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faceyneck
Legitimate Philosopher



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Cleaning up a culture - and perplexed
#14169600 - 03/23/11 12:01 PM (12 years, 10 months ago) |
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I start all my cultures from agar dishes these days.
I recently blended up some plates into some LC. I now blend up a plate into LC, and then test the LC before I consider it a "clean culture."
One of the cultures was PE, from spore, which grew out clean from the get-go. The other culture was a PF Redspore clone which had bacteria on the first plate it was transferred to.
After a couple transfers from the bacterial plate, the culture appeared completely clean.
Now here's the clincher. Check out this picture of the LCs that I made:

The PF Redspore clearly shows bacteria, as evidenced by the overall cloudy appearance.
...but look at the wedge I placed into a master test jar at the same time:

Those little specks in there are gypsum by the way. Oh, and that's the BACKSIDE of the plate that's facing the camera. So yeah, growing REALLY nice and healthy.
The ONLY conclusion I can think of is that the bacteria has been growing the whole time underneath the mycelium, and so when I transfer a wedge to a jar, the bacteria is still held captive, and also the grains are too dry for the bacteria to thrive anyhow.
...but in the LC, the agar is completely broken down, and the bacteria is released into the ideal environment for bacteria to thrive in, and so it takes off.
I welcome any worthwhile input, as I'd really like to solve this conundrum.
Thoughts?
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cyanara
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Re: Cleaning up a culture - and perplexed [Re: faceyneck]
#14169686 - 03/23/11 12:18 PM (12 years, 10 months ago) |
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one of several things could of occured. 1) your LC could of been dirty 2) your culture could of had contaminate and on or in the mycelium and upon breaking it up released the endospores. 3) certain cube types resonde differently to mediums, I did some lc with the red boy and it's still cloudy and growing thick satelite colonizes similare to that, so until you streak it or sacrifice a jar I wouldn't call it contaminated. Another phenomenon I've noticed is that nutritionaly dense subs then to allow the mycelium to pronounce it's true atributes without sigificant cell division, meaning if your culture is not a true iso it will start to seperate quite noticibly and alot of times the sectores that are not rhizomorphic and week in growth are either carriers of contaminants or the mycelium it's self is too weak to support growth and fight of attack from invading spores. just
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faceyneck
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Re: Cleaning up a culture - and perplexed [Re: cyanara]
#14170201 - 03/23/11 01:40 PM (12 years, 10 months ago) |
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Quote:
1) your LC could of been dirty
That's just silly. I sterilized some LC mix - light malt extract and dextrose - and blended the plate up into that. The LC was not inoculated with LC. It was inoculated with a plate.
Quote:
2) your culture could of had contaminate and on or in the mycelium and upon breaking it up released the endospores.
Yeah, I could see that. That begs the question though - how the fuck would one clean up the culture if it looks great on agar? 
Quote:
3) certain cube types resonde differently to mediums, I did some lc with the red boy and it's still cloudy and growing thick satelite colonizes similare to that, so until you streak it or sacrifice a jar I wouldn't call it contaminated.
I suppose that's true. However, the PE jar is expanding a lot better than the PF Redspore jar. The PFRS jar doesn't look like it's growing into the solution. Frustrating I REALLY wanna grow with this clone, and I also wanna have another inoculant-type other than just grains for G2G.
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OoBYCoO
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Re: Cleaning up a culture - and perplexed [Re: faceyneck]
#14170253 - 03/23/11 01:48 PM (12 years, 10 months ago) |
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Quote:
faceyneck said: The ONLY conclusion I can think of is that the bacteria has been growing the whole time underneath the mycelium, and so when I transfer a wedge to a jar, the bacteria is still held captive, and also the grains are too dry for the bacteria to thrive anyhow.
...but in the LC, the agar is completely broken down, and the bacteria is released into the ideal environment for bacteria to thrive in, and so it takes off.
This makes sense to me. It makes me think of a BRF cake of Redboy that I had. It was 95% colonized and low and behold the green monster reared its ugly head for the other 5%. Anyways, I removed it from my incubation area b/c I didn't want to get any cross contamination w/ my other healthy jars. I set it on a shelf somewhere else just to see what happens and what do ya know, the healthy myc overtook it and "encapsulated" it. It is now fruiting w/ no signs of trich. If you noticed myc is pretty resilient and forms a tough barrier, that's why we dunk our cakes for 24 hours b/c it's even hard for water to penetrate it. So in my mind, the scenario you described could very well be the case. But then again, I'm just postulating. lol
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LeopardMan
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Re: Cleaning up a culture - and perplexed [Re: faceyneck]
#14170299 - 03/23/11 01:56 PM (12 years, 10 months ago) |
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Quote:
faceyneck said: So yeah, growing REALLY nice and healthy.
Yes, it's possible. But I would wait until full colonization. How do you know that there are no bacteria in the jar?
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TranscendingLife
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Re: Cleaning up a culture - and perplexed [Re: faceyneck]
#14170335 - 03/23/11 02:02 PM (12 years, 10 months ago) |
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Quote:
faceyneck said:
Yeah, I could see that. That begs the question though - how the fuck would one clean up the culture if it looks great on agar? 
Quote:
RogerRabbit said: You drop the mycelium-covered agar right into the grains. Don't try to separate the mycelium from the agar. It needs the agar there to get a good jump off into the grains.
Mycelium will grow on the surface of agar, and this can be used to advantage. If you have bacteria on a plate, you can pour piping hot agar over the top of it, and when the mycelium climbs up through the new layer to reach the surface, shave it off and transfer to a new dish. This is a great way to separate mushroom mycelium from bacteria. RR
From this thread
There ya go FN...I got yo back brotha
-------------------- AMU: We Quickly Answer Questions Here "One must accept the probability of failure to experience the elation of success." - TranscendingLife “A man of genius makes no mistakes; his errors are volitional and are the portals of discovery.” - James Joyce
      How I Do EVERYTHING      "Your vision will become clear only when you can look into your own heart…. Who looks outside, dreams; who looks inside, awakes."- Carl Jung "Anything that can be done chemically can be done by other means."- William S. Burroughs "You are as dead now as you will ever be" - Seth
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OoBYCoO
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Yeah, greysrdbest told me about that a long time ago. Has anyone ever tried that?
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cyanara
jedi in training



Registered: 12/22/09
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Re: Cleaning up a culture - and perplexed [Re: OoBYCoO]
#14170796 - 03/23/11 03:20 PM (12 years, 10 months ago) |
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ok so back to the nutrient aspect. I bought some lab grade MEA, and i don't feel like rewriting the damn formula out for the 11th time but I noticed right away that the RB is growing beautiful mycelium but very slowly however the PE is responding to the medium alot better not only than all the other types that I've put on this particular agar, but it actually responding in forming rhizomorphic mycelium. It was pointed out by another member that he could not maintain agaricus bisporus on PDA/PDYA but oyster loved it.. I would suggest maybe trying a LC that would be more closely resembling PDA. the RB really like it and I just got done doing some sweet potato agar and the response is phenomenal.
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faceyneck
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Re: Cleaning up a culture - and perplexed [Re: LeopardMan]
#14171149 - 03/23/11 04:23 PM (12 years, 10 months ago) |
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Quote:
LeopardMan said:
Quote:
faceyneck said: So yeah, growing REALLY nice and healthy.
Yes, it's possible. But I would wait until full colonization. How do you know that there are no bacteria in the jar?
I think there IS bacteria in the jar, but I think it's more or less imprisoned by the Cubensis mycelium.
-------------------- Anything posted here, is total bullshit. My Meyers-Briggs Personality: INTJ New growers, or anyone else just needing help; I'm always glad to help right here.
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faceyneck
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Quote:
todlow said:
Quote:
faceyneck said:
Yeah, I could see that. That begs the question though - how the fuck would one clean up the culture if it looks great on agar? 
Quote:
RogerRabbit said: You drop the mycelium-covered agar right into the grains. Don't try to separate the mycelium from the agar. It needs the agar there to get a good jump off into the grains.
Mycelium will grow on the surface of agar, and this can be used to advantage. If you have bacteria on a plate, you can pour piping hot agar over the top of it, and when the mycelium climbs up through the new layer to reach the surface, shave it off and transfer to a new dish. This is a great way to separate mushroom mycelium from bacteria. RR
From this thread
There ya go FN...I got yo back brotha 
I forgot to mention this. Thanks for bringing that up, TL! 
I was hoping it wouldn't come to this. Like, damn. I thought that technique was for when transfers weren't taking because of the bacteria?
Oh, well. Time to do some more digging.
-------------------- Anything posted here, is total bullshit. My Meyers-Briggs Personality: INTJ New growers, or anyone else just needing help; I'm always glad to help right here.
We give cultivation advice here. AMU Q & A - We're glad to help My Doggy Door Greenhouse! First Ever Shmuvbox Tek! Do Manure Right!
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faceyneck
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Re: Cleaning up a culture - and perplexed [Re: cyanara]
#14171228 - 03/23/11 04:35 PM (12 years, 10 months ago) |
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Quote:
cyanara said: ok so back to the nutrient aspect. I bought some lab grade MEA, and i don't feel like rewriting the damn formula out for the 11th time but I noticed right away that the RB is growing beautiful mycelium but very slowly however the PE is responding to the medium alot better not only than all the other types that I've put on this particular agar, but it actually responding in forming rhizomorphic mycelium. It was pointed out by another member that he could not maintain agaricus bisporus on PDA/PDYA but oyster loved it.. I would suggest maybe trying a LC that would be more closely resembling PDA. the RB really like it and I just got done doing some sweet potato agar and the response is phenomenal.
I've done a lot of LCs. Any time they've gotten cloudy in the past, they had bacteria in them. That's not to say I haven't had LCs that looked okay but weren't also. 
If it actually colonizes enough for me to take some out and test it, we'll find out for sure. It's not looking so good at the moment though.
Also, it wasn't cloudy when I first made it up. This happened over the course of about a day and a half. Cubensis mycelium simply does not make an LC murky, rhizomorphic or not.
One last thing - if I made a PDY-type LC, it would be cloudy to begin with, so like, I wouldn't be able to see the bacteria. PDA for plates didn't ever do much for me either. It performed a little worse than MEA, and smelled like... well... boiled potato farts.

I do appreciate your input, cyan. It's gotten me thinking more about this, so I appreciate your input.
-------------------- Anything posted here, is total bullshit. My Meyers-Briggs Personality: INTJ New growers, or anyone else just needing help; I'm always glad to help right here.
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cyanara
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Re: Cleaning up a culture - and perplexed [Re: faceyneck]
#14171324 - 03/23/11 04:53 PM (12 years, 10 months ago) |
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I did ham's PDA and shit works really good. as far as an LC is concerned, If you soak something with starch and strain it you should be able to get a fairly clear LC, and as soon as the mycelium colonizes it will clear it up, cause this has happened with malt style lc's.
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Javadog
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Re: Cleaning up a culture - and perplexed [Re: cyanara]
#14172203 - 03/23/11 07:35 PM (12 years, 10 months ago) |
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Hey Facey,
Whatever the mechanism of introduction, I think your explanation of why the LC fails when grains do not is sound.
As to this mechanism, either the bacteria came with the agar or it was introduced by your technique. (yes, let's omit the "it survived the PC" idea) You put this in a PC'ed blender bottle and blended it? (eberbach)
I have read of fungal colonies that defied cleaning through transfers. I cannot recall if this was something specific to a certain species of fungus, but I seem to recall that it was. There was some physical interaction, so that the fungus seemed to carry the bacteria with it.
Good luck!
JD
-------------------- Boyd Rice told my brother that life is a corny pack of freesakes Myco-tek.org
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TranscendingLife
I Don't Need a Life to Live



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Re: Cleaning up a culture - and perplexed [Re: Javadog]
#14172844 - 03/23/11 09:47 PM (12 years, 10 months ago) |
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Quote:
Javadog said: Hey Facey,
Whatever the mechanism of introduction, I think your explanation of why the LC fails when grains do not is sound.
As to this mechanism, either the bacteria came with the agar or it was introduced by your technique. (yes, let's omit the "it survived the PC" idea) You put this in a PC'ed blender bottle and blended it? (eberbach)
I have read of fungal colonies that defied cleaning through transfers. I cannot recall if this was something specific to a certain species of fungus, but I seem to recall that it was. There was some physical interaction, so that the fungus seemed to carry the bacteria with it.
Good luck!
JD
The first thing I would check would be the culture, which you said was contamed w/ bacteria.
The next thing, if you're absolutely 100% certain the bacteria did NOT transfer, would be sterile technique on the transfer, but I know you have a flowhood. Therefore, I do not acknowledge this problem.
Lastly, as JD said, I would suspect some kind of symbiotic relationship between the fungus & the bacteria. Well, more a bacteria that sits inside of a fungal body then presents itself when more "food" is introduced, which would explain the explosion during grain transfer.
I don't feel as though bacteria is that big of a problem w/ mushrooms, unless it's a "serious" one that grows as yours did. I've seen bacteria/yeast infected jars fruit OK, not great but ok, in bulk substrates.
That being said, I've also seen mushrooms that've eaten trich fruit fine. I guess it's all a toss up. I wouldn't touch at trich ridden jar/tray, but I wouldn't have a problem fucking w/ a bacteria/yeast ridden tray.
My motto goes something like this:
Trich = the flu (as it's an AIRBORNE Pathogen) Bacteria = Herpes (as you have to come in DIRECT contact w/ it)
-------------------- AMU: We Quickly Answer Questions Here "One must accept the probability of failure to experience the elation of success." - TranscendingLife “A man of genius makes no mistakes; his errors are volitional and are the portals of discovery.” - James Joyce
      How I Do EVERYTHING      "Your vision will become clear only when you can look into your own heart…. Who looks outside, dreams; who looks inside, awakes."- Carl Jung "Anything that can be done chemically can be done by other means."- William S. Burroughs "You are as dead now as you will ever be" - Seth
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faceyneck
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Quote:
The next thing, if you're absolutely 100% certain the bacteria did NOT transfer, would be sterile technique on the transfer, but I know you have a flowhood. Therefore, I do not acknowledge this problem.
The only way I can think of to check to see if the bacteria did or didn't transfer would require a microscope, which I would LOVE to have, but can't afford at the moment. 
I at least have good evidence. Perfectly prepared, dry grains = not conducive to bacterial growth, and the bacteria does not appear to be present. In a liquid culture which is the ideal environment for bacterial growth, I have a cloudy LC. It's not a perfect litmus test, but it'll do for a poor cultivator like myself. 
Quote:
That being said, I've also seen mushrooms that've eaten trich fruit fine
I'd need to see that to believe it. Being as the life cycle of cubensis lasts 1-2 months, and the life cycle of trich is measured in days, I don't think it's biologically possible for cubensis to overtake trich. But like I always say; whether or not it looks good in theory, occasionally one should look at the results.
I did recently see a picture from Morelman of a bag of rye with "trich" in it, that was clearly penicillium. It's in PJ's journal, if you wanna take a look. Not too long ago I had a jar with a lid that failed, and some penicillium started growing in it. It looks a lot like trich actually. Anyway, I let the two grow out on the grains to see what would happen. They kind of got into a standstill, and then very slowly, the cubensis mycelium would grow a little bit over some of the grains with the penicillium growing on it. I doubt it was eating it or whatever. In 3-dimensional space, they were more or less blending together though. Like I said - very small-scale difference, but if the cubensis had larger infantry from the get-go, it probably could've grown right over the whole damn penicillium colony.
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faceyneck
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Re: Cleaning up a culture - and perplexed [Re: Javadog]
#14173808 - 03/24/11 01:18 AM (12 years, 10 months ago) |
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Quote:
You put this in a PC'ed blender bottle and blended it?
Indeed. The PE LC is growing faster than any LC I've ever made from spore or LC inoculation. It's the fastest I've ever seen. I'll definitely be perfecting this procedure, as it's the best thing I've come up with for starting a culture out. If I had a stir plate, it'd probably be almost done by now. 
The initial clone definitely had bacterial problems. It was first cloned to grains; from grains to a plate, which showed a weak strain of some sort of bacteria - so weak the mycelium ended up growing over the puddles of snotty bacteria, actually; from that plate I made another transfer to a clean plate; from that plate I took pieces to the LC and grain jar. I did allow the plate I used as inoculant to grow out too much also. All-in-all, I wasn't really on top of it, I've realized after much thought. I thought I had isolated away from the bacteria when none of it was visible on the plates I used for inoculation. 
I think I have three options left:
1. Take a transfer from one of the other plates I have of this clone; make yet another transfer from the leading edge of the culture while it's growing ferociously, but before it colonizes the whole plate; take pieces of that plate to make yet another plate(s) for the purposes of inoculation; use that newest plate to make another LC, and see if it clouds up.
2. Take a transfer from one of the plates I have; allow that plate grow and recover a bit, maybe to the size of a quarter; pour hot agar over the culture, and follow the RR procedure to a new plate; make one more transfer from the leading edge to a new plate; use that newest plate to make another LC, and see if it clouds up.
3. Wait until full colonization of the master jar pictured in the first post; place pieces of that grain onto fresh plates; make transfers from the leading edge of those cultures to new plates; use that newest plate to make another LC, and see if it clouds up.
I'd start over, but: 1. this is a typical dilemma when trying to isolate a clean cloned culture from contaminates, so it's not like I can just start over with another fruit and avoid this problem, and; 2. what I'm learning will come in handy when I need/want to clone other fruits. The PE culture I'm developing and will grow out soon in the LC jar comes to mind.
-------------------- Anything posted here, is total bullshit. My Meyers-Briggs Personality: INTJ New growers, or anyone else just needing help; I'm always glad to help right here.
We give cultivation advice here. AMU Q & A - We're glad to help My Doggy Door Greenhouse! First Ever Shmuvbox Tek! Do Manure Right!
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Javadog
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Re: Cleaning up a culture - and perplexed [Re: faceyneck]
#14179360 - 03/24/11 11:17 PM (12 years, 10 months ago) |
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Hey bros,
I was reading up on some edible threads when I saw where a grower referred to hitchhiker bacteria.
I thought to add a ref to it: https://www.shroomery.org/forums/showflat.php/Number/12731338#12731338
Take care,
JD
-------------------- Boyd Rice told my brother that life is a corny pack of freesakes Myco-tek.org
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hamloaf
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Re: Cleaning up a culture - and perplexed [Re: faceyneck]
#14179541 - 03/24/11 11:52 PM (12 years, 10 months ago) |
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Quote:
The ONLY conclusion I can think of is that the bacteria has been growing the whole time underneath the mycelium, and so when I transfer a wedge to a jar, the bacteria is still held captive, and also the grains are too dry for the bacteria to thrive anyhow.
...but in the LC, the agar is completely broken down, and the bacteria is released into the ideal environment for bacteria to thrive in, and so it takes off.
This seems like a hypothisis I can subscribe to. Only problem I can foresee is that even though your grains are self-proclaimed too dry to support the germination and thriving of the invader spores, the invader spores are by your own hypothesis held captive by the mycelium under the agar and are still present.
Seems like if this were the case, as soon as you transfer your mother jar's mycelium into their receiving jars, the invader spores will then be released and have made contact with air upon being transfered, stimulating the invader spore to germinate, infecting your receiving jars.
Either that or you goofed on your sterile procedure when inoculating the LC over the grain master jar. Another way to be sure is to make more transfers of the agar onto other agar plates and see if a contamination occurs. Another option would be to inoculate another LC with another wedge of your agar culture, being more focused about your sterile technique and seeing if that LC contaminates. I'd go with the transfer of wedges to freahly prepared agar plates to see if contamination is present.
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faceyneck
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Re: Cleaning up a culture - and perplexed [Re: hamloaf]
#14180786 - 03/25/11 07:50 AM (12 years, 10 months ago) |
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Quote:
even though your grains are self-proclaimed too dry to support the germination and thriving of the invader spores, the invader spores are by your own hypothesis held captive by the mycelium under the agar and are still present.
Odd thing is this is the second time this has happened. First time, a plate contaminated in the refrigerator. A transfer to grains worked great, and the master jar yielded jars which I'm still G2Ging today. I made a LC from a wedge of a plate made from the contaminated one, which grew out pretty and everything, but I grew nothing but penicillium and trich from the LC. 
I gotta get some parafilm. I've heard maybe that the static charge on GladWrap attracts contaminates. 
I do enjoy that dry grains aren't conducive to bacterial growth, but I really gotta get my shit together, is what it comes down to.
Quote:
Either that or you goofed on your sterile procedure when inoculating the LC over the grain master jar.
I actually have yet to feel like I've done a clean, crisp transfer from an agar plate to a jar. I don't get my hands in between the jar and the laminar flow, but maybe the blender blade catches on the outside of the jar for a fraction of a second, or maybe the agar wedge won't cooperate and get the fuck out of the dish in a timely manner, or maybe I feel I didn't wipe the plate down' with alcohol well enough, etc.
That one will just need to remain forever something I need to improve on.
I'm pouring plates today. I'll make some new transfers, but they can't be from the plate I used as I promptly threw away what was left after use. I do have some plates that came from the same original plate though - the plate that I KNOW had bacteria on it. It's odd those transfers didn't exhibit any signs of bacteria until being placed in LC. Unless of course I'm just a clumsy bastard.
-------------------- Anything posted here, is total bullshit. My Meyers-Briggs Personality: INTJ New growers, or anyone else just needing help; I'm always glad to help right here.
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Doc_T
Random Dude




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Re: Cleaning up a culture - and perplexed [Re: faceyneck]
#14180797 - 03/25/11 07:54 AM (12 years, 10 months ago) |
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Try practicing with blank media. No myc, just blank (or used) plates, a jar with a little water in it. Practice cutting and dropping wedges on your kitchen table or someplace comfy until you can do that part reliably. Then do it in front of the flowhood, with good hand placement etc.
But LC is sketchy. Just stop using it and the problem goes away.
-------------------- You make it all possible. Doesn't it feel good?
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