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Offlinemicki_t
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Registered: 07/19/12
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Loc: Northumberland, UK
Last seen: 11 years, 15 days
Contamination, home aseptic technique, and fractional sterilisation, your opinions please!
    #16819442 - 09/09/12 08:02 PM (12 years, 4 months ago)

Hi,

I am a newbie grower, and am worried about the sterility of my substrates, and also general home aseptic technique and contamination's. 

Since inoculating my first bags (1 week ago), I have lost 4 out of 12 bags/jars of rye grain spawn to Trichoderma, or alike blue/green mould, and am not sure whether my technique or equipment have let me down.

I am using a glove box with neoprene gloves sealed to it, and other general aseptic cleaning measures to reduce contamination.  I have just built a sealed? polythene tent/box within a cupboard to help further reduce contact with household air, which will hopefully help (it's really damp here this year and am in an old cottage). 

The spawn that I bought was not seemingly packaged adequately (commented on in a previous post), which I don't think has helped matters, but what do you think I could do in my limited situation to improve my new war with moulds?

I am using an old pc, and don't currently have the funds to buy a better one, so wondered if fractionally sterilising my substrates (rye grain, sawdust etc) would help guarantee no contamination's;

Today I have just put some rye grain on soak, and intend to do this for  24 hours, when I will then boil it off, drain, etc...  I will then put in bags/jars and perhaps put a little extra water/not dry grains quite as much before bagging to allow for extra evaporation?, and then pc for 90mins?.  These will all be naturally cooled/depressurized in the cupboard still tent, and then sat in the glove box for 1 or 2 days to germinate endospores etc, then pc'd again, incubated, and finally pc'd.  Do you think this would work/help?

Also, do you think that one of the small hepa air filters (like the home air cleaning units, apparently screen down to 0.2um) would serve a functional laminar hood when used in conjunction with a sealed tented glove box, or alike?

Your advice will be most appreciated!

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Offlinesystemz
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Registered: 04/13/12
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Last seen: 9 years, 7 months
Re: Contamination, home aseptic technique, and fractional sterilisation, your opinions please! [Re: micki_t]
    #16819521 - 09/09/12 08:12 PM (12 years, 4 months ago)

Where I used to live I had leaky faucets and sinks and just generally mold everywhere. I couldn't do grain teks no matter how sterile I made my still box and how careful I was. I ended up buying presterilized grain jars with inoculation ports. You reminded me to make a review in the suppliers section because those jars literally saved my grow hobby. I almost gave up before I tried the jars.  Anyway, just comparing because you live in an old cottage and I'm sure the mold content is higher than a normal growers house.

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Offlinemicki_t
Male

Registered: 07/19/12
Posts: 12
Loc: Northumberland, UK
Last seen: 11 years, 15 days
Re: Contamination, home aseptic technique, and fractional sterilisation, your opinions please! [Re: systemz]
    #16821961 - 09/10/12 08:06 AM (12 years, 4 months ago)

I have had a better read through this forum and it has indeed answered most of my above questions!

I am new to this forum, or any forum, so apologise to all for asking without thorough research of the information available in front of me!  Please forgive me fellow mycologists (experienced and aspiring).

Thanks :smile:

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Offlinetwistedty
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Re: Contamination, home aseptic technique, and fractional sterilisation, your opinions please! [Re: micki_t]
    #16821975 - 09/10/12 08:12 AM (12 years, 4 months ago)

dont add water to your bags before you pc.  dont use incubator.  no hepa with your glove box.  point of glove box is so there is no moving air.  and  you pc if its doesnt leak it shouldnt matter how old it is.  and dont take the jars out of your pc until it has cooled down.  which can take a VERY long time

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Offlinemicki_t
Male

Registered: 07/19/12
Posts: 12
Loc: Northumberland, UK
Last seen: 11 years, 15 days
Re: Contamination, home aseptic technique, and fractional sterilisation, your opinions please! [Re: systemz]
    #16822005 - 09/10/12 08:26 AM (12 years, 4 months ago)

Quote:

systemz said:
Where I used to live I had leaky faucets and sinks and just generally mold everywhere. I couldn't do grain teks no matter how sterile I made my still box and how careful I was. I ended up buying presterilized grain jars with inoculation ports. You reminded me to make a review in the suppliers section because those jars literally saved my grow hobby. I almost gave up before I tried the jars.  Anyway, just comparing because you live in an old cottage and I'm sure the mold content is higher than a normal growers house.




Thanks for the advice systemz, I am definitely looking into making injectable port jars, and using liquid cultures.  My agar work has been working well, so I do question the sterility of the initial grain spawn I purchased, but this is still assumption. 

I need a laminar flow, and will not now stop until I get one!

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Offlinemicki_t
Male

Registered: 07/19/12
Posts: 12
Loc: Northumberland, UK
Last seen: 11 years, 15 days
Re: Contamination, home aseptic technique, and fractional sterilisation, your opinions please! [Re: twistedty]
    #16822024 - 09/10/12 08:37 AM (12 years, 4 months ago)

Quote:

twistedty said:
dont add water to your bags before you pc.  dont use incubator.  no hepa with your glove box.  point of glove box is so there is no moving air.  and  you pc if its doesnt leak it shouldnt matter how old it is.  and dont take the jars out of your pc until it has cooled down.  which can take a VERY long time




Thanks twistedty!  I am picking up on a lot of school boy errors, in the making, with my ideas.  The PC is old, but it doesn't leak, and does rock a bit when it's raging, so perhaps I have questioned its function without good reason.

I don't want to use a hepa filter with a glove box, but was thinking of placing one in a cupboard/clean area, with the flow pointing towards me, blowing out of the door, which is very similar to how the laminar flow hoods are at the university I studied at.  I'm aware of the need for the air flow not to create any back draft, ie the air needs to flow out of the cupboard, not around it, but am unsure whether scaling the hepa down to a home aircleaning model would be sufficient for mycology purposes.

I will be posting my findings when I have something worth posting, including the negatives.

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OfflineRogerRabbitM
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Registered: 03/26/03
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Re: Contamination, home aseptic technique, and fractional sterilisation, your opinions please! [Re: micki_t]
    #16822546 - 09/10/12 11:18 AM (12 years, 4 months ago)

Quote:

micki_t said:

I am using an old pc, and don't currently have the funds to buy a better one, so wondered if fractionally sterilising my substrates (rye grain, sawdust etc) would help guarantee no contamination's;

Today I have just put some rye grain on soak, and intend to do this for  24 hours, when I will then boil it off, drain, etc...  I will then put in bags/jars and perhaps put a little extra water/not dry grains quite as much before bagging to allow for extra evaporation?, and then pc for 90mins?. 




Fractional sterilization is bunk.  It will fail a vast majority of the time.  Use your PC per instructions with the rye grain prep tek.  Don't add extra water either.  Follow the tek.
RR


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Offlinemicki_t
Male

Registered: 07/19/12
Posts: 12
Loc: Northumberland, UK
Last seen: 11 years, 15 days
Re: Contamination, home aseptic technique, and fractional sterilisation, your opinions please! [Re: RogerRabbit]
    #16824516 - 09/10/12 05:15 PM (12 years, 4 months ago)

Quote:

RogerRabbit said:
Quote:

micki_t said:

I am using an old pc, and don't currently have the funds to buy a better one, so wondered if fractionally sterilizing my substrates (rye grain, sawdust etc) would help guarantee no contamination's;

Today I have just put some rye grain on soak, and intend to do this for  24 hours, when I will then boil it off, drain, etc...  I will then put in bags/jars and perhaps put a little extra water/not dry grains quite as much before bagging to allow for extra evaporation?, and then pc for 90mins?. 




Fractional sterilization is bunk.  It will fail a vast majority of the time.  Use your PC per instructions with the rye grain prep tek.  Don't add extra water either.  Follow the tek.
RR




Thanks RR, I have just finished pc'ing the grain spawn, as per your video, which is cooling in the clean area.  I have given up on fractional sterilisation, except for an experimental batch, which I have steamed in the other pc with a poor seal (certainly not 15psi, but seal not dripping). I intend to allow them to incubate until tomorrow, and then pc in old but working pc.

In conclusion, I need a bigger pc, or autoclave, and will not stop until I have one (and a laminar flow!) :smile:

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