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Amazon Shop for: ½ Pint Jars, Agar, Autoclave, HEPA Filter, Laminar Flow Hood, Malt Extract, Paul Stamets, Pink Floyd, Pressure Cooker, Rye Grain

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Anonymous

Re: WOW what a loss, any more words on rye
    #85099 - 04/25/00 05:02 PM (16 years, 7 months ago)

first off- how did you sterilize the grain? did you cook it first? liquid innoculant never seems to work for me- bacteria galore! but when you use actual mycelia, it works wonders!
what ive done with my grains (including rye) is cook them first in a rice cooker with a mix of water and hydrogen peroxide (4:1 or so) i let the grains cook and then load them into jars with filter disc lids. they are pressure cooked @ 15lbs for no less than an hour. i let them cool and then take mycelium scrapings from mea cultures in a bleach bombed DRAFT FREE kitchen with the a stove burner and oven running. this whole process takes a heap of motivation, but is worth the tedious effort. hope you find the motivation again!

~nati shroom

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



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Offlinevts1134
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Re: WOW what a loss, any more words on rye
    #85101 - 04/25/00 07:02 PM (16 years, 7 months ago)

how do you accomplish mea cultures without a laminar flow hood? I went out today to see if I could build a hood for myself but could not find any of the neccesary equipment. I am going to try and use a fully colinized rice cake for innoculation a bag of rye tonight, I will let you know how it goes. Oh and you mentioned that you turn on the oven and a burner, do you do your work directly over these things? Thank you for your feedback.


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Anonymous

Re: WOW what a loss, any more words on rye
    #85102 - 04/26/00 02:04 AM (16 years, 7 months ago)

You could wait till I can get someone to photograph mine into computer pictures or just build a big old sealed glove box and spray lysol if you want a low tek.

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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InvisibleHippie3
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Re: WOW what a loss, any more words on rye
    #85103 - 04/26/00 07:52 AM (16 years, 7 months ago)

a simple glove box made out of a cardboard box will eliminate 99% of contams but rye can be a bitch anyway. i soak it 24 hours then cook it an hour then load jars and pressure cook it another hour.

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Offlinevts1134
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Re: WOW what a loss, any more words on rye
    #85104 - 04/26/00 05:03 PM (16 years, 7 months ago)

Hippie that is basicly methodology but I am going to start to sterilize for 2+ hours instead of one. $480 range is a little steep for my wallet have any of you constructed your own laminar flow hood? I am trying to track down materials right now. If I find a reasonably priced suplier I will let you guys know.


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Anonymous

Re: WOW what a loss, any more words on rye
    #85105 - 04/26/00 07:40 PM (16 years, 7 months ago)

I sourced parts for my charged electrostatic system for near 100. Its the labor that would kick the price high into that sky if I had to build it for people. And mine from the outside looks almost like the $4800 commercial lab ones except you can tell my chamber is a clear 60 quart tupperware box, everything else on it looks like the commercial units.

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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InvisibleHippie3
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Re: WOW what a loss, any more words on rye
    #85106 - 04/27/00 10:39 AM (16 years, 7 months ago)

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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InvisibleLanaV
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Re: WOW what a loss, any more words on rye
    #85107 - 04/27/00 10:46 AM (16 years, 7 months ago)

Hi vts1134,
My little suggestion is along the lines of what Hippie3 said, "soak for 24 hours".

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



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Anonymous

Re: WOW what a loss, any more words on rye
    #85108 - 04/28/00 12:17 PM (16 years, 7 months ago)

quote:
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 ?

Actually my hepa chamber :smile:... 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!



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Anonymous

Re: WOW what a loss, any more words on rye
    #85109 - 04/28/00 12:18 PM (16 years, 7 months ago)

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

Lana


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



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Offlinevts1134
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Re: WOW what a loss, any more words on rye
    #85110 - 04/29/00 11:33 PM (16 years, 7 months ago)

Learner, you were talking about an electrostatic system and, pardon my ignorance, but it sounded like a glove box. If this is infact the case then could you explain it in greater detail as I have never heard of such a thing nor can I figure out how you would use a filter on a glove box. If this is not the case, however, and you are using it a lamflow hood then I can not see how you could get away with just using an electrostatic filter and not a hepa filter. Either way could you explain your set up in greater detail to me as I am willing to spend alot of time on a solution.


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Anonymous

Re: WOW what a loss, any more words on rye
    #85111 - 04/30/00 08:39 AM (16 years, 7 months ago)

McMaster-carr supply company www.mcmaster.com
They carry every thing needed to build a flow hood + other things that fungi perfecti sells for alot less money. I have their 3000 page catalog and it kicks ass.

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Anonymous

Re: WOW what a loss, any more words on rye
    #85112 - 04/30/00 10:54 AM (16 years, 7 months ago)

vts1134 - growing on rye isn't hard, you either contaminated it or didn't have decent spores or a viable solution. If you don't want to loose so much rye, don't try and start out so large. Try using 5-10 jars and just inject sterile water/spore solution. Incubate them and when you have some nice colonzed jars, then you can go to the 8 pound expansion from your master jars. Also, a 15psi Presto (or other), normal kitchen pressure cooker is highly sufficient to sterilize grain or anything else. As for laminars and hepas, not really needed. If you have a sealed glove box and you use Lysol and or alcohol to properly wipe everything, you'll be fine. Just my eperience.


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Offlinevts1134
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Re: WOW what a loss, any more words on rye
    #85113 - 05/01/00 11:16 AM (16 years, 7 months ago)

15 psi maybe okay for you but I live above nine thousand feet and 15 psi for an hour is not enough as I have learned the hard way. And I am not worried about loosing that much rye I just thought that it would get alot of replies. As for a glove box vs a lfr I have not yet made up my mind. Oh and yes I did contaminate the rye myself because the solution is viable I have bags that are succesfull to.

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



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Anonymous

Re: WOW what a loss, any more words on rye
    #85114 - 05/01/00 03:33 PM (16 years, 7 months ago)

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

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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Offlinevts1134
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Re: WOW what a loss, any more words on rye
    #85115 - 05/01/00 05:49 PM (16 years, 7 months ago)

It was my understanding that electrostatic filtration is not sufficient for sterile work with higher fungi.


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Anonymous

Re: WOW what a loss, any more words on rye
    #85116 - 05/01/00 07:39 PM (16 years, 7 months ago)

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

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 :smile:.. 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).]



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OfflineElektrolurch
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Re: WOW what a loss, any more words on rye
    #85117 - 05/03/00 12:21 AM (16 years, 7 months ago)

Hi vts1134,

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

quote:
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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Travelling by telephone
Hey ho here we go
Ever so high"

Flaming, 1967 Pink Floyd



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The doll's house, darkness, old perfume
And fairy stories held me high on
Clouds of sunlight floating by.", Pink Floyd '67


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Anonymous

Re: WOW what a loss, any more words on rye
    #85118 - 05/03/00 01:52 PM (16 years, 7 months ago)

Wow. Now that's an intelligent post! Welcome aboard, who ever you are. Hope to hear more from you.
P.S. I use a small spray bottle filled with iso alcohol to spray my hands and work surfaces. I strongly suggest you get a red bottle or label the alcohol, or you may do what I just did!
------------------

What little I know....

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



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Offlinevts1134
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Re: WOW what a loss, any more words on rye
    #85119 - 05/03/00 04:44 PM (16 years, 7 months ago)

Thank you for those pieces of advice it is very helpfull. I have always used bleach water and not Isopropl alcohol solution, I think that I will change to that now. Also you said that 30 psi would be an overkill for rye, I say why not an overkill as long as I don't sterilize my mea at those tempretures I think that I will be alright. Again thank you for those words of advice.


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Amazon Shop for: ½ Pint Jars, Agar, Autoclave, HEPA Filter, Laminar Flow Hood, Malt Extract, Paul Stamets, Pink Floyd, Pressure Cooker, Rye Grain

Mushrooms, Mycology and Psychedelics >> Advanced Mycology

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