~nati shroom

[This message has been edited by shroominati (edited April 25, 2000).]

Stamets sells a kit or two for HEPA flow hoods out of wood (hrmm???) that start I think in the $480 range and go to just about $1000. Which you have to assemble/build yourself. Which of course is a cheaper alternative than a lab supply which I've seen easily in the $2800-$4800 range.

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I am 98% done with mine. Just got to lay down a special foam strip seal on the inner lid surfaces and I will have a perfect sealed system. As it stands I did full fan CFM tests throughout the range and it had a pretty lock tight seal so I could technically start using it but will wait until my next free day off to finish it properly.

Only difference between mine and a cardboard box and seran wrap is cost, an actual filtered clean air flow, negative pressurization, last beyond my lifetime vs. a few uses. I can give it to my prodigy and last through his lifetime, looks like some serious lab unit to have your hands inside.

It just comes down to how much effort and knowledge you want to put into it.

I'll have to find another free day to hook up with a friend with a Hi-8 system and computer digitizer as it would take better snaps than my shitty web cam. Then you can see what I've built.

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I would encourage you to soak a.k.a steep your rye for AT LEAST 24 hours, even a bit longer if need be. I wouldn't go under the 24 hour mark though. As you probably know, all those endospores and other fun things floating around on the rye need to be ...hmmm is germinated the right word?

Anywho....try letting your rye soak for at least 24 hours:-)

Lana

quote:

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Originally posted by Hippie3:
one other consideration to ponder...
if you get caught,
which looks worse,
a cardboard box or
a full-blown custom made laminar flow/hepa filtered chamber ?

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Actually my hepa chamber ... because I am near the end of my psillycybe reign and I'm growing gormets pretty soon from here on out; unless I move to Amsterdamn or some shit. There's nothing much new for me to learn cultivating cubies, maybe a few things to learn with some wood lovers like azures but thats about it, I've almost got my way through boot camp and want to start playing in the bigs. Of course I'll still help everyone out with their questions and spread what I have learned, it's only right!

quote:

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Originally posted by Lana:
...hmmm is germinated the right word?

Lana

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Hmm let it start sprouting and converting starches into sugars? malted rye! heh.. give your substrate a sweet tooth.

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[This message has been edited by vts1134 (edited May 01, 2000).]

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Originally posted by vts1134:
Learner, it sounded like a glove box.

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Its both, I combined the concept of laminar hood and glove box together. Laminar Flow coming into a large sealed glove box so there is no human skin cells/hairs blowing around in the wind flow. It comes in clean, passes over the instruments and containers inside and is vented out the side.

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Originally posted by vts1134:
It was my understanding that electrostatic filtration is not sufficient for sterile work with higher fungi.

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ULPA filters are electrostatic and put HEPA filters to shame. I recall Stamets lists HEPA down to .3 microns and the ULPAs are at least in the .01 micron range.

What I have is not even ULPA and is almost in the HEPA specs range. Good enough for ya .. You can also buy a spray for the filter to really lay down a static charge across the filter and boost the ratings. The inside of the container including the back of the filter is sprayed with Lysol.

Trust me, its pretty damn kick ass.

[This message has been edited by The Learner (edited May 01, 2000).]

I read through the replies and saw that some possibilities were not taken into account.

You wrote that you live at about 9000 feet, thats about 2750 m (I use the metric system). A short calculation using thermodynamics shows that you only loose about 5 C (9 F) because of the height. At sea level a difference of 10^5 Pa (15 psi) in pressure would give you about 121 C (250 F). Normaly 1 hour at 10^5 Pa are enough to autoclave the rye, now 1.5 hours would be more than enough to contrarest the 5 C you loose because of the height.

Actually 15 min at 121 C (250 F) is enough to kill any kind of endospore, especially those of Bacillus(Naevke & Tepper, "Einfuehrung in die mikrobiologischen Methoden"). Rye needs 1 hour at 10^5 Pa because it needs at least 30 to 45 min to reach 121 C, while agar is autoclaved after 20 min at 10^5 Pa because it has a much smaller Warmthcapacity than rye. Of course the time the rye needs to get the right temperature depends on the mass of rye.
2*10^5 Pa (30 psi) would be actually an overkill (130 C or 266 F) and COULD even harm media like MEA because of caramelization.

I have myself autoclaved rye mixed with other seed at about 2300 m (7550 feet) without any contamination problems.

Like many people wrote presoaking helps a lot against contamination because the warmth unsensible endospores, especially from Bacillus, germinate, i.e. change to the warmth sensible vegetative form. But you shouldn't forget that if you wait to long the vegetative form could build new endospores and make things worser. So 24 hours soaking at room temp. should be enough, much longer wont help and could be even harmfull.

You didn't mention which kind of contamination your rye got. If you sterilized 1.5+ hours and the problem would come from your sterilization method, you would mostly have the slimy, like rotten apple smelling bacteria, shortly a Bacillus. Because that baby is the only common microorganism which would survive such an sterilization. So if the contams you have are not a Bacillus, e.g. a green mold or something like that, then your problem doesn't come from the sterilization.

Some rye sorts are extremely full of spores. So changing the type of rye could help to reduce chances of getting a contamination. One of the best rye one can use is the one designed for human consumption.

You didn't mention how you stored/incubated the rye. Sometimes a jar which isn't closing good can bring a lot of contamination problems.

You wrote something about using bags. Are you using autoclavable bags to sterilize and incubate the rye in them? How much rye are you autoclaving in one bag? I'm new to this forum, so maybe you have told the others about that.
Maybe you should quantisize the rye you are autoclaving, especially if you autoclave a lot of rye at one shot. Big amounts of rye need of course a lot more of time to reach 121 C or in your case 115 C, especially the center. If you don't autoclave in the bags, then the bags could be a source of contamination. The best method is to use some sort of jar to make the spawn. Jars automatically quantisize the rye amount, are also autoclaved, so you won't have to handle the sterilized rye to much, exposing it to contams.

A very easy way to get out, if the problem comes from a bad rye sort or from the sterilization or from the way you incubate your rye, is to sterilize a little bit of rye the way you normally do and put it to incubate without inoculating. If you get again a rainbow effect then you know that it comes from one of the above mentioned things. The rye should at least be incubated 3 or 4 weeks and within that time it shouldn't get contams.

You should check the cleaness of your sporewater. You wrote that you have bags which worked. Thats maybe not the best method to get out if your sporewater quality is good. The best way is to use agar to check for contams. Contams are spotted very easily on agar.
For using agar you don't need a laminar flow hood. You only need a free and clean surface and a room without air currents. Keep the surface, on which you are working, wet with a solution of 60% vol.to 70% vol. ethanol (alcohol). You don't need drinkable alcohol use the one that you can buy for cleaning or for alcohol torches, i.e. use denatured alcohol. It normally has 94% to 96% ethanol, so you'll have to dilute it. It is important to remark here that 60% to 70% is much more effective than a higher concentration, although many people wouldn't think so. Ethanol is good for sterilizing because it coagulates proteins. A high concentrated solution doesn't work as good as a 70% solution, because a high concentrated solution coagulates only the proteins of the suface, while the 70% solution gets much deeper.

Agar needs only about 20 min at 10^5 Pa, at your height take 30 min. After it cools down and solidifies put some drops of the sporewater on the agar. Don't fully open the petridishes, just pull up one side of the cover, so it still is over the dish and you have access to the inside. Make it as fast as you can without making an error. If the points where you inoculated with the sporewater shows contams, then your sporewater is not clean. If you get contams somewhere else in the dish, then it could come from not working alright when you inoculated.
Agar is much easier than many people think, and it always lets you select contamination free inocula. If the petridish shows contamination, then you always can take a piece of agar which hasn't contamination und separe the mycelia from the contams. If the contams are fungi and produced spores, then it can be difficult to separate it without loosening the spores of the contam.

A very easy method to produce a home made chamber for inoculation is given at the "9er method", which can be found here at the Shroomery. I have one that is very similar and it works great.

As a last word always use ethanol or isopropanol (70% solution) to desinfect your hands, the surface on which you are working, the things you are using to make the process of inoculation, etc. And if you use something like an inoculation loop or scalpel always put them into a flame to sterilize them.

I'm sorry this post got so long, but I hope I could give you some advice,

Elektrolurch

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Originally posted by vts1134:

Well I lost over 8 pounds of rye today, damn that hurts. I tried both honey solution and spore water, soked the rye for 12-24 sterilized for 1.5 hours+ and I ended up with a very beautiful ranbow effect in my bags. I think that I am done untill I build/purchase a laminar flow hood, agar, and an autoclave that sterilizes to 30+ psi. If there is any one out there with any feed back on rye please let me know. Thank you

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"Streaming through the starlit skies
Travelling by telephone
Hey ho here we go
Ever so high"

Flaming, 1967 Pink Floyd

What little I know....

[This message has been edited by Dr. Charles Lindeman (edited May 03, 2000).]