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Offlineupupup
guardian

Registered: 08/25/01
Posts: 889
Loc: George "I love Hitler" Bu...
Last seen: 13 years, 7 months
Contam free MEA
    #1131806 - 12/11/02 08:44 AM (14 years, 10 months ago)

Frank tried something the other day I would like to share and see if anyone else has done this. He borrow from several teks to do this. He has been getting contaminated MEA but he is admittedly new to the whole agar thing.

Basically what he did was took two coffee filters and foled them into quarters. He placed them on top of my flask after he mixed in the MEA. On top of those he placed a micron filter disc that was also folded into quarters and secured it with rubber bands. Since he felt that most of his contams were happening during cooling he thought this might be a good remedy to try to keep contams out during the cool down proccess.

He pc'd like normal then removed the flask from the pc'er as soon as he could open it. He opened the filter set up and dropped in a thermometer sprayed with 90% isopropyl alcohol and replaced the filters. He let it cool with a rag on top soaked with h2o2 until the agar had cooled 135 and then injected h2o2 right through all the filters then immediatly poured his plates. Out of the half sleve he did only one has contamed and that has been more than a week now.

I know that sounds like overkill but it looks like he solved his problem with this.....


--------------------
Support bacteria - they're the only culture some people have.


Edited by upupup (03/15/03 09:06 AM)


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OfflineH2O2shrooms
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Re: Contam free MEA [Re: upupup]
    #1133978 - 12/12/02 03:42 AM (14 years, 10 months ago)

This guy is a local mushroom grower (oysters and the like) who has a PhD in Biochem and a Masters in molecular Bio. Here is his way to reduce contam in agar.



I am clipping the text from the following source
"Growing mushrooms the easy way, Home Mushroom cultivation with Hydrogen Peroxide Volume 1" R.Rush Wayne PhD

To be on the safe side with my plate cultures, I use the lowest concentration of peroxide that I have found effective in agar
medium, which is about 0.018%, or 6 mls per liter of medium. (You can add twice this much without visible harm to the
mycelium of the species I have grown, but note that very slow-growing species such as Stropharia may be more sensitive to
peroxide exposure.

1) I add all the ingredients to a jar with the desired amount of water. The jar should hold about twice the volume you will
actually use, to keep the agar from boiling over when it cooks.
2) I adjust the pH with a little baking soda (my water is acidic, but you could use vinegar if yours is alkaline. Also, see my
"Note on Measuring pH of Substrate" below in the section on preparing bulk substrate).
3) I then use my ordinary kitchen pressure cooker to melt and sterilize the medium. (I use tap water and have not had any
problems with it. In fact, when I grew mushroom mycelium on medium prepared with distilled water, growth was
noticeably slower). I put lids loosely in place and pressure cook at 15 psi for no more than 10 minutes, allowing an initial
ten minutes of steaming to melt the agar before putting on the pressure regulator. (If you are using ready-mixed MYA
medium, the instructions may say to pressure cook for much longer times, for example, 45 minutes. Don't do it! 20 minutes
is the most you?ll need, and any longer will produce carmelization products in the medium that are harmful to the
mycelium). I also sterilize a set of petri dishes along with my medium, placing the dishes in a large tomato can covered
with aluminum foil (I use plastic reusable petri dishes, and a liter of medium fills up about 30 plates).
4) When the cooking is finished, I gently slide the cooker off the burner and allow the pressure to drop. I carefully remove
the jar containing the medium and let it cool in the open air on my counter top. There is no need to avoid entry of
unsterilized air, assuming there is not a great deal of heavy dust, since the peroxide will kill the airborne contaminants
when it is added.
5) When I can handle the jar quite comfortably, I usually put the jar of agar in a pot of warm water for the last part of the
cooling process, since the agar is close to solidifying at this temperature.
6) Then I add my peroxide solution with a pasteurized pipette and quickly mix the peroxide into the medium by moving the
jar with a circular motion, reversing directions a couple of times (but doing my best to avoid making a lot of bubbles, which
will end up on the surface of the agar).
7) Once I've added the peroxide, I go straight to my petri dishes, which I have set out on a clean counter, and I free-pour the
medium into the dishes, closing the lids as I finish.


--------------------
Mycology is a lot like quantum mechanics in that we don?t have causal relationships like in Newtonian physics, only probabilities of various outcomes.<=== Misapropriated from Mycofile


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OfflineH2O2shrooms
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Registered: 12/11/02
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Re: Contam free MEA [Re: H2O2shrooms]
    #1133980 - 12/12/02 03:43 AM (14 years, 10 months ago)

Forgot to mention he always refers to 3% H2O2 for adding to stuff.


--------------------
Mycology is a lot like quantum mechanics in that we don?t have causal relationships like in Newtonian physics, only probabilities of various outcomes.<=== Misapropriated from Mycofile


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Offlineupupup
guardian

Registered: 08/25/01
Posts: 889
Loc: George "I love Hitler" Bu...
Last seen: 13 years, 7 months
Re: Contam free MEA [Re: H2O2shrooms]
    #1135575 - 12/12/02 02:28 PM (14 years, 10 months ago)

Thanks for that and I will be sure to send Dr. Wayne some cash and get those manuels....


--------------------
Support bacteria - they're the only culture some people have.


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