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InvisibleBridgeburner
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Re: is my TiT contaminated? [Re: shaggydogman]
    #7834511 - 01/05/08 05:04 AM (16 years, 27 days ago)

Quote:

shaggydogman said:
When it worked before what temperature were the jars left in to colonise?




27C-30C


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Invisibleshaggydogman
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Re: is my TiT contaminated? [Re: Bridgeburner]
    #7834530 - 01/05/08 05:30 AM (16 years, 27 days ago)

It gets odder and odder.

So the facts so far are:
- 1/2 pint brf/verm jars
- steamed for 1 1/2 hours
- inoculated with multispore syringe
- inncubated in tit at 26-27C
- some myc growth at inoculation points
- Bacterial contam found in middle of cake

I think the crux of the issue lies with where you said you found the contams and the type of contams, Bacterial in the middle of the jar.

This would imply the bacteria was there after sterilisation and didn't enter the jar during inoculation or incubation.
If it had entered during inoculation it would be at the inoculation points.
If it had entered during incubation it would more likely be at the top of the jars not in the centre.

It has got to be to do with either:
- the sub having more bacteria before it was steamed (different brf,verm or water???)
- the sub being to wet
- steaming didn't get the bacteria in the middle.

Going forward the best way to find out for certain is the 4 jars 2 controls test. Then you'll know for sure where the problem lies and can fine tune your procedures.


--------------------
Rye -- WBS | Grain LC -- G2G | Bulk -- Monotub | 50/50 -- Late Casing -- A Pinning Strategy
Disclaimer: My opinion is subject to change at short notice subject to but not limited by new information and knowledge being made available.


Edited by shaggydogman (01/05/08 05:31 AM)


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InvisibleBridgeburner
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Re: is my TiT contaminated? [Re: shaggydogman]
    #7834627 - 01/05/08 06:52 AM (16 years, 27 days ago)

Quote:

shaggydogman said:
It gets odder and odder.

So the facts so far are:
- 1/2 pint brf/verm jars
- steamed for 1 1/2 hours
- inoculated with multispore syringe
- inncubated in tit at 26-27C
- some myc growth at inoculation points
- Bacterial contam found in middle of cake

I think the crux of the issue lies with where you said you found the contams and the type of contams, Bacterial in the middle of the jar.

This would imply the bacteria was there after sterilisation and didn't enter the jar during inoculation or incubation.
If it had entered during inoculation it would be at the inoculation points.
If it had entered during incubation it would more likely be at the top of the jars not in the centre.

It has got to be to do with either:
- the sub having more bacteria before it was steamed (different brf,verm or water???)
- the sub being to wet
- steaming didn't get the bacteria in the middle.

Going forward the best way to find out for certain is the 4 jars 2 controls test. Then you'll know for sure where the problem lies and can fine tune your procedures.





i'm making a few new jars and using a different heater. my normal room temps are 18C-22C so colonization is gonna take forever.

no, my prepping of jars and syringes was fine, i'm confident about that.


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Invisibleshaggydogman
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Re: is my TiT contaminated? [Re: Bridgeburner]
    #7834630 - 01/05/08 06:56 AM (16 years, 27 days ago)

I recon you'll be good if you up the steaming to 2 hours then.
Make sure for certain the middle of those jars get done.

Good luck.


--------------------
Rye -- WBS | Grain LC -- G2G | Bulk -- Monotub | 50/50 -- Late Casing -- A Pinning Strategy
Disclaimer: My opinion is subject to change at short notice subject to but not limited by new information and knowledge being made available.


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OfflineNibin
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Re: is my TiT contaminated? [Re: shaggydogman]
    #7834667 - 01/05/08 07:35 AM (16 years, 27 days ago)

Mebbe a bad batch of rice with lots of endospores in it?

Try buying the rice from another store and a different brand.


--------------------
Newcomers guide-----> For all things shroomy


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Offlinexeallos
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Re: is my TiT contaminated? [Re: Nibin]
    #7834822 - 01/05/08 09:36 AM (16 years, 27 days ago)

First off I think you know you will find better success when you acquire a PC. It is unfortunate they are not readily available to you, but I think you would benefit from making it a priority even if you have to mail order. You can get better deals online anyway, so it's not all bad!

I only use steam to pasteurize subs in poly bags, and have never boiled jars with temps in sterilization range as a goal, fwiw.

What I do know from experience with Incubators, and something that is constantly repeated by RR is the acceptable temperature range. If you go over 81F, you are going to get bacterial bloom on any uncolonized grain. Another thing that's just as important is temperature fluctuations, I find even if I have a 5 degree flux that doesn't go over ramifications (75 to 80 for example) you will also approach these conditions due to more air exchange between the filtered jars and the ambient air in the tub. I have also read and presume it to be true that colonized material throws off a few degrees of heat itself, a healthy jar could do as much as 5F... for this reason I keep my TiT at not much higher than room temperature... 70-72F. That way if it spikes it doesn't go to 90 and stall, ruining all of your work in short order.

If you can keep a temperature gauge in with the jars that will record min/max it can help you read the fluctuations daily, also topping off the water level can help prevent some of these in my experience. It will evaporate off eventually and I assume this is when things get off balance and the heater overcompensates and goes out of range.

I always just nuke mine with alcohol/lysol between jar sets, never had problems with ambient spores or anything afaik.


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Invisiblefastfred
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Re: is my TiT contaminated? [Re: Nibin]
    #7834904 - 01/05/08 10:13 AM (16 years, 27 days ago)

Remember that steaming does not equal sterilization.

Sterilization requires the entire substance to be heated to 121°C for 15 min.

A general rule is to double your sterilization time for every 10C difference. So you really need to use close to 2 hours for PF style cakes. Even then you won't achieve true sterilization, but hopefully it will be good enough.


-FF


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Invisiblefastfred
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Re: is my TiT contaminated? [Re: fastfred]
    #7834916 - 01/05/08 10:20 AM (16 years, 27 days ago)

> I keep my TiT at not much higher than room temperature... 70-72F.

That kind of defeats the purpose of an incubator now, doesn't it? Maybe you should write a new tek called "the room temp incubator". LOL

85-86F is optimal temperature. Jars generate perhaps 5F tops, so 80-81F is the temperature you should be incubating at.


-FF


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Offlinexeallos
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Re: is my TiT contaminated? [Re: fastfred]
    #7835525 - 01/05/08 01:09 PM (16 years, 27 days ago)

Quote:

fastfred said:
That kind of defeats the purpose of an incubator now, doesn't it? Maybe you should write a new tek called "the room temp incubator". LOL




Not if the room temp is 55F... like a basement... LOL

I've kept my inc at 81F before, accounting for everything else I've mentioned it has proved to be more fatal than helpful in that range.


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Invisiblefastfred
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Re: is my TiT contaminated? [Re: xeallos]
    #7838398 - 01/06/08 07:46 AM (16 years, 26 days ago)

Incubation temp really has nothing to do with sterility. Your containers are either sterile or they're not. Raising the temp will not magicaly introduce contams, nor will a lower temp somehow remove them. Myc will grow faster at 80F than 70F.


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OfflineNibin
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Re: is my TiT contaminated? [Re: fastfred]
    #7838415 - 01/06/08 07:57 AM (16 years, 26 days ago)

Quote:

fastfred said:
Incubation temp really has nothing to do with sterility. Your containers are either sterile or they're not. Raising the temp will not magicaly introduce contams, nor will a lower temp somehow remove them. Myc will grow faster at 80F than 70F.




BUT, no media is 100% sterile. A few endospores might survive or get introduced via inoculation. And contaminants are generally more thermophillic than cube mycelium.

That means thaw while mycelium growth is increased by say 2x, contaminant growth is accelerated x5 (numbers are chosen randomly to illustrate the fact, I don't know what the numbers are really), which can give the contaminant an advantage over the cube mycelium allowing it to get a foothold on the substrate.


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Newcomers guide-----> For all things shroomy


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Invisiblefastfred
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Re: is my TiT contaminated? [Re: Nibin]
    #7839103 - 01/06/08 12:34 PM (16 years, 26 days ago)

Quote:

Nibin said:
BUT, no media is 100% sterile. A few endospores might survive or get introduced via inoculation. And contaminants are generally more thermophillic than cube mycelium.




Sure it is. If the entirety of your media reaches 121C for 15 minutes, it's sterile. The occurrence of the rare endospore here or there on certain types of media are quite overblown. Very few species of bacteria can produce endospores that survive sterilization, and there are ways to deal with those if you run into them.

The rare batch of rye may be full of heat-resistant endospores, but in general you shouldn't have to worry much if you follow reasonable procedures.

PF substrate is the topic of this thread and will almost never contain any endospores. In any case your jars should be sterile. It's not a bad idea to leave sterilized jars for awhile to check for completeness of sterilization.

You should easily be able to attain +99% sterilization with a PC or autoclave. If you're not getting that you really need to adjust your procedures before you worry about anything else.

Incubation should have no effect on contamination rate. And you want to identify and throw out those cultures anyways. Almost every contam will grow faster than myc at any temp and you don't want to be growing contaminated cultures regardless of weather it was able to colonize completely.

A properly sterilized jar can sit until the end of time with no growth.


-FF


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InvisibleBridgeburner
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Re: is my TiT contaminated? [Re: fastfred]
    #7977227 - 02/04/08 10:44 AM (15 years, 11 months ago)

this is where i'm at:

i replaced the TiT with house temperatures (a friend took the jars and put them on his shelf where it's warm) and it still developed the same symptoms.

so i have to conclude that:

a) the prints i made half a year ago have all been exposed to contaminants somehow: all to the same type.

b) the boiling process now fails to sterilize.

i'm thinking of ordering a print from one of the sponsors and try it again. if i STILL get the same contam then it must be the sterilization proccess: and i never use the same inocc syringe again.


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Invisibleshroober
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Re: is my TiT contaminated? [Re: Bridgeburner]
    #7977319 - 02/04/08 11:13 AM (15 years, 11 months ago)

i would suggest investing in a PC, many people disagree with me but I don't trust microwave or boiling pot sterilization techniques, to me a PC is the only way to properly sterilize substrate

if all your contams seem to be coming from the middle of the cakes and not your inoculation points then perhaps your spores arent the problem, again, invest into a PC, you will save yourself a lot of time in the long run


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InvisibleBridgeburner
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Re: is my TiT contaminated? [Re: shroober]
    #7977376 - 02/04/08 11:28 AM (15 years, 11 months ago)

way a head of you, already saving up for one.


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Invisibledumbfounded1600
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Re: is my TiT contaminated? [Re: Bridgeburner]
    #7977421 - 02/04/08 11:43 AM (15 years, 11 months ago)

You can never be to sterile...I take extra sterile procedures when inoculating since I don't have a flowhood... yet.....I take a shower and shave my head....I deoderant...brush teeth and use Listerine mouthwash 2 times...I floss before all this just because its good to...I shave all arm...face hair off...I then spray myself naked with bleach water solution...then put clothes on and spray myself down again with bleach/water solution head and all....I then brush my teeth and mouthwash again....use gloves...70% iso because it evaporates slower...I then scrub my floor with ammonia...use lysol and a can of ousts of my bathroom...I find the lysol to stink really bad when using excessivly so be sure to use a mask if you can...otherwise bear with it

BTW my pc has already been done pcing and is just sitting since overnight...

Next I get the stuff from the pc and wipe it down with alcohol...as well as my syringe and inoculate...wipe once again with alcohol and put in room temperature in as clean as possible place....

Thank You...That Is All.


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Invisibleshroober
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Re: is my TiT contaminated? [Re: dumbfounded1600]
    #7977459 - 02/04/08 12:01 PM (15 years, 11 months ago)

Quote:

dumbfounded1600 said:
You can never be to sterile...I take extra sterile procedures when inoculating since I don't have a flowhood... yet.....I take a shower and shave my head....I deoderant...brush teeth and use Listerine mouthwash 2 times...I floss before all this just because its good to...I shave all arm...face hair off...I then spray myself naked with bleach water solution...then put clothes on and spray myself down again with bleach/water solution head and all....I then brush my teeth and mouthwash again....use gloves...70% iso because it evaporates slower...I then scrub my floor with ammonia...use lysol and a can of ousts of my bathroom...I find the lysol to stink really bad when using excessivly so be sure to use a mask if you can...otherwise bear with it

BTW my pc has already been done pcing and is just sitting since overnight...

Next I get the stuff from the pc and wipe it down with alcohol...as well as my syringe and inoculate...wipe once again with alcohol and put in room temperature in as clean as possible place....

Thank You...That Is All.




that's amazing, I have the same exact procedure but instead of shaving my body hair I routinely burn off the skin from my face, hands and arms, it prevents hair from growing and the scar tissue is much easier to clean than regular skin, i may look like a monster and I have not been laid in about 15 years but I hardly ever get contams so its obviously worth it


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Invisibledumbfounded1600
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Re: is my TiT contaminated? [Re: shroober]
    #7977485 - 02/04/08 12:10 PM (15 years, 11 months ago)

hahaha...Order yourself a whore...I dunno about going that extreme...but ok lolololol


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InvisibleBridgeburner
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Re: is my TiT contaminated? [Re: dumbfounded1600]
    #8138298 - 03/12/08 06:49 PM (15 years, 10 months ago)

this is driving me nuts! i can't believe the contams got it again. i put one jar on a shelf, the second one to another room and the third into a cupboard - same fucking contam. used different syringes.


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