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Invisiblejellyfish


Registered: 10/02/05
Posts: 7,457
Need some advice UPDATED WITH PICS ADVICE APPRECIATED GREATLY * 1
    #23824055 - 11/11/16 08:20 PM (7 years, 2 months ago)

So I have recently got back into the hobby after a 3 year hiatus. I am going from spores from 3-1 years old onto malt extract agar in jars. I have a flowhood and did my best to be sterile. My goal was to immediately cut out healthy looking growth, but it all looks a bit fluffy. I did 4 jars each of ps. galindoi, ps. tampanesis, ps. mexicana and p. cubensis (golden teacher). It's been 2 weeks and most plates either have no growth or very fluffy white growth (although some stuff that's crept up the side of the jar looks like it could be rhizomorphic. I was experiencing some obvious non-white contams that I would make doing transfers easy, I did a control where I didn't inoculate and just opened them to air and they got nasty blue mould I'm going to nuke in my PC and then throw out.

The issue is these jars all have designs and stuff on them, showing a photo will be hard. I'm considering taking off the lids in front of the flowhood to take quick picture to get some opinions. I have never grown anything but cubes and I'm not sure how fluffy/w.e. the growth should be, it all looks like cobweb to me but I'm surprised that some jars showed no growth and some jars bright white growth, doesn't make it easy for me to know what part to cut out and transfer to new agar.

If you guys think it's a good idea, I'll sanitize the area, run the flow hood and take quick pictures to show you, ideally I'd like to transfer the healthiest portions to new agar plates. I feel like I might have mycellium and bright white mould and I'm unable (because of the designs on the jar) to know for sure) I see no yellow spots/myc piss indicating bacteria but some of these fluffy growths don't seem to merge with others. The growth all started from places where I had swapped spores but the prints could have not been the cleanest. The cube print I took 3 years ago.

So should I wait for the plates to fully colonize? Or would you advise that I open them up under a flow of laminar HEPA filter purified air and post pictures? I feel maybe these are all failures, but in my younger days, when I messed up, I would see all sorts of mould. Everything I see is bright white, just different morphologies. I have time Sunday to prepare more plates and do transfers I'm just not sure if I'm just cultivating some kind of white mould.

If you check my sig, you can see my flowhood and agar plates. The fluffiness makes me think cobweb but it's hard to take a photo through the patterns in the glass.

Any help would be greatly appreciated.


Edited by jellyfish (02/25/17 10:48 AM)


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InvisiblePirateSwazey
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Re: Need some advice [Re: jellyfish]
    #23824095 - 11/11/16 08:40 PM (7 years, 2 months ago)

I would take a picture after you do your next transfers.

It sounds like you have cube myc, it's not uncommon at all for growth from spores to be mostly tomentose. Make a few rapid transfers and look for rhizomorphs, then transfer from those a couple more times.

As far as working with the jars go, why not order some plastic petris and make it easier on yourself in a few weeks?


Since you got mold on your control plate, when is the last time you changed your pre filter on your flowhood?


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OfflineA.RichardTrickle
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Registered: 11/04/16
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Re: Need some advice [Re: PirateSwazey]
    #23824104 - 11/11/16 08:47 PM (7 years, 2 months ago)

I concur, I have used MS for most of my experiments over the past few years and found new growth from spores is almost always fluffy.
Best wishes
Dick


--------------------
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[quote]Niffla said:
[quote]A.RichardTrickle said:
Dick[/quote]
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Invisiblejellyfish


Registered: 10/02/05
Posts: 7,457
Re: Need some advice [Re: A.RichardTrickle]
    #23824120 - 11/11/16 08:55 PM (7 years, 2 months ago)

I opened the control in open air, not in front of flow hood. The jars that show no growth at all (despite having spores swabbed on them, were opened in front of the flow hood which makes me think it's working fine. The prefilter is just a bunch of cheese cloth over the top of the fan. I read some rule of thumb that the flow should only be strong enough to make the flame on a lighter bend but not extinguish but I don't remember where I read it and if it was supposed to bend at a 45 or 90 degree angle. I've never changed the prefilter but I also haven't ever used the flow hood too many times in total. Probably less than 20/30 times.

edit: no point in waiting till full colonization for transfers? Also, can anyone let me know what I should be looking for for the truffle forming species? I know how to pic out nice rhizomorphic cubensis growth but this is my first time with truffles. On Sunday I will make a new batch of agar and then take photos and do transfers (can't find my scalpel damn it). I'm guessing it makes no sense to let it fully colonize. I am just unsure in this bright fluffy white mess what sections would be best to transfer and what good tamp/mexicana/galindoi myc looks like. I was under the impression it was more greyish and also, it looks kind of clumpy like clumpy white fluff. I don't use petri dishes because I find it easier to PC the agar already in the jar. I should just get clear regular jars. Thanks a lot guys. All input is appreciated.


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OfflineGrundalizer
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Registered: 03/15/15
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Re: Need some advice [Re: jellyfish]
    #23824148 - 11/11/16 09:10 PM (7 years, 2 months ago)

Any white mycelia, fluffy or not, which is derived from a known spore addition to a sterile media under sterile or semi-sterile conditions and is growing out of the area you doped the media with, is 99.99% what you are looking for.  Cobwebby growth does not equal contamination.  I'm a microbiologist and mycologist by trade and I'm amazed at how often I see people on hear freaking out about contamination and throwing things out (not trying to call you out, I just don't post much, and am trying to start to more).  If you have anything growing on a plate, just pick a corner with a sterile scalpel or sterile toothpick and re-plate...subculture till purity. 

Any moulds are easy to see because of their almost instantaneous rapid color changes due to conidia with spores forming which change color often due to their age.

Any true basidiomycete will stay in a white mycelial growth, and yes, often if I regenerate old spores or super old dried our desicated agar plates, initial growth is very very very wispy. 

I found sub-culturing doesn't help, but taking some and dropping it into sterile broth, like potatoe dextrose broth, yields very healthy and dense mycelial growth...which I then re-plate onto solid agar.


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InvisiblePirateSwazey
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Re: Need some advice [Re: Grundalizer]
    #23824165 - 11/11/16 09:20 PM (7 years, 2 months ago)

The tamp mycelium will look fainter and stringier than what you're used to with cube myc.

Sounds like your flowhood is working fine... 45-90 degrees should be on.

Yes it's perfectly fine to transfer before the plate grows all the way out.

Sorry I just don't get why you have a flowhood but do no pour agar? :tongue:


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Invisiblejellyfish


Registered: 10/02/05
Posts: 7,457
Re: Need some advice [Re: PirateSwazey]
    #23824221 - 11/11/16 09:51 PM (7 years, 2 months ago)

Basically I started as a newb with pf tek. Now I have a punch of jars, with lids on them that have 4 holes poked in them for pf tek style inocs, that I've converted to tyvek lids. So everything I use is stuff I've converted via tyvek from newb times before I built my flowhood. I don't have any petri dishes, maybe 2, I remember I broke one. I guess  I should get petri dishes. I just like for whatever reason, pressure cooking jars with tyvek lids and then inoculating them in front of the flow hood. That way if something goes wrong, it only goes wrong with one plate as opposed to if I poured the molten agar in front of the flow hood and something went wrong, I would ruin the whole batch.


I can't tell you guys enough how grateful I am for the fast responses. I joined this site in 2005 boiling pf tek jars and inoculating in my kitchen with a syringe. I finally built a flow hood in 2012 then got distracted with life and now I'm back and trying to figure shit out.


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InvisiblePirateSwazey
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Re: Need some advice [Re: jellyfish]
    #23824360 - 11/11/16 10:42 PM (7 years, 2 months ago)

Hey it's just matter of preference.

Pouring plates is easy in front of a hood though.

Best luck man!


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Invisiblejellyfish


Registered: 10/02/05
Posts: 7,457
Re: Need some advice [Re: PirateSwazey]
    #23824381 - 11/11/16 10:49 PM (7 years, 2 months ago)

I'll be back with pics soon because honestly I'm rusty. I went from pf tek to pf tek with casing to really shitty glove box with rye which I could never spawn successfully. I only had 2 grows with my flowhood, so my experience with agar is limited. Since I built the flow hood my grows have been amazing, no spore syringes, just prints, no contams at all and spawning to bulk was a piece of cake as well but it's been 3 years since I went from print, to grain, to coir/verm and I have forgotten a lot. I'll probably post pics sunday, and make a few transfers and hopefully I can get some more input then. I need to see how much tyvek I have left, the stores near me don't sell those tyvek paint suits anymore. And jars, I want to make as many agar plates Sunday as possible. Thanks again everyone. I'll be back with pics/news. I will be very generous with prints if this all works out and I can generate some.

edit: one more question, the plates that I swabbed over 2 weeks ago that still show no growth, I don't exactly have a ton of tyvek lids/half pint jars, should I nuke them along with the contamed plates or is it possible I will see growth after 2 weeks. I need to make new agar jars tomorrow and to do so, I need to at least free up some of the jars I have, so I am going to nuke the 2 contamed jars (out of good practice to kill any potentially dangerous mould), then I'm going to make as many agar jars as I can, do the transfers and take pics while I do them.


Edited by jellyfish (11/12/16 11:45 AM)


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InvisibleBig Bear
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Registered: 06/11/14
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Re: Need some advice [Re: jellyfish]
    #23825930 - 11/12/16 01:23 PM (7 years, 2 months ago)

I just had some wikidzons take a little longer than  2 weeks to germ.  I'd say say get a sleeve of petris or more no pour  plates rather than having to clear cut for new upscale mycelium luxury condos.


I fucked myself by not having enough plates earlier in the year,  I took some mold with me somewhere along the line, and then everything got fucked.  These days when I take transfers and open plates and start spores I go crazy with it and make way more than I need. 

I like petris a lot.  I do better with them personally.  Just do yourself the favor and get a 500 pack for ~ 100 bucks and it'll last you a really long time. 

The User bodhisatta has a really good writup on agar, that I pretty much go by.  Only difference is I use parafilm cuz I like it

Good luck :thumbup:


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Invisiblejellyfish


Registered: 10/02/05
Posts: 7,457
Re: Need some advice [Re: Big Bear]
    #23830255 - 11/13/16 07:35 PM (7 years, 2 months ago)

Thanks a lot. I'm taking a look at bodhisattas tek now. I have a nice 500 mL erlenmeyer flask and I'm going to buy some petri dishes. I found 2 at home today so I just put the agar in them first and PC'd them, I know it's not the best way to do it but hopefully I can get some growth.

I don't know why I thought I'd have pics to show you all today. I did all the agar stuff hours ago and the jars are still warm and the agar is still molten (last I checked anyways). The person I live with recently got over lung cancer and I didn't want to open any contam'd jars so I had to run the PC once first just to eradicate the contams before cleaning the jar out to reuse it. Now I have about 16 jars/plates in total so once they are cool I'm going to open up my cultures, take photos and do transfers. I couldn't find my scalpel blade so I pressure cooked my nail clippers (because they have a little knife thing on them and they're metal), and a pair of tweezers.

Is 70% IPA enough to clean the tweezers/knife between transfers in front of my flow hood or should I get 99%?


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InvisibleBig Bear
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Re: Need some advice [Re: jellyfish]
    #23830371 - 11/13/16 08:10 PM (7 years, 2 months ago)

70 is good

Edit: didn't read well. I thought u meant normal sanitation for which 70 is fine.  Flame for transfers


Edited by Big Bear (11/14/16 12:51 AM)


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InvisiblePastywhyteMDiscord
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Registered: 09/15/12
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Re: Need some advice [Re: Big Bear] * 1
    #23830844 - 11/13/16 11:11 PM (7 years, 2 months ago)

You should be flaming tools in between transfers. . .


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InvisiblePirateSwazey
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Re: Need some advice [Re: Pastywhyte]
    #23830865 - 11/13/16 11:21 PM (7 years, 2 months ago)

:whathesaid:

Only time you don't need to is when you're using multiple wedges from the same plate to inoculate the same grain master.


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Invisiblejellyfish


Registered: 10/02/05
Posts: 7,457
Re: Need some advice [Re: PirateSwazey]
    #23831359 - 11/14/16 07:03 AM (7 years, 2 months ago)

Yea I flame my inoc loop on my alcohol lamp I just thought maybe the knife and tweezers are a bit too thick of metal for me to see that nice red glow that let's me know they've been under the flame long enough. I haven't posted the pic in this thread but in the journal linked in my sig there's a pic of my flow hood with my alcohol lamp burning which I use for flaming.

Edit: when I was an undergraduate student we would dip our innoc loop in alcohol then flame it. I kind of forgot about that technique and I'm afraid of causing a fire but should I do it that way or just hold tools over my flame


Edited by jellyfish (11/14/16 07:45 AM)


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InvisiblePirateSwazey
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Re: Need some advice [Re: jellyfish]
    #23831469 - 11/14/16 08:17 AM (7 years, 2 months ago)

Buy a scalpel with disposable blades. The blades will be thin and turn red very quick. I like the #7 handle with #11 blades. Very cheap on eBay!

If you like to dip your tools in alcohol before you flame them it isn't going to hurt anything. Guessing they taught that in your classes to clean off any soot or agar that may get on there.


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Invisiblejellyfish


Registered: 10/02/05
Posts: 7,457
Re: Need some advice [Re: PirateSwazey]
    #23831518 - 11/14/16 08:42 AM (7 years, 2 months ago)

I figured it was because it would burst into flame which was a visual indicator that they had gotten hot enough. I modified my scalpel to hold a razor blade for taking cuttings of cacti and now I can't find the original scalpel blade so I will get a new one. The growth in my dishes looks a lot healthier than I thought and I think I even see some rhizomorphic growth so the only issue now is that I'm not sure how to select healthy mycellium from the non cubensis species I'm growing. I have about 16 agar plates/ jars to use tonight and like 12 jars of healthy looking mycellium of tamp, cubes, and mexicana. My galindoi jars still show no growth. I really appreciate all the advice and will have some pics coming soon but I'm actually quite hopeful. Building a flowhood was the best move I ever made in terms of this hobby. Contams have become a rarity. It cost me $500 CAD to build the flowhood and the confidence it gives me in terms of successful inoculations makes it worth every penny.


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InvisiblePirateSwazey
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Re: Need some advice [Re: jellyfish] * 1
    #23831667 - 11/14/16 09:38 AM (7 years, 2 months ago)

Quote:

Building a flowhood was the best move I ever made in terms of this hobby. Contams have become a rarity



:lookingslick:


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Invisiblejellyfish


Registered: 10/02/05
Posts: 7,457
Re: Need some advice [Re: PirateSwazey]
    #23834853 - 11/15/16 09:28 AM (7 years, 2 months ago)

Didn't have time for transfers yesterday. Hydro store nearme sells 100 plastic petri dishes for 30 bucks which I assume can't be pressureduced cooked. also have expensive and in my experience, very dilute spores syringes and bags of Rye and brown rice flour yoi could get at bulk store down the street for 10x cheaper but it's cool they're catering to the noobs for the hobby. I bought a presterilied scalpel but I don't wanna open it but I feel the handle might be plastic therefore I can't pc it either.gonna try to do transfers and post pics tonight if I get home early enough from work.


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Offlinespore-ty
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Registered: 01/21/16
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Re: Need some advice [Re: jellyfish]
    #23835121 - 11/15/16 11:09 AM (7 years, 2 months ago)

Dam I wish my local hydro stores hooked us up


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