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Offlinedeathblade
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Registered: 06/25/06
Posts: 302
Last seen: 8 months, 10 days
WTF is happening????????
    #26469862 - 02/05/20 08:30 AM (4 years, 12 days ago)

So the old mean green is back once again. This is so frustrating I dont know what to do. I've tried everything to be as sterile as possible. I'm hoping that someone can tell me what stupid little thing I've missed in my process so I can laugh about how dumb I was at not noticing it sooner.

I will describe my whole set up and process.

I'm using a culture I isolated several years ago and saved in a slant tube in a fridge.
My grow area is a heated shed that I attempt to keep at 70f. Unfortunately if it gets really cold my heater cant keep up and temps did drop to around 40f for about 8 days recently.

I use mono tubs with a black garbage bag liner and micropore tape to maintain FAE. I wash the tubs down with bleach water after every grow attempt.

I start my sterile work by spraying my flowhood with bleach water. I also have a plastic sheet that covers the surface I will have my plates or jars on that gets sprayed at the same time. Then I turn it on and let it run for a few hours before I do anything. I also turn off any other fans so theres no other air currents.

Next its shower, brush teeth and mouth wash. Change of clothes from my closet and then outside I go. I put on shoulder length vet gloves and surgical gloves and a surgical mask. 70% ISO on paper towels and I have a propane torch just outside of the flowhood zone for sterilizing scalpels. I wash hands and halfway up my forearms with alcohol. Then I wipe the scalpel and set it aside. I wipe down my pasty plates with alcohol and grab the scalpel  and heat the blade red hot, plus run the flame half way up and down the shaft several times to hopefully make sure theres no friends on it. Open the recieving dish and cool the blade in the agar. Open the colonized plate and cut a small piece out and move to other plate. If I'm making a Master jar I would then cut the agar in half and transfer pieces to a grain jar that has been wiped down. I open the lid and drop in agar pieces. Replacing the lid and opening between pieces. Then I monitor the jars and plates as they colonize to look for intruders. If I suspect theres issues into the garbage.
G2G process is very similar. ISO on paper towels. Wipe down every crack I can. Wipe filter discs on lids. Keep jars as close to middle of flow as possible. Crack lid on master and next jar. Remove recieving jar lid and lay it face up on jar beside it. Open master and pour small amount into jar. Master lid stays off for transfers.(just like RogerRabbits video)

Sub is Hpoo, coir, vermiculite and a handful of gypsum. Hydrate to field capacity and load into jars with foil caps. Each jar is loaded to within a few grams of each other.
Then into the pots. Fill with water to the shoulder on the jars. Medium heat till temp climbs 10f over starting temp(usually 90f)
Once temp reaches 140 start timer for 90min. Usually temp tops out at 159. After time is reached I pull jars out, empty water and then put jars back into pots with the lids on to cool for about 18 hours before I can make up tubs.

The tubs get a fresh bag as liner and 3 jars of sub, 3 jars of grain. I smell each jar before adding to tubs. With rubber gloves on I mix tub and level sub.
I then cover all air holes with non breathable tape until fully colonized.(about 10/12 days). Then replace tape with micro pores tape and then wait for the magic. Lights on a 12 and 12 schedule. I mist the surface if I see the sides of the tub drying off.

WTF am I missing? I will add pics later today of all my gear and set up.


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Offlinedeathblade
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Registered: 06/25/06
Posts: 302
Last seen: 8 months, 10 days
Re: WTF is happening???????? [Re: deathblade]
    #26515510 - 03/03/20 09:18 PM (3 years, 11 months ago)

Sorry it took so long. Had some technical issues that kept me from getting any pics.


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OfflineBarrel
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Registered: 10/27/18
Posts: 90
Loc: USA
Last seen: 3 years, 9 months
Re: WTF is happening???????? [Re: deathblade]
    #26515608 - 03/03/20 10:53 PM (3 years, 11 months ago)

How old are all of your filters in your flow hood, on your grain jars, and on your pasty plates?

Is there a mold colony in your closet?


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Offlinedeathblade
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Registered: 06/25/06
Posts: 302
Last seen: 8 months, 10 days
Re: WTF is happening???????? [Re: Barrel]
    #26517077 - 03/04/20 06:33 PM (3 years, 11 months ago)

Flowhood is 6 years I believe. Filters on jars are 7 months, only 10 cycles I would say. The pastyplates get new micro pore every use. The mold colony I doubt. Brand new grow room.

I thought maybe the flowhood filter to but I only lose a few plates and that definitely my fault for sticking my gloves where they shouldn't be. Sometimes I've noticed i dont do the G2G as close to the filter as my agar transfers, but I thought that was the beauty about using a flowhood is there was a little bit of wiggle room. Maybe I should video my process for critique?


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OfflineBarrel
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Registered: 10/27/18
Posts: 90
Loc: USA
Last seen: 3 years, 9 months
Re: WTF is happening???????? [Re: deathblade]
    #26522814 - 03/07/20 08:14 PM (3 years, 11 months ago)

Go ahead and stack 2-3+ layers of micropore on the pasty plates. The hole is only there for sterilization pressure equalization, not FAE. There's no real need to replace it between uses.

A 6 old flowhood filter is a bit suspect. Pull it out and inspect it for damage, and make sure you've got a good fit on it in the housing.

There's a little wiggleroom with a flowhood, but it won't correct for bad technique. A video would likely be helpful.


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Offlinedeathblade
Long time lurker


Registered: 06/25/06
Posts: 302
Last seen: 8 months, 10 days
Re: WTF is happening???????? [Re: Barrel]
    #26540667 - 03/17/20 12:07 PM (3 years, 10 months ago)

Here's one of the tubs that hasnt shown any mold. Still looks ugly though. And it isnt producing like it used to.


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