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mike-corrhizae
Nerd

Registered: 12/21/09
Posts: 131
Loc: Canada
Last seen: 11 years, 6 months
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sigh... where is my vector of contamination?
#15418535 - 11/24/11 05:30 PM (12 years, 7 months ago) |
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Well for awhile I was doing pretty good with a very low contam rate, but lately its been god-awful. procedure:
-Rinse grains well, then soak anywhere from 6 to 16 hours in water with a re-run of yesterdays coffee (a second pressing from a french press) and a little gypsum.
--bring to a boil at low heat over a 45 minute period (big pot/lots of water) continue boiling for approximately 10 to 15 minutes or until grains are partly soft in the middle.
---Load up jars 2/3 to 3/4 full, put on lid with polyfil balled up and stuffed in a 3/8" hole from the top down cover with foil and load into pc
----With about an inch of water in the bottom of the PC I let it boil and steam up with the rocker weight off until the safety pops up after two more minutes or so I put the weight on and let the pressure build, once it starts to rock i turn the stove down to just above medium where the rocker goes off every 5 or 10 seconds and then let it do its thing for 90 minutes no less.
-----I let jars cool completely over night in the PC. Next morning I prepare my glovebox by wiping all the surfaces down twice with Caviwipes and then spraying lysol and wait for 20 minutes. Jars are lifted out of PC with sanitised gloves, tinfoil is removed and entire jar is wiped down with the caviwipes before I crack the GB and put the jar in. rinse and repeat for all jars and clean agar culture(125ml jars) Once im done another quick blast of lysol into the glovebox and then wait for around 15 minutes.
------I don gloves and tyvek wrist sleeves and sanitise them both then insert arms into glovebox. all jar lids are loosened and I open a package with a sterile scalpel in it and get to work.
-------I'm very careful not to let my hands or fingers pass over any jars, and rye jars are only open for the 2 seconds it takes to throw my speared wedges in(usually try and get 3 or four wedges in each to speed up colonization)
-------- the jars then sit in a clean cupboard at around 22C or 72f until their fate is decided. usually takes two weeks with one shake around the week mark for full colonization.
Lately at least half of my jars have been contaminating with bacteria, it usually shows up around the 10 day mark 50 to 75% colonization. I have NEVER had any mold problems and all of my agar work in the glovebox is clean (60 some jars later and no contamination yet-knock on wood) Lately I have been doing the shotgun approach as I call it and just making twice the jars that i need expecting half to be lost to bacteria, but thats no way to do it -its worth noting that faster growing cultures such as oyster(1 week for full colonization) have NEVER contaminated
HELP ME OUT SHROOMERY! WHERE IS THE VECTOR OF CONTAMINATION!!! starts like this
 ends like this

- Mike
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San
Novice


Registered: 10/30/11
Posts: 646
Last seen: 9 years, 9 months
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Re: sigh... where is my vector of contamination? [Re: mike-corrhizae]
#15418804 - 11/24/11 06:36 PM (12 years, 7 months ago) |
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You have to let your rye dry out a little before pressure cooking. Those look greasy and too much moisture will help bacteria. The rye should be tested by placing a sample after boiling onto a paper towel or toilet paper. If it's wet when the rye is removed then the grain is too damp.
If you're using polyfill on your jars then that's probably it. Get some filter disks and if you are still using the polyfill then be careful when shaking. Don't get the stuff wet.
Disposable sterile scalpels are useful, but you have to be careful about cross contamination. Flame in between jars and if flaming isn't an option then get a better scalpel or open a new one for each jar.
--------------------
Actually not everyone was a noob. Being a noob is a very new phenomenon. Many people, the great majority in fact, were simply "beginners", "novices" or "new to mushroom growing". Being a "noob" is reserved, and in fact created specifically for and by, the newer, much more lame generations coming about. -Shpongle1
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mike-corrhizae
Nerd

Registered: 12/21/09
Posts: 131
Loc: Canada
Last seen: 11 years, 6 months
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Re: sigh... where is my vector of contamination? [Re: San]
#15418920 - 11/24/11 06:58 PM (12 years, 7 months ago) |
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The greasy look is the bacteria. the first jar is a week old, second is two weeks. I use the paper test every time, and usually let them dry a bit more before jarring them up.
I have been using polyfil all along, and have had control jars sit for months without a problem. when I shake I am very careful not to let the grains touch the filter, but theres usually enough of it on the inside part of the lid that I couldn't imagine any residual moisture wicking up through it...
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SKrink
KING MOB



Registered: 01/29/11
Posts: 1,042
Last seen: 11 years, 4 months
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Re: sigh... where is my vector of contamination? [Re: mike-corrhizae]
#15419026 - 11/24/11 07:19 PM (12 years, 7 months ago) |
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Quote:
mike-corrhizae said: Lately at least half of my jars have been contaminating with bacteria, it usually shows up around the 10 day mark 50 to 75% colonization.
Have you tried leaving a jar to sit without inoculating anything? Depending on how long it takes to contaminate (or whether it contams at all), this might give you a clue about the contam vector.
Also, which cultures, when inoculated in the grain, fell to bacteria?
--------------------
SWEET POTATO HOME FRIES
HOW TO USE A PENIS ENVY SPORE SWAB
... Oh mighty masticator, salivator, vocalizer, swallower, licker biter sucker brow-knitter looker blinker rubbernecker thumber prodder up-yours fingerer ringwearer nosepicker waver drinker armlifter bodybender hipswiveler kneer springer runner ZERO::::::::OOOOOOOOO:::::::: RUN!!!
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Frontchasack
Sativa Cyborg



Registered: 04/02/11
Posts: 163
Loc: Mt.Mycology
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Re: sigh... where is my vector of contamination? [Re: mike-corrhizae]
#15419034 - 11/24/11 07:20 PM (12 years, 7 months ago) |
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Hard to tell you man, could be the syringe/culture that you are using. If that is the case, you will have set your self up for failure every time. Do you perhaps have another syringe/print/culture you could test? If all your jars are doing this, that could very well be the problem. If you got the syringe from a vendor, most offer a 30 day viability guarantee. Your sterilization/soaking technique seems to be up to par, so I could tell you either you are introducing the contams to the spawn via a bad syringe, or contams in the air are finding their way onto your needle before inoculation.
What sterility precautions are you taking during inoculation?
-------------------- "I once was here, but now I'm not. I went away to smoke some pot. I left this here to prove a point, that life ain't shit without a joint." R.I.P. J.M.B. 5.26.06
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vanthonyv
Stranger
Registered: 08/17/11
Posts: 60
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Re: sigh... where is my vector of contamination? [Re: Frontchasack]
#15419566 - 11/24/11 09:07 PM (12 years, 7 months ago) |
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I'ma have to agree with frontchasack here. You mentioned a control jar of PC'd rye grains lasting a month... So it would seem to me that that the bacteria contams would come from your inocculating procedures (Which dosn't seem likely if you havn't changed them and they worked well before) or the syringes/LC you are using is contamed with some fashion of bacteria.
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Pandeist
FriendlyStranger


Registered: 01/12/09
Posts: 310
Last seen: 12 years, 6 months
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Re: sigh... where is my vector of contamination? [Re: vanthonyv]
#15419615 - 11/24/11 09:20 PM (12 years, 7 months ago) |
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What are your living arrangements like? How clean is the rest of your place?
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RogerRabbit
Bans for Pleasure



Registered: 03/26/03
Posts: 42,214
Loc: Seattle
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Re: sigh... where is my vector of contamination? [Re: Pandeist]
#15419893 - 11/24/11 10:31 PM (12 years, 7 months ago) |
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Quote:
-----I don gloves and tyvek wrist sleeves and sanitise them both then insert arms into glovebox. all jar lids are loosened and I open a package with a sterile scalpel in it and get to work.
You really want your scalpel sterile for every transfer. If not, the first dish or jar etc., with bacteria that you touch, your scalpel cross-transfers it to all the rest. Get an alcohol or butane torch to keep outside the glovebox. Pull the scalpel out between each dish or jar to heat it red hot before doing the next transfer. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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mike-corrhizae
Nerd

Registered: 12/21/09
Posts: 131
Loc: Canada
Last seen: 11 years, 6 months
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Re: sigh... where is my vector of contamination? [Re: RogerRabbit]
#15421264 - 11/25/11 09:39 AM (12 years, 7 months ago) |
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I have been wiping the scalpel with the caviwipes between each transfer and have never had a contam with agar to agar, could it be the addition of the coffee thats getting me into trouble? I would have thought that would have encouraged mold and not bacteria Coffee = Bacteria? what kinds of contaminants does coffee favor? the only other thing that has changed in my technique was running my PC to the maximum jar capacity but ive always used 90 minutes.
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SomeGuy
I feel better now :)


Registered: 04/18/10
Posts: 7,496
Loc:
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Re: sigh... where is my vector of contamination? [Re: mike-corrhizae]
#15421300 - 11/25/11 09:57 AM (12 years, 7 months ago) |
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I'm not the leading expert, but I'd say your grain is too wet. If I were you, after soaking, I would get the water to a rolling boil and THEN add your rye and only boil 15 minutes.
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deucedbi9
Stranger


Registered: 10/24/06
Posts: 4,647
Loc: UK
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Re: sigh... where is my vector of contamination? [Re: mike-corrhizae]
#15421312 - 11/25/11 10:03 AM (12 years, 7 months ago) |
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Quote:
mike-corrhizae said: I'm very careful not to let my hands or fingers pass over any jars, and rye jars are only open for the 2 seconds it takes to throw my speared wedges in(usually try and get 3 or four wedges in each to speed up colonization
You could try using just one wedge on a few jars. The longer/more often the petri's/grain jars are opened the more chances there are for a contaminant to enter.
Edit:Are the gloves you use the variety that are 'powdered' inside? if so, the powder could very easily act as a vector.
-------------------- whether low pressure sucks or high pressure blows... it's a bugger to cycle in. even though I'm feeling good Something tells me I'd better activate my prayer capsule
Edited by deucedbi9 (11/25/11 10:06 AM)
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tahoe
Noob Slayer



Registered: 11/26/03
Posts: 6,274
Loc: N38.93829W119.98108
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Re: sigh... where is my vector of contamination? [Re: mike-corrhizae]
#15421321 - 11/25/11 10:07 AM (12 years, 7 months ago) |
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Before I read your procedure I figured that I would easily be able to tell where you went wrong. Then I read it and thought that you go through more trouble than I do. If you are good with working with agar then a transfer should be no problem. I will tell you my concerns. The half inch of water in the pc, probably doesn't matter since you are not drying it out during the cooking but I would put at least an inch of water in there. Does your pc get hot enough. Some of those weight only types only go to 10 psi. Since you are using a polyfill lid have you considered using spore syringes or better yet mycelium syringes? Take your good petri and boil up a syringe full of water. Let cool, crack open the petri and squirt a cc of water on it. Work under that bubble of water and cut the mycelium up with the needle. Suck it back into the syringe. If you can see mycelium in the syringe then you have more than enough to do inoculations with. Stick the needle through the polyfill and give each jar a cc or more. Works great. Now the last thing that i will say will make others mad. You say that you usually have good luck but recently you are not. Does recently mean since you bought your new bag of rye. Did i mention that i fucking hate rye. It contams on me all the time. I can pc it for 3 days at 20psi and it will contam. Okay i am exaggerating, a little but i hate rye enough to never use it. It always contams on me. Try birdseed, its a lot less expensive and not as prone to contams as rye.
-------------------- Stop experimenting half way through your first grow. Grow it to maturity, watch it, learn from it. Do this a few times then experiment with different ideas and figure out what works best for you.
My Legacy https://www.shroomery.org/forums/showflat.php/Number/22140987#22140987 Teh=The I need to proofread
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SKrink
KING MOB



Registered: 01/29/11
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Re: sigh... where is my vector of contamination? [Re: tahoe]
#15421887 - 11/25/11 12:46 PM (12 years, 6 months ago) |
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Quote:
tahoe said: Take your good petri and boil up a syringe full of water. Let cool, crack open the petri and squirt a cc of water on it. Work under that bubble of water and cut the mycelium up with the needle. Suck it back into the syringe. If you can see mycelium in the syringe then you have more than enough to do inoculations with.
This sounds like a great idea I gotta try it for my next batch.
The time I take to empty the agar into the jar makes me nervous (I like to use most of the plate for super fast colonization) plus agar sticks to the sides of the jar when shaken... so annoying.
Mycelium syringe from agar plate it is!
--------------------
SWEET POTATO HOME FRIES
HOW TO USE A PENIS ENVY SPORE SWAB
... Oh mighty masticator, salivator, vocalizer, swallower, licker biter sucker brow-knitter looker blinker rubbernecker thumber prodder up-yours fingerer ringwearer nosepicker waver drinker armlifter bodybender hipswiveler kneer springer runner ZERO::::::::OOOOOOOOO:::::::: RUN!!!
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mike-corrhizae
Nerd

Registered: 12/21/09
Posts: 131
Loc: Canada
Last seen: 11 years, 6 months
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Re: sigh... where is my vector of contamination? [Re: SKrink]
#15426777 - 11/26/11 03:29 PM (12 years, 6 months ago) |
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UPDATE~
- A week ago I inoculated 3 jars with my old culture and 4 with a MS culture (both on agar of course) looking at it today the original culture is about 20% colonized and two of them look like they might end up getting the bacteria.
-The MS culture is at around 75% and no sign at all of them greasy wet grains!
it seems as if the bacteria had been hitching a ride undetected from agar transfer to agar transfer. The jars that succeeded simply had more wedges, better distribution and faster growth, overcoming the bacteria.... sigh now i must go through all that work to find another isolate from MS. I feel alot better knowing what was up though
-thanks for all your great suggestions shroomery
- Mike
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RogerRabbit
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Re: sigh... where is my vector of contamination? [Re: mike-corrhizae]
#15427153 - 11/26/11 05:27 PM (12 years, 6 months ago) |
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I suspected inoculum. Next time flame sterilize the scalpel between each dish. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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hamloaf
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Re: sigh... where is my vector of contamination? [Re: tahoe]
#15538363 - 12/19/11 05:32 AM (12 years, 6 months ago) |
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Quote:
tahoe said: Now the last thing that i will say will make others mad. You say that you usually have good luck but recently you are not. Does recently mean since you bought your new bag of rye. Did i mention that i fucking hate rye. It contams on me all the time. I can pc it for 3 days at 20psi and it will contam. Okay i am exaggerating, a little but i hate rye enough to never use it. It always contams on me. Try birdseed, its a lot less expensive and not as prone to contams as rye.
If your rye is contaminating on you, something is wrong with your rye prep./set-up, sterilization procedure, gas-exchange filtration is thwarted or, the inoculum used to inoculate said rye mediums was contaminated.
Are you using organic rye berries? Mushroom spores won't germinate on rye treated with fungicides. Already established myce. cultures will run through rye treated with an anti-fungal. Also, for me, rye is cheaper then WBS.
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