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Offlinemadchemist1
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help with agar work please
    #21825811 - 06/18/15 10:06 PM (8 years, 7 months ago)

I am having trouble with my agar work and think it is some kind of problem with my heppa filtration setup and was needing some help. Let me first explain my sterilization technique I start out with a shower were i completely clean off soap shampoo the works then i run naked about 10 feet to my built decontamination room which is 4x4x6 and then completely spray myself with Lysol then put on my dust mask and gloves which i then sterilize then i go into my main room which has had my heppa filter running for about 2 hours then cover my arms with alcohol and then begin doing my work with my agar flame sterilize the syringe then put a few drops of solution on the agar then follow steps that are in RR videos to a tee came in about five days later and it looks like they are contaminated with green mold but has not turned color yet anyone got any tips on what i am doing wrong


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OfflineGreenRabbit
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Re: help with agar work please [Re: madchemist1]
    #21825831 - 06/18/15 10:11 PM (8 years, 7 months ago)

You are running a hepa filter and trying to filter an entire room?

A Still Air Box (SAB) might be more effective.


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Offlinemadchemist1
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Re: help with agar work please [Re: GreenRabbit]
    #21825851 - 06/18/15 10:15 PM (8 years, 7 months ago)

it is a 6x6x6 foot room with a two foot hepa filter constructed exactly like the one from fungifun on google 6 inch inline fan pushing air through is my fan strong enough


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InvisibleNumeroEno
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Re: help with agar work please [Re: GreenRabbit]
    #21825865 - 06/18/15 10:19 PM (8 years, 7 months ago)

Hold a lighter up to the hepa while it's running. The fan is correct if it blows the flame perfectly sideways without blowing it out.


--------------------

:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
What shall we say, shall we call it by a name
As well to count the angels dancing on a pin
Water bright as the sky from which it came
And the name is on the earth that takes it in

DOG FOOD AGAR

MY ELECTRIC INOCULATION LOOP


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OfflineGreenRabbit
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Re: help with agar work please [Re: madchemist1]
    #21825868 - 06/18/15 10:20 PM (8 years, 7 months ago)

I'd recommend a Laminar Flow Hood. I think the problem is that you have too much air moving. Any contaminant in that air has a chance to get on your plates. It's really hard to sterilize an entire room. You are making a great effort and it still seems it's not enough.


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Offlinemadchemist1
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Re: help with agar work please [Re: GreenRabbit]
    #21825870 - 06/18/15 10:21 PM (8 years, 7 months ago)

i have been successful with brf and went to agar but had no luck kept getting contams in the still air box so decided to quit for a while but do not like to be defeated LOL so came back with the heppa and a completely sealed off room and still getting my assed kicked just need some tips


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Offlinemadchemist1
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Re: help with agar work please [Re: NumeroEno]
    #21825876 - 06/18/15 10:21 PM (8 years, 7 months ago)

i will check that thank you


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Offlinemadchemist1
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Re: help with agar work please [Re: GreenRabbit]
    #21825886 - 06/18/15 10:24 PM (8 years, 7 months ago)

is there a way to build one around my filter without spending that kind of money LOL


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OfflineGreenRabbit
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Re: help with agar work please [Re: madchemist1]
    #21825905 - 06/18/15 10:30 PM (8 years, 7 months ago)

Quote:

madchemist1 said:
is there a way to build one around my filter without spending that kind of money LOL




Probably...
http://www.instructables.com/id/Portable-Laminar-Flow-Hood/?ALLSTEPS
Get creative.


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InvisibleNumeroEno
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Re: help with agar work please [Re: madchemist1]
    #21825906 - 06/18/15 10:31 PM (8 years, 7 months ago)

Dude, it sounds like everything you have is fine except for the fan. Did you do the lighter test yet?

Also, where did you source your hepa, and what are the dimensions (height, width, and thickness)?


--------------------

:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
What shall we say, shall we call it by a name
As well to count the angels dancing on a pin
Water bright as the sky from which it came
And the name is on the earth that takes it in

DOG FOOD AGAR

MY ELECTRIC INOCULATION LOOP


Extras: Filter Print Post Top
Offlinemadchemist1
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Re: help with agar work please [Re: NumeroEno]
    #21825919 - 06/18/15 10:34 PM (8 years, 7 months ago)

24x18x12 fungifun  bought from amazon it was 228


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InvisibleNumeroEno
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Re: help with agar work please [Re: madchemist1]
    #21825931 - 06/18/15 10:36 PM (8 years, 7 months ago)

No wonder. 12" thick is double what you normally use for a flowhood. You're gonna need a fan that's twice as powerful. The preferred hepa for sterile work is a 6" 99.99% filter. The 12" thick ones are mostly used for air scrubbers.


--------------------

:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
What shall we say, shall we call it by a name
As well to count the angels dancing on a pin
Water bright as the sky from which it came
And the name is on the earth that takes it in

DOG FOOD AGAR

MY ELECTRIC INOCULATION LOOP


Extras: Filter Print Post Top
Offlinemadchemist1
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Registered: 06/18/15
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Re: help with agar work please [Re: NumeroEno]
    #21825980 - 06/18/15 10:49 PM (8 years, 7 months ago)

i just did the lighter trick the flame did not go out and blew  all the way to one side could it be the syringe got from spores 101


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Offlinemadchemist1
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Re: help with agar work please [Re: NumeroEno]
    #21826030 - 06/18/15 10:59 PM (8 years, 7 months ago)

i am almost positive it it mold it is a clear circle with white around edges looks like rhizo but the whole inside of the circle is clear but has not turned green yet i know one is for sure it got white specks everywhere and i have seen that pattern before ugh


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OfflineGreenRabbit
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Re: help with agar work please [Re: madchemist1]
    #21827717 - 06/19/15 11:13 AM (8 years, 7 months ago)

Non sponsor syringe? Yea that could be your issue...

Your sterile technique is miles beyond mine and I don't have the issues you are having.


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Offlinemadchemist1
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Re: help with agar work please [Re: GreenRabbit]
    #21830025 - 06/19/15 08:56 PM (8 years, 7 months ago)

whats a good sponsor with reliable syringes?


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Offlinethoraxx
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Re: help with agar work please [Re: madchemist1]
    #21830462 - 06/19/15 10:39 PM (8 years, 7 months ago)

How long has it been since inoculation?
I started a pan cyan plate over a week ago and im just now seeing the first proper mycelium growth, the water from the syringe can make it look like bacterial contam
white specks are just what you would expect starting with syringe, let it grow for a few more days and worry about your already overblown technique later when you actually confirm contam
Also try and make a few empty control plates next time, where you just open the dish up and expose to the airflow, if you see growth there, then you can start to worry


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