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Offlineleo_spaceman
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Bottom of PF Tek Jar
    #22268648 - 09/21/15 10:05 AM (8 years, 4 months ago)

A couple weeks ago, I inoculated 5 BRF jars. They were steam sterilized for two hours; however, three of them are definitely contaminated. The fourth has a small spot of contamination on the bottom of the jar, but is otherwise mostly colonized:





This is the second batch of inoculations in this house; the previous batch had a similar success rate: 2/10. I felt like this time my procedure was better, but I guess not.

They had been incubating in a damp (and probably moldy) basement, but I've got a dehumidifier down there now so I'm hoping that will help get the mold under control for future batches. Oh, I should also note that the two uncontaminated cakes from my previous batch never fruited in the basement. Perhaps they were too busy fighting off mold spores that were landing on them?

Questions:

  • Can the pictured jar be salvaged or should I give up on it?
  • Is there anything to be learned from the fact that the contamination is on the bottom of the jar?
  • I kept the jars from my last contaminated batch and washed them well prior to sterilizing for this batch. Should I just throw out the jars this time? The jars aren't exactly cheap, so I'd like to save them unless that is ill advised.
  • What would be the next step if I wanted to up my sterilization game? A SAB?


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Invisiblemicro
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Re: Bottom of PF Tek Jar [Re: leo_spaceman]
    #22268667 - 09/21/15 10:12 AM (8 years, 4 months ago)

Did the other jars' contamination look similar to that?

Where did you inoculate them?

The PC should sterilize them as long as you keep it at full pressure for the proper amount of time.

A dehumidifier might help curb future spore growth but will also help anything that's there already to become airborne. If that's contamination (which I'd be worried it is -- it almost looks the colour of bruising but a single spot like that probably isn't) then it's definitely a mould. If you're worried about the jars you could always pressure cook them twice and put a little water in the first time with the lids loosened a bit.


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Offlinemycolangelo4po
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Re: Bottom of PF Tek Jar [Re: micro]
    #22268869 - 09/21/15 11:05 AM (8 years, 4 months ago)

Yea sorry, but that looks contaminated. you said you steam sterilized, I'm assuming that means without the PC, I would suggest getting one they can be found very cheap at second hand stores. Did you inoculate in the basement, that would explain the 2/10, I have been lazy with mine in the past, but I would turn off the air, Lysol bomb the room, take a shower and use gloves with alcohol, but I was getting 9-10/10. It takes practice when mine first got contaminated I thought it was the end of the world,but I started holding on to the questionable ones and segregate them, and watch what happens, it helped me to be able to tell if a jar is contaminated or if I'm just worried. Oh and yea definitely make yourself a sab, they at priceless. Best of luck to you


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Offlineconfuzzed
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Re: Bottom of PF Tek Jar [Re: mycolangelo4po]
    #22269652 - 09/21/15 02:10 PM (8 years, 4 months ago)

What's your temp for your steam sterilization???

Most steam sterilization would not heat the sub evenly and your middle may not be getting hot enough for long enough depending on the temperature.

Proper sterilization will mean ur jars are not the problem.  I don't think you should throw them out.

What is your steaming method? Think ur running into ur problem there.  As for that jar in particular only time will tell if it's safe to keep. If it's bacterial you should be ok. If it produces spores you need to get rid of it asap or it may infect others.  Is it fully colonized?


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Offlineleo_spaceman
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Re: Bottom of PF Tek Jar [Re: confuzzed]
    #22269987 - 09/21/15 03:16 PM (8 years, 4 months ago)

Quote:

micro said:
Did the other jars' contamination look similar to that?





Here is one of the others:



The yellowish part is grainy, almost like pollen on a flower.

Quote:

micro said:
Where did you inoculate them?





Upstairs in a room that I cleaned out and disinfected. I flame sterilized the needle after each jar.

Quote:

confuzzed said:
What's your temp for your steam sterilization???...What is your steaming method? Think ur running into ur problem there.





I elevated the jars in a pot w/ some water on the bottom and simmered the water for two hours. I'm not sure how hot it got but the water was bubbling.

If my steaming method is the problem that's great because that should be easy to rectify.

Quote:

confuzzed said:
As for that jar in particular only time will tell if it's safe to keep. If it's bacterial you should be ok. If it produces spores you need to get rid of it asap or it may infect others.  Is it fully colonized?




How will I know if it produces spores? It is fully colonized in the sense that there is mycelium everywhere but that spot. However, I normally wouldn't birth it for another week or so. How will I know if it is safe to birth?

Thanks for the help guys!


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Offlineconfuzzed
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Re: Bottom of PF Tek Jar [Re: leo_spaceman]
    #22270078 - 09/21/15 03:34 PM (8 years, 4 months ago)

Bacterial will be slimy or moist looking.  Most of the time anyway!  Sometimes comes with a bad smell...  I refer to it as colored looking snot.  but that may not be the best description.

If it looks fuzzy then it's most likely something that will sporulate.  There are always acceptions to every rule, but this the most generalized way I can describe it.

So which one do you think.
Looks trichoderma like to me, cuz it appears green and fuzzy-ish, but I'm not there in person.
That second jar confirms it to me that it doesn't appear your sterilization/inoculation methods are gettin it done.

The contam being at the bottom of the jar also is a slight indicator of this, since it's the last place to be exposed to bad air and the needle normally doesn't make down to the bottom of the jar, the needle is a "point source" of contam considering it's the only thing to be put directly inside the jar.

Hope that helps:thumbup:


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InvisibleMad Season
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Re: Bottom of PF Tek Jar [Re: confuzzed]
    #22273841 - 09/22/15 12:32 PM (8 years, 4 months ago)

First set of pics are aspergillus. Also I guarantee the steaming would have killed any green molds. They die easily in pasteurization temps. 130F for 30 minutes is more than plenty. If you have a dry verm layer, it didn't come from the outside (and if it did, it would appear near the holes, not the bottom).

Do you flame the needles between transfers? Inoculate in a sab? Please tell me you aren't wiping the syringe in alcohol after flaming it... if yes to all of those, inoculate in agar and clean it up. It's quite common to have contams in syringes. This one is from 1 drop of a vendors syringe..
luckily I streaked the single drop, or else all 5 of those contams would have germinated in 1 spot.


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Invisiblemicro
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Re: Bottom of PF Tek Jar [Re: Mad Season]
    #22277389 - 09/23/15 03:31 AM (8 years, 4 months ago)

pressure cook

steaming isn't enough

it looks like it's all the same contaminant so it either came from the syringe, or the substrate

i don't think it was from the house unless it's from when you were inoculating it

is that green stuff under one of the holes in the lid?


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InvisibleMad Season
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Re: Bottom of PF Tek Jar [Re: micro]
    #22277549 - 09/23/15 06:24 AM (8 years, 4 months ago)

Steaming is part of the pf tek. Basically just boil the jars for 90 minutes. It's because brf has no endospores. We only pc for bacteria since almost all molds die ridiculously easy. Like I said, green dies at 130F. Boiling temps are 212F. Going for 15 psi just makes it 250F. All of those are much higher than what temps the molds will die at. That's why for the OP it's either getting in during the inoculation due to bad techniques, or the syringe is filled with contams


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Re: Bottom of PF Tek Jar [Re: Mad Season]
    #22278500 - 09/23/15 12:12 PM (8 years, 4 months ago)

no, i pc'ed for everything

there are spores and bacteria everywhere

spores can often survive temperature extremes before germination

best to sterilize them by pressure cooking or autoclaving

as far as green molds, Penicillium duponti is a thermophile and thrives at that temp, for example

hard to blanket it like that as there are many different kinds of green mold


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InvisibleMad Season
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Re: Bottom of PF Tek Jar [Re: micro]
    #22278564 - 09/23/15 12:29 PM (8 years, 4 months ago)

Yeah spores and bacteria are everywhere, but almost none of them will be thermophilic or endospores.. There's a reason why people have been doing straight boiling of the pf tek for the longest time with no problems. There's a reason why pasteurization at lower temps than what calls for in say dairy is much better in this hobby.

Cakes (brf and verm) are extremely clean, and hard for things to germinate on. Will a pc be better at sterilizing? Definitely! Is it needed for grains? Definitely! Is it needed for pf tek? Definitely not, since it seems everyone and their dog started without a pc. RR and all the other TCs will advocate that you don't need a pc for pf tek. I can guarantee if op used a clean lc/LI with proper technique, or had clean spores (almost impossible), he'd not be having these problems. And tbh i doubt pcing things will fix the problem at hand.


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Re: Bottom of PF Tek Jar [Re: Mad Season]
    #22278659 - 09/23/15 12:47 PM (8 years, 4 months ago)

i see lots of people having problems with the pf tek

pf tek also says to use a pc:

http://www.fungifun.org/English/Pftek

i won't jump to conclusions about what caused it; it's speculation

like i said, i was just giving an example of a green fungi that lived in hotter environments

but as far as sterilising, a quick google search gave me this:

https://www.emlab.com/s/sampling/env-report-03-2006.html

Hot dry air sterilization
170 °C (340 °F), 1 hour
160 °C (320 °F), 2 hours
150 °C (300 °F), 2.5 hours
140 °C (285 °F), 3 hours
Kills virtually all spores

Penicillium spore death in water
54.4 °C (130 °F), 30 minutes

Ascospores activation in grape juice
70 °C (158 °F), 30 minutes
This is higher than the temperature which kills Penicillium in water

Germination of chlamydospores stimulated by moist heat
75 °C (167 °F), 30 minutes
This is higher than the temperature which kills Penicillium in water


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InvisibleMad Season
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Re: Bottom of PF Tek Jar [Re: micro]
    #22278753 - 09/23/15 01:04 PM (8 years, 4 months ago)

http://www.mushroomvideos.com/BRF-Pf-Tek

Quote:

Part 1 also gives the recipe and demonstrates the procedure for grinding the brown rice using a coffee grinder. You’ll see how to modify the lids of your mason jars to allow for inoculation and air exchange, and you’ll see how we sterilize the brown rice flour above a kettle of boiling water.

Part 2 of the brown rice flour technique (pf tek) picks up where part 1 left off. You’ll remove the sterilized jars from the steaming kettle, and once they’ve cooled to room temperature, inoculate them with either a mushroom spore syringe or liquid culture syringe using sterile procedures as demonstrated by well-known mycologist Jim Read of Seattle, Washington.




Do you know how many people use mushroom videos with success compared to failures? I'm just saying that boiling jars has been the norm for longer than the both of us have been registered lol. It's not been a problem back then, and it shouldn't be a problem now. Although I will still agree that a pc is something that would be better in the long run. But for the small time users who use pf tek, they definitely will succeed without needing a pc.

Besides spores are almost never clean.


This was from 1 drop of a spore syringe swiped on the plate.

Another recent syringe


Of course there are clean ones..

I'm just saying that syringes that performed amazingly on cakes have been pretty damn bacterial on agar. I'd say 50% of the syringes I've had are dirty somewhere in them.


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contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
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Invisiblemicro
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Re: Bottom of PF Tek Jar [Re: Mad Season]
    #22280568 - 09/23/15 08:19 PM (8 years, 4 months ago)

they weren't for me

why do you have agar in an open plastic container

also, they are wrong it doesn't sterilise it

only thing i would pasteurise is straw inoculated with colonised substrate

also you can get a pc from a thrift store for like 20 bucks


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InvisibleMad Season
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Re: Bottom of PF Tek Jar [Re: micro]
    #22280648 - 09/23/15 08:29 PM (8 years, 4 months ago)

I took the pic post transfer. Normally it has a lid on it with a filtered gas exchange hole. And it's the new thing man :P

http://www.shroomery.org/forums/showflat.php/Number/19208976

either way if op was to do agar or what you're saying, he'll still need to get a pc :lol:


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contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


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Invisiblemicro
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Re: Bottom of PF Tek Jar [Re: Mad Season]
    #22280747 - 09/23/15 08:40 PM (8 years, 4 months ago)

you have a 50% contamination rate

i guess if you don't care then it's all good

i never had that; the syringes i got from one of the vendors here were always sterile

i think anyway, we had contamination every now and then, more so my roommate

also:

Quote:

You can now safely inoculate any media like grains or LC without the issue of invisible contams.




I've been saying this for a while now but there is another way that's possibly easier:

use bags
inject sterile water into the bag with a syringe (thicker needle, min 12gg)
mix it up without opening the bag then suck it back up

sterile inoculum

i got the ones with the self healing injection ports


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InvisibleMad Season
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Re: Bottom of PF Tek Jar [Re: micro]
    #22280871 - 09/23/15 08:52 PM (8 years, 4 months ago)

From my syringes/prints, yeah. At least 50% contam. After transferring I have very little contams, like 1 contam every 30-50 plates, so I know it isn't my sterile techniques.

Grain LI is pretty good, but it's still not quite as clean as say...


Which is the same idea, but utilizes pp5 agar dishes. There's also this tek which uses a blender:
http://www.shroomery.org/forums/showflat.php/Number/20429745

Just food for thought :thumbup:


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


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Invisiblemicro
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Re: Bottom of PF Tek Jar [Re: Mad Season]
    #22281023 - 09/23/15 09:02 PM (8 years, 4 months ago)

I always used LB media...

Yeah, 50% is bad. If it's from the syringes I'd find another source.


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