it :shrug:

Afraid to Start Growing From Your Own Prints? Drop it Like a Tiger!
No Pouring. No Syringes. No Cutting. No flaming. No Contamination. No Bullshit.

"The best thing to do while your waiting is to start more stuff. I usually got so much happening that I have tossed projects simply because I didn't have time for them. -Pastywhite QFT

Pastywhite's Easy Agar Tek (PastyPlates)

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EZEKIEL 23:20


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OfflineNunyabiz
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Re: Agar...more like Ughar - need expert interpretation [Re: elasticaltiger]
    #25246397 - 06/03/18 02:24 PM (5 years, 7 months ago)

Is that bacteria in the pics? I get a few moldy plates and one or two with mycelium. The syringes are from Micro Supply. I also have syringes from Ralphsters. Same results.

I heat up the 500cc agar/mea mix on the stove. Bring it to a boil while stirring to make sure it mixes well. Then I transfer it to a 1000cc beaker and pour it into a 500cc Pyrex media bottle. Then I put it in the PC (Presto 23qt) on high until it purges for 10 mins. I then pressure to 18psi and turn down the heat so it cooks at 18psi for 20 mins. I then turn off the burner and let it depressurize. I take it out of the PC and wipe with alcohol and place in front of the Flowhood (with the blower on high). I have an IR temp gun. I check the temp and pour when it drops to around 140.

Rarely one turns out great



And I also get a few with mycelium and the green mold that I will try to save with a transfer using the edges away from the moldy side but turning the moldy side away from the flowhood so that I don't blow the mold into the clean stuff.



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InvisiblebodhisattaMDiscordReddit
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Re: Agar...more like Ughar - need expert interpretation [Re: Nunyabiz]
    #25246407 - 06/03/18 02:28 PM (5 years, 7 months ago)

Technique is off getting mold around the edges of plates like that

Practice is the best thing you can do and your trying so :thumbup:


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OfflineGeinstein
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Re: Agar...more like Ughar - need expert interpretation [Re: bodhisatta]
    #25246426 - 06/03/18 02:38 PM (5 years, 7 months ago)

OK wow 1 cc per dish is way to much of course your going to fuck up your looking for like 1 drop
I like to place 1 drop at the far end of the dish then use inoculation loop to make a zig Zak pattern to fin out the liquid even more.
Agar work at it's core is 1 to clean things up and 2 to isolate strains
That's way more spores mean leas success(and typically more water means more room for contam)


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Nothing breads nothing


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OfflineMycillyum
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Re: Agar...more like Ughar - need expert interpretation [Re: Geinstein]
    #25246545 - 06/03/18 03:47 PM (5 years, 7 months ago)

I too have had this problem recently with a redboy and mazatapec syringes I recently received. The maz germinated something besides the yellow shit but the redboy has failed on 8+ dishes so far. One drop of a shaken syringe on the loop and streaked. Not sure if the syringe is just fucked or what.


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OfflineNunyabiz
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Re: Agar...more like Ughar - need expert interpretation [Re: Geinstein]
    #25246551 - 06/03/18 03:53 PM (5 years, 7 months ago)

I use .1 cc per plate. Instead of using a 10cc with the huge 18ga needle and trying to barely push the plunger to get off one drop, I take a 1cc syringe with either a 25ga or 22ga needle and draw off one cc from the 10cc syringe. I then use .1cc for each petri or 1cc for 10 plates. I am going to use my inoculating loop and do some smear patterns later today. I ordered some parafilm that is being delivered today. Thought I would wait and try that instead of clingwrap to eliminate another variable.


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Offlineverum subsequentis
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Re: Agar...more like Ughar - need expert interpretation [Re: Nunyabiz]
    #25246607 - 06/03/18 04:25 PM (5 years, 7 months ago)

cling wrap works just fine. i think you need to work on your tek and you might have bad syringes. did you make them or order them? if you right up a detailed description of your sterile tek we'll analyze and try to help. it can be frustrating finding the problem but it's always possible. one step gone wrong nullifies all others done properly.


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OfflineNunyabiz
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Re: Agar...more like Ughar - need expert interpretation [Re: bodhisatta]
    #25246674 - 06/03/18 05:12 PM (5 years, 7 months ago)

Fortunately, I have a few plates to practice with. :grin: A case of 500 100mm plates on ebay were only $49 with free shipping. Score!



I was wondering if you have tried using micropore tape for your spawn jars instead of polyfil? I drilled a 9/16" hole in the lid and covered with 3 layered squares of micropore. Seemed easier than tyvek or polyfil and wayyy cheaper than the filter discs. So far, my jars are clean with the micropore. I bought a big bag of polyfil nonetheless. I have a week or two before I need to make more oats. Thought I'd ask before then. Thanks for all your great vids and teks. Helped a lot.


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OfflineNunyabiz
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Re: Agar...more like Ughar - need expert interpretation [Re: verum subsequentis]
    #25246705 - 06/03/18 05:25 PM (5 years, 7 months ago)

I suspect that I am using too much spore solution. Even .1cc is much more than a drop. I am going to use a loop with a syringe solution drop and streak accordingly. I thought this was a good reference along with all the other posts I have read on here. That in conjunction with Bod's tek and streak technique will hopefully solve the problem.

http://vlab.amrita.edu/?sub=3&brch=73&sim=212&cnt=2

If I get the same results, I will write up my detailed tek and probably order some new syringes. I haven't tried sporeworks because their pricing is nearly double micro supply and ralphsters (I'm using purchased not produced syringes). But, I have spent countless hours making plates. So, it is pretty stupid to cheap out on the syringes if they are the problem. Thanks for all of your input. Greatly appreciated.


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Offlineverum subsequentis
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Re: Agar...more like Ughar - need expert interpretation [Re: Nunyabiz]
    #25246759 - 06/03/18 05:55 PM (5 years, 7 months ago)

streaking is nice for a couple of reasons but one drop is totally fine. i've done plenty of ms plates by just dropping a drip.


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OfflineMycillyum
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Re: Agar...more like Ughar - need expert interpretation [Re: Nunyabiz]
    #25246952 - 06/03/18 08:16 PM (5 years, 7 months ago)

Post a link for them plates bro!


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OfflineNunyabiz
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Re: Agar...more like Ughar - need expert interpretation [Re: Mycillyum]
    #25247015 - 06/03/18 09:12 PM (5 years, 7 months ago)



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OfflineNunyabiz
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Re: Agar...more like Ughar - need expert interpretation [Re: Nunyabiz]
    #25247022 - 06/03/18 09:15 PM (5 years, 7 months ago)



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Offlinescab eater2
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Re: Agar...more like Ughar - need expert interpretation [Re: Nunyabiz]
    #25247033 - 06/03/18 09:23 PM (5 years, 7 months ago)

Here is how i do my agar with great results.

Half cup water
1 teaspoon agar (make sure the only ingredient is agar)
2 teaspoons potato flakes (think im using Idahoian)
1 drop karo
1 drop food coloring.


This makes about 8 plates.

Mix it all into a sauce pan and stir for a minute or 2 on low heat.

Spoon about a teaspoon into glad mini rounds or the smallest jars you can get. Just enough to cover the bottom.

Pressure cook them for 45 mins.

Try to keep one untouched as a control. This will help you figure out if it's your process that's failing.


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Offlineverum subsequentis
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Re: Agar...more like Ughar - need expert interpretation [Re: scab eater2]
    #25247042 - 06/03/18 09:27 PM (5 years, 7 months ago)

45 minutes is a long pc time for that little amount. 20 should do.

i love getting good deals on petris but i hate shitty flimsy ones. celltreat is a great company and their 100x15 petris can be had for about 80 dollars for 500 on amazon. free shipping with prime.


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OfflineMycillyum
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Re: Agar...more like Ughar - need expert interpretation [Re: verum subsequentis]
    #25247130 - 06/03/18 10:29 PM (5 years, 7 months ago)

Gonna check that out now. I really like EZ bioresearch but they are not available right now.


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OfflineKizzle
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Re: Agar...more like Ughar - need expert interpretation [Re: Mycillyum]
    #25247158 - 06/03/18 11:05 PM (5 years, 7 months ago)

If you have inoculation loop use it. Just squirt a little inoculant into it. It makes inoculating with a liquid far easier. That way the amount of fluid you use is perfectly controlled and you can make nice little streaks with the droplet if you want.


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Offlineverum subsequentis
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Re: Agar...more like Ughar - need expert interpretation [Re: Kizzle]
    #25247220 - 06/04/18 12:10 AM (5 years, 7 months ago)

nice loop, kizzle


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Offlinemullugh
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Re: Agar...more like Ughar - need expert interpretation [Re: verum subsequentis]
    #25247243 - 06/04/18 12:42 AM (5 years, 7 months ago)

Did you make prints from your pf grow? If so, try using those spores on the plates as an alternative to these syringes you've bought.


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