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DinkinFlicka84
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Registered: 01/19/14
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Petri to Grain = Disaster. Advice?
#21710958 - 05/22/15 03:18 PM (8 years, 8 months ago) |
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I had 6 tri-sectioned petris, which I had transfered 4 times to get healthier myc. However, after transferring a petri to WBS jars, it went to shit and got trich within 4-5 days.
This happened despite the fact that 32/36 G2G transfers went off perfectly and fully colonized in 7 days. It was great! I want to know what I could've done wrong.
I used a simple SAB which worked for my regular G2G (2nd attempt ever), flame-sterilized scalpel, tyvek sleeves (with straps to keep the wrists close and I even spray down my sleeves with Lysol and spray Neutra-Air that kills airborne bacteria.
The wedges I used per jar were the full triangular sections. So, I figured that was plenty. On about half of them I cut the wedge with a flamed red-hot scalpel into 3 smaller pieces to help with breakup when I shake the jars. I'm obviously fairly new and eager with agar, but I want to know what I can do differently last time.
Please guide me with your infinite wisdom, oh great site!!!!
  
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mushpunx
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Registered: 04/20/14
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Well the bright side is you caught it early, and not when you go to fruit your substrate!
Trich does hide so well in myc, there is a possibility it was hiding in your culture, but if thats not the case, it just sounds like it got in during your A2G man.
It happens to all of us man and it sucks.
Make sure you wipe everything down very well with ISO, your plates gloves reciving jar, get under the rim very well etc.
Just try again man youll get it!@
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FriedEgg



Registered: 09/22/14
Posts: 2,536
Loc: Taiwan
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Hmmmmmm.... Besides just being unlucky, When you put the agar slices into the jars did the handle of your scalpel touch the jar at all? And did you wipe down your entire scalpel (handle and all) with alcohol?
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PinPornProducer
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Registered: 08/23/14
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How many transfers did you make? Its always best to transfer at least 3 times to assure the culture is clean
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DinkinFlicka84
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Re: Petri to Grain = Disaster. Advice? [Re: FriedEgg]
#21711048 - 05/22/15 03:41 PM (8 years, 8 months ago) |
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It is likely that the handle accidentally touched the jar lid. I try to avoid ALL contact like that. The scalpel was wiped with alcohol before made the whole blade red hot.
7/12 got trich. My guess, after reading these posts, is that my agar was contammed. I couldn't see anything out of the ordinary, but it was actually hard to tell because the agar jar sat around for a couple of weeks before the last transfer.
I have the RR DVD and follow his sterile tek to the T as best I can. I've only done it once before. The regular G2G transfers were 32 for 36. I wish i had a better eye for what means contaminated in agar plates.
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DinkinFlicka84
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That was my 4th transfer.
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DinkinFlicka84
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I have one really important question because I'm about to spawn a lot of subs. Before I do this, I would like to make a master out of 1 or 2 of these aggressive mycelium jars I had. 5 jars colonized exceedingly quickly and I want to hold onto them.
2 things:
1. Is each colonized jar only 1 strain? So, I was thinking of getting a smaller tub for the 1 jar and spawn it to half the sub I normally do. This way the ratio wouldn't be off and it would still be about 3-4" of sub and I would have 1 strain of aggressive mycelium or at least see how well it fruits.
2. Keeping a jar as a master? I don't get how that works if you have to use the grains inside to inoculate other jars, you would waste it in the 1st inoculation of about 10-20 jars. Hopefully that makes sense. Solution?
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PinPornProducer
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Registered: 08/23/14
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If it's ms then no, there are many different strains in a jar, even a clone jar has many different strains but much less than ms obviously.
As far as masters go, again if it's ms then just take that master and make 10-20 more from it and store those. If you had an isolate or mono culture you could take the master and just drop a few grains from it on some agar plates and expand from there
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mushpunx
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Registered: 04/20/14
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I dont keep jars as masters, I keep plates and slants.
Ill take a piece of my slant (or master plate) and knock up as many plates as I need for my spawn run, then inoculate all my master jars via agar wedge.
Right now I use spawn bags so 1 pint master goes into a 4.5qt spawn to make one tub. But before that I would take each master jar and knock up 5-10 more jars for each tub.
Ill use one plate into 3 pints or quarts but you can be more stingy if you like..
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DinkinFlicka84
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Re: Petri to Grain = Disaster. Advice? [Re: mushpunx]
#21712488 - 05/22/15 11:13 PM (8 years, 8 months ago) |
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I had the tri-sectioned 100mm plates and I used each section to inoculate 1 pint each. However, being my first time with agar, 7/12 jars went bad within ~4 days. Interestingly enough, 32/36 of my regular G2G transfers went fine and are now 100% colonized.
I follow RR's tech on flame sterilizing, never touching the under part of the lid and laying the lid on it's back on top of another pint so that I don't touch anything that would effect the colonization. Do I need to cool the scalpel off somewhere else before cutting int the agar?
Is it ok for a large agar wedge (2" wide x 2" long) to be placed in the pint without breaking it up first? When I shook my jars inoculated with agar, the wedges didn't really come apart because they're essentially a jello-substance.
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DinkinFlicka84
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Registered: 01/19/14
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Re: Petri to Grain = Disaster. Advice? [Re: mushpunx]
#21712498 - 05/22/15 11:19 PM (8 years, 8 months ago) |
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I have always been curious about this in the couple of years I've had this hobby. Here are 2 "fully" colonized jars. They're from the same strain, but one looks differently than the other. one has a little more white colonization to it, while the other shows the seeds a little more. Can someone help me identify if one of these isn't going to fruit and is already contaminated? Also, if they're the same strain (essentially), why would they look different when 100% colonized?
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mushpunx
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Registered: 04/20/14
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You cool the scalpel un the blank (reciving) dish before you cut into the culture plate.
Unfortunately just because a jar is fully colonized there is almost no way to know if it is contaminated with mold untill its spawned and usually fruited.. trich hides so well its terribly frusterating.
Yes, I use large wedges all the time and no they dont need to be broken up, just burried in the grains some.
The two jars arent the same strain. They might be the same variety (B+, golden teacher etc) but not the same strain. If you isolated down to a single sector culture, and used the same isolate in both jars then they would be the same strain. But even then the jars might look different if one jar was like say prepped a little drier than the other
You can give the jars a shake at full colonization and watch to see how thry recover, an experienced eye might be able to detect a contam that way
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DinkinFlicka84
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Re: Petri to Grain = Disaster. Advice? [Re: mushpunx]
#21712813 - 05/23/15 01:39 AM (8 years, 8 months ago) |
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So a final test would be to shake the jar when fully colonized to see if I see contaminants? I knows that a lot of times, I won't see the mean green until I shake at 20%
Also, with G2G I'm hoping that should be less chance of contams since I'm not using a MS syringe. Is it a safer way to inoculate since there's no extra water and it colonized almost twice as fast? I would usually squirt 1-2cc's into each jar, which in hindsight led me to offsetting the water content and giving trich and/or bacteria more or a fighting chance.
I've had a bad spawn issue for a long while, which is why I'm doing agar and G2G. I have 5 aggressive mycelium jars and I'm wondering if I should spawn each individual jar to it's own much smaller monotub with the same ratio OR if I should use 3 of the aggressive mycelium jars into one regular monotub. What would be the difference in spawning on it's own or with others with the same aggressive trait? Thanks for you answers in advance!!
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mushpunx
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It doesnt always indicate anything but it can help sometimes, its *something* anyways.
Really for innoculating via syringe you only need 1-2 drops, not 1-2 ccs! But the trich usually comes from syringes rarely being totally clean. So yea, agar and G2G Is a much safer way to inoculate.
Of course its also a vector to let contams in, you need to use impeccable sterile technique! I use agar bur frequently get trich in during G2G. You just need to learn to use perfect tecnique, and never cut corners.
To answer your question, sometimes its certainly safer to make 5 mini tubs over 1 big one because if one jar is contaminated then only one mini tub will go over one big one. But it might be more work.
If its not a clone or isolate it shouldnt really matter how you mix them cause its MS
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