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OfflineCue
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Repeated failure with flow hood
    #22472811 - 11/03/15 07:14 PM (8 years, 2 months ago)

Been having repeated g2g transfers with my flow hood. Agar transfers have been about 75%. This has been going on for about a month now. I've been trying to clean up my sterile procedure in that time to try and nail down what the problem is.

I currently work in a full tyvek suit, including the hood, with a face mask, and alcohol washed gloves. I also spray down the hood, and let it run for at least an hour before I work in front of it.. I wipe everything down, and do transfers just as I would in an SAB, only a little bit faster because I'm not stuck inside a box.

Last time I tried PCing my wbs jars for 90min instead of 60. I tossed some wedges in those jars. So far so good, but I have my doubts given my past record of issues.

I should mention that before the hood, I had about a 5-10% contam rate with the SAB.

Im debating doing a comparison of the SAB to the hood, after relocating the hood to another room. Maybe the room I'm working in now is just not clean enough?

Frankly, I'm about to go back to the SAB and ditch the hood, despite the limited workspace.


Ideas?


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OfflineCue
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Re: Repeated failure with flow hood [Re: Cue]
    #22472931 - 11/03/15 07:36 PM (8 years, 2 months ago)

G2g transfers have been about 25% clean, or less, while others Agar transfers have been about 75-80% clean. Sorry if that wasn't clear. This includes spore prints, syringes, and wedges to new plates.


Edited by Cue (11/03/15 07:39 PM)


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Offlinemushpunx
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Re: Repeated failure with flow hood [Re: Cue]
    #22472951 - 11/03/15 07:40 PM (8 years, 2 months ago)

Hah. I know EXACTLY how you feel. My agar work is stellar, never see so much as a blob so I know the hoods working.

I cleaned up my room, found a better table , put a big sheet of glass under the hood to make wiping it down easier.

Definitely keep it up with the 90 min cook times, that helps.
Are you G2G into bags or jars?

I did the stop cock upgrade on my PC so it acts as a sterilizer now. I can pull my jars/bags out in front of the hood and they will be sterile, no having to wipe, hopefully that mAkes a difference.

I have no idea where Im going wrong either, seems no matter how much I step it up it doesnt maje a difference. So I feel your pain :lol:


Try innoculating your spawn all wedge to grain for a while. Split a plate to 4-5 jars and only use spawn specific to each plate for your tubs.
If the A2G fails, then you know it could either be your cultures or your master jars causing contams and not G2G.
I know you said your agar work isn't perfect so it very well could be that.

This seems the best way to pinpoint and its where Im at right now too man


Oh one more thing, throw your tyvek in a spawn bag and run it thru a PC cycle. That or try using freshly laundered clothes instead and clean tyvek sleeves.
My results go a *little* better when I started cleaning my tyvek regularly. I was usinh a suit for a while and since I couldn't throw them in the wash I think it picked up a lot of crud


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Offlinethebitterbuffalo26
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Re: Repeated failure with flow hood [Re: mushpunx]
    #22472987 - 11/03/15 07:47 PM (8 years, 2 months ago)

need more pc?... probably. If you had perfect grain prep and sterilization, I would bet you could get 75% success in an open room.


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OfflineDrCrumbs
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Re: Repeated failure with flow hood [Re: mushpunx]
    #22472990 - 11/03/15 07:48 PM (8 years, 2 months ago)

You working with stuff from before?

I've had issues build and build. I blamed moving, but now I wonder.


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Offlinethebitterbuffalo26
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Re: Repeated failure with flow hood [Re: DrCrumbs]
    #22473001 - 11/03/15 07:51 PM (8 years, 2 months ago)

a bad wedge of agar that goes undetected will ruin all subsequent jars, maybe the bad jars came from one wedge of agar?


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Invisibleblindingleaf
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Re: Repeated failure with flow hood [Re: thebitterbuffalo26]
    #22473132 - 11/03/15 08:16 PM (8 years, 2 months ago)

a HEPA isn't magic…needs to be built right…blower and all that.

if that is dialed in, i would check ur plates man.


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Offline99.99
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Re: Repeated failure with flow hood [Re: blindingleaf]
    #22473439 - 11/03/15 09:15 PM (8 years, 2 months ago)

I bought one from fungi perfect and I improved it as a vertical hood
It helped a lot with the contaminants and I've recently found this about vertical versus horizontal laminar flow

http://www.shroomery.org/forums/showflat.php/Number/22162570/page/2




http://www.terrauniversal.com/blog/all-terra-blogs/horizontal-vs-vertical-laminar-flow-hoods/
I've also added a UV-c light to keep contaminants down to a minimum when not in use


Edited by 99.99 (11/04/15 06:38 PM)


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Offlineinvitro

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Re: Repeated failure with flow hood [Re: Cue]
    #22473558 - 11/03/15 09:41 PM (8 years, 2 months ago)

I do 2 hours in the pc as a standard.  If I had contams I might up the time or split it into 2 cooks... pasty does that from time to time with dirty grain. 

Get medieval on them spores.





Edited by invitro (11/03/15 09:48 PM)


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InvisiblebodhisattaMDiscordReddit
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Re: Repeated failure with flow hood [Re: invitro]
    #22473601 - 11/03/15 09:51 PM (8 years, 2 months ago)

A flow hood is no substitute for sterile technique either. It requires more precision and dexterity than it otherwise does in a SAB. Contamination falls down vs not being able to sterilize the outside of jars and your hands and then working with flowing air. Flow hoods are excellent if you know how to use them and their application. If people built adequately sized SABs lots of hoods would have went unbuilt but most people get good enough to use a FH by the amount of practice they've had by the time they build one but mistakenly attribute success to things you buy not your own skill development


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OfflineRJ Tubs 202
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Re: Repeated failure with flow hood [Re: bodhisatta]
    #22473921 - 11/03/15 11:03 PM (8 years, 2 months ago)

Quote:

bodhisatta said:

. . .  but mistakenly attribute success to things you buy not your own skill development






Great great point.

Looking back on my hobby, I realize there were points I made progress and improved quality and yield, and made a conclusion, but years later, I realized my positive progress was based on a false concept.

Put another way, if things go well, don't assume the parameters you observe have anything to do with your success. There might be other factors.


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Re: Repeated failure with flow hood [Re: Cue]
    #22474005 - 11/03/15 11:30 PM (8 years, 2 months ago)

Ditch the tyvek suit. I'm sure it's covered in contams.
Shower before working and either put on freshly washed clothes or work naked or in underwear.
Soak your grain for 18-24 hours. PC for 2 hours.
Be sure you aren't moving your hands (or anything else) in between the filter and the plate/jar.


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OfflineCue
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Re: Repeated failure with flow hood [Re: azur]
    #22474441 - 11/04/15 03:50 AM (8 years, 2 months ago)

I've gone g2g to  both jars and bags. All other points duly noted. I may record myself with the GoPro to critic my technique post transfer. Will try to remember to report back after another few attempts. Thanks!


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Offlinemushpunx
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Re: Repeated failure with flow hood [Re: azur]
    #22474501 - 11/04/15 04:56 AM (8 years, 2 months ago)

Quote:

azur said:
Ditch the tyvek suit. I'm sure it's covered in contams.
Shower before working and either put on freshly washed clothes or work naked or in underwear.
Soak your grain for 18-24 hours. PC for 2 hours.
Be sure you aren't moving your hands (or anything else) in between the filter and the plate/jar.





That's what I was saying man, I had gotten a bunch of suits from a trade but after a while I started getting worried about whats clinging to them because you can't really wash empty.

I run my tyvek sleeves thru PC cycle. They can handle it, I use them in my spawn bags over and over.

I used to be a phlebotomist so I have a ton of scrubs around, so I usually use those, fresh oug of the wash.


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Offlinemushpunx
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Re: Repeated failure with flow hood [Re: bodhisatta]
    #22474536 - 11/04/15 05:22 AM (8 years, 2 months ago)

Quote:

bodhisatta said:
A flow hood is no substitute for sterile technique either. It requires more precision and dexterity than it otherwise does in a SAB. Contamination falls down vs not being able to sterilize the outside of jars and your hands and then working with flowing air. Flow hoods are excellent if you know how to use them and their application. If people built adequately sized SABs lots of hoods would have went unbuilt but most people get good enough to use a FH by the amount of practice they've had by the time they build one but mistakenly attribute success to things you buy not your own skill development





The stopcock sterilizer upgrade goes pretty awesome with a FH for that reason. At least the outside of your recieving jars/bags will be sterile. Im hoping that should help some.
Can't do much about your masters except wipe them down before work, same with your gloves/hands.
But if you move the masters from sterilizer to FH to somewhere nice and clean then you can at least reduce the nasties on the outside of the jars some yea?


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InvisibleJuiceh
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Re: Repeated failure with flow hood [Re: azur]
    #22474796 - 11/04/15 07:27 AM (8 years, 2 months ago)

Quote:

Cue said:
Been having repeated g2g transfers with my flow hood.



Details on your flow hood? If you're having this much bad luck with a flow hood there is either something wrong with it or there is a flaw in your inoculation procedures.


Quote:

azur said:
Ditch the tyvek suit. I'm sure it's covered in contams.
Shower before working and either put on freshly washed clothes or work naked or in underwear.
Soak your grain for 18-24 hours. PC for 2 hours.
Be sure you aren't moving your hands (or anything else) in between the filter and the plate/jar.



All good stuff here, especially that last tidbit!!

In my experience showering and the cleanliness of my clothes haven't made any difference that I can tell. As long as all parts of you and your clothes are downstream and your flowhood is functioning properly it shouldn't matter. However, it is good practice so I try to do this before flowhood work too but it doesn't always happen.

You can even do agar transfers ungloved with a flowhood. As long as you keep your hands downstream it's pretty easy. I still use gloves frequently soaked in alcohol and do not recommend ungloved flowhood work though.

I also use a surgical mask while working with the flowhood. That way any coughs or sharp exhales get diffused by the mask and don't have a chance to make it back into the flow of air and into my work.


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Re: Repeated failure with flow hood [Re: Juiceh]
    #22474974 - 11/04/15 08:29 AM (8 years, 2 months ago)

When I do jars or plates, I won't always wear sleeves, but with bags I do, larger opening and all.
I always wear gloves, but obviously many good people on here don't so it's more preference/technique.

I think it's his hood or his plates.


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Offlinemushpunx
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Re: Repeated failure with flow hood [Re: blindingleaf]
    #22476117 - 11/04/15 11:47 AM (8 years, 2 months ago)

Quote:

blindingleaf said:
When I do jars or plates, I won't always wear sleeves, but with bags I do, larger opening and all.
I always wear gloves, but obviously many good people on here don't so it's more preference/technique.

I think it's his hood or his plates.





He can always use new cultures, but how would you sugguest he tests his hood? Leave a blank plate in frony of it? Flame test?


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Invisibletahoe
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Re: Repeated failure with flow hood [Re: mushpunx]
    #22476467 - 11/04/15 12:52 PM (8 years, 2 months ago)

I suspect the hood also. Assuming other things are good.
A tyvek suit is not needed. Your body should not be in your flow hood. Maybe wear sleeves but washing your arms/hands will do the trick


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OfflineCue
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Re: Repeated failure with flow hood [Re: tahoe]
    #22477101 - 11/04/15 03:45 PM (8 years, 2 months ago)

My hood consists of an astrocel I, 2x2, with a Dayton 1TDT5


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