Methods of Disinfecting Substrate???

Alright haters, have at you!

Let's reinvent the substrate disinfecting and inoculation process so that you don't need an airtight room, glovebox, or even a pressure cooker. A way that you can inoculate your solid substrate jars with the lid off Still all very theoretical at this point.
Substrate, how do I disinfect thee? Let me count the ways:

Pasteurization:
-Heating the substrate to varying temperatures for a set amount of time then cooling it quickly in order to slow down or reduce the growth of unwanted bacteria. There is a similar method to the pressure cooker (autoclave), referred to as Pascalization that has to do with pressure and steam but applied intermittently.

Pretty basic, doesn't have to be a high temp either, can be an extremely low temp too (Like cooking from freezer to boiling water). I think the idea here is the speed of change in temp and pressure that is important.

Many methods of pasteurization just aren't feasible (unless maybe if you're using liquid culture).

Sterilization:
-Heating the substrate either though direct heat (oven) or Autoclave steam (pressure cooker) to temperatures of 121–134 °C (250–273 °F) for around 15-25 mins. This is the most used process for sterilizing substrate in jars.

-Using chemical disinfectants to kill bacteria: Phthalaldehyde, Glutaraldehyde, formaldehyde, Bleach, Ozone, Peracetic acid, and Silver are some used in food and/or household cleaning.

Let's stay away from those ones though, as many are not only hard to come by but also (like Silver and Bleach) Will prevent the culture we're trying to build from growing and many are very poisonous even to humans.

Some of the more SAFER chemicals to use for our purposes would include: Hydrogen Peroxide (H2O2), 70%-100% Ethanol (140-200 Proof grain Alcohol), And Isopropyl Alcohol (Rubbing alcohol, ISO).

-Radiation through electron beams, X-rays, gamma rays, subatomic particles, and non-ionizing electromagnetic radiation.

Now again, most of these are not what we want to be using... But that last one, the "non-ionizing electromagnetic radiation" is the radiation given off by your everyday microwave oven.


-What we are basically left with is: Temperature, Steam, Pressure, Hydrogen Peroxide, Ethanol, ISO, and Microwaves.
-What we want: (maybe?) Temp, Steam, - (Definitely) Hydrogen Peroxide, Ethanol, ISO, and Microwaves.

you forgot bleach.

I'm thinking of a process that can be used with both large particle and powder form substrates. As stated above, this is more a focus on solid substrate than liquid cultures.

Sort of cooking, rinsing (if needed like rice cakes and rye seed), mixing, then heating, then mixing again.
There already exists the lesser known hydrogen peroxide method for medicinal mushrooms but hardly layman to keep it "simple" and doesn't remove the pressure cooker.

For instance: Assume we are using 500 ml jars = 2 cups
-DO NOT add holes to lids

-Measure half jars of rice about 250 ml or 1 cup (because it doubles in size when cooked).
If you're using other sized jars, the best way to do this is to fill one jar completely and then pour that into a measuring cup to find out how much one jar holds, divide by half.

-Cook the rice. Adding a lot of water unless you want sticky rice? In my mind the mycelium needs Oxygen and sticky rice won't allow for that...

-Rinse the rice by pouring ice cold water?

-Add 1/4 or 125 ml water to jars and microwave for 4-5mins. (WITHOUT LIDS ON!!)

-While the jars are in there:
*Put rice in a bowl
*Mix together a solution of Grain alcohol, Water, and H2O2. Parts 2:1:1 Respectively ~ Part=25 ml per 2 cups of cooked rice? (A Dunno Maf)
*Mix solution into cooked rice with a spoon or by hand

-Remove one jar from the microwave at this point and pour out any remaining water

-Add rice to jar until full, a little under full (allowing for casing?), about 4/5 of the way full (no casing but good if you want to shake during incubation?), any amount full really...

-Inoculate by syringe (with lids off) but of course first sterilizing the tip with flame.

-Clean lids with solution from above

-Add lids, Incubate, Repeat until all jars are done.

polyarcturus said:

you forgot bleach.

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Nope it's in there, but it's one of the ones I'd want to steer clear of as too much will inhibit ANY life growing in the jars...

Snakebyte said:

-Using chemical disinfectants to kill bacteria: Phthalaldehyde, Glutaraldehyde, formaldehyde, Bleach, Ozone, Peracetic acid, and Silver are some used in food and/or household cleaning.

Let's stay away from those ones though, as many are not only hard to come by but also (like Silver and Bleach) Will prevent the culture we're trying to build from growing and many are very poisonous even to humans.

Some of the more SAFER chemicals to use for our purposes would include: Hydrogen Peroxide (H2O2), 70%-100% Ethanol (140-200 Proof grain Alcohol), And Isopropyl Alcohol (Rubbing alcohol, ISO).

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Higher fungi mycelium is extremely resiliant against grain alcohol and peroxide whereas the lower form fungi moulds (and bacteria) without fruiting bodies that we try to protect our culture from are not.

I want opinions, Experiences, and other creative ideas.
Be as harsh as you can, take it and change it, criticize, bitch, whine, complain, innovate!
Even predictable old timers who want to tell me to "stick to old ways" have had thoughts and have thoughts about it.
I cannot be the only one haunted by this?

I have been looking for a lot of answers to substrate nutritional value, nutrition uptake by mycelium, and ways to keep mould infections of substrate down.
I think it would be really interesting to be as self sufficient as possible and am looking into growing some of my own medicinal mushrooms.
And if it'll work for psilocybin, it sure work for other Fungi too. Psilocybin would just be a plus

Wouldn't mind having more input than "You forgot bleach"...

IF it were to work, does this seem "simple"?
Are there other variables to consider - If so, what are they?
Maybe you want to discuss different substrates?
Maybe you want to discuss different amounts?
Any other similar methods of "dry" sterilization?
What if we mix the solution with the substrate and put THAT in the microwave?
Any alternate solutions we can use besides H2O2 and Ethanol?
Maybe we should use a glovebox just the same?
Is this idea more feasible with bags?
Will bags still need to be microwaved or boiled or simply washed with the/a solution?

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no offense but your reinventing the wheel here. pressure cooking/autoclaving is really the most efficient way to sterilize ATM, any other method is going to be subpar.

Aha, I knew at least one person would say that. No offense taken.

However even the wheel itself has been reinvented dozens of times.
When in reality, the autoclave is the most efficient way to sterilize that we currently know about.
Furthermore, The autoclave is actually very slow when inoculating bulk substrate. It makes sense for small jobs.

What if I want to make a bulk so big it won't fit in a pressure cooker - Go out and buy a larger pressure cooker? Not very ideal having different sized pressure cookers all over the house...
I also mentioned about the hydrogen peroxide aerobic method to sterilize bulk wood substrates for oyster mushrooms and such. So THIS is pretty much a change on THAT process, not really the PC autoclave method.

Snakebyte said:

Sort of cooking, rinsing (if needed like rice cakes and rye seed), mixing, then heating, then mixing again.
There already exists the lesser known hydrogen peroxide method for medicinal mushrooms but hardly layman to keep it "simple" and doesn't remove the pressure cooker.

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SnakeByte said:

Aha, I knew at least one person would say that. No offense taken.

However even the wheel itself has been reinvented dozens of times.
When in reality, the autoclave is the most efficient way to sterilize that we currently know about.
Furthermore, The autoclave is actually very slow when inoculating bulk substrate. It makes sense for small jobs.

What if I want to make a bulk so big it won't fit in a pressure cooker - Go out and buy a larger pressure cooker? Not very ideal having different sized pressure cookers all over the house...
I also mentioned about the hydrogen peroxide aerobic method to sterilize bulk wood substrates for oyster mushrooms and such. So THIS is pretty much a change on THAT process, not really the PC autoclave method.

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I admire your efforts and your enthusiasm but poly is correct. Go back and do some research on the longevity and toughness of the critters you have to contend with. Endospores are the closest things we have in nature to indistructable. When they germinate, some bacteria can reproduce every 20 minutes. So, all it take is for one to survive. And they can withstand temperatures of over 150 degrees - for ever. They can withstand damp heat of 250 degrees for many minutes and you can float them on bleach or alcohol. Damp heat in excess of 212 degrees for at least half an hour is one of the few things that are dependable - the other is the kind of heat that makes metals glow red.

Now what you are doing is trying to subvert the natural course of events in nature. Don't do that. You want nature working for you as often as you can.


Pasteurization is a natural event. You are not looking to wipe out all organisms in your substrate, you are looking to kill only the ones that compete directly for the resources your mycelium wants. Believe me please - brute force is not the way to grow mushrooms and certainly not to do so sustainably.

Pasteurization will eliminate molds, bacteria, seeds and bugs that are capable of growing faster than your mycelium. Kill them and leave the ones that do nothing untoward - and possibly help your mycelium along while surpressing the uglies until your myclium is capable of taking care of itself. That is all you need to do.

Never forget that mushrooms do just fine in the forest where there is an endless number of other sucessful organisms in competition, all you are looking to do is tilt the environment in your particular organism's favor for long enough to allow it to gain an advantage.

SnakeByte said:

I have been looking for a lot of answers to substrate nutritional value, nutrition uptake by mycelium, and ways to keep mould infections of substrate down.
I think it would be really interesting to be as self sufficient as possible and am looking into growing some of my own medicinal mushrooms.
And if it'll work for psilocybin, it sure work for other Fungi too. Psilocybin would just be a plus

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Nope, I have managed to domesticate a grand total of 7 distinct species of mushrooms in 40 years. No two are alike. I enjoy the fact that p. Cube folks figure they can conquar the world after they have managed to crack the P.C code but the others are far - FAR more complex Please let me know when you have managed to domesticate morels, chanterelles, boletus or truffles.


Now the secret to the black morel was stumbled upon by an avid P. cubensis grower who holds the patent on the process - they sell for what/ 250 bucks a lb dry? And a very lucky few have managed to grow white truffles in oregon which they sell for about 600 a lb wet. And we think pot is expensive. Growing mushrooms is one of the most difficult and tedious endeavors you can begin and the road runs much farther than any of our life spans

A bit of topic, but I used the oven door trick. Turn it on low and leave the door open and do all the work on the door with rubber gloves, n95 respirator, and haste. I didnt have any contamination issues. I'm not exactly sure why this worked for me or if it was just a placebo effect. I just thought it might be worth sharing.

canndo said:

Go back and do some research on the longevity and toughness of the critters you have to contend with.

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Well there are ways we can trick the endospores like "Bacillus sp." in certain substrates. Grain for instance can be soaked 12 to 24 hrs before sterilizing. This allows them to germinate and become vulnerable. The other bacteria I've looked at are just too weak to withstand the chemical sanitizing of alcohol and peroxide.
These steps I've described are just merely off the top of my head and can easily be amended if you like. For one: The strength of peroxide was never determined. Would it be best to use 5% or 35% food grade hydrogen peroxide?

canndo said:

Now what you are doing is trying to subvert the natural course of events in nature. Don't do that. You want nature working for you as often as you can.
Pasteurization is a natural event. You are not looking to wipe out all organisms in your substrate, you are looking to kill only the ones that compete directly for the resources your mycelium wants. Believe me please - brute force is not the way to grow mushrooms and certainly not to do so sustainably.

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But what else are you doing when we sterilize with the pressure cooker? We're humans, that's what we do - It's our thing. We change our environment to meet our needs.
What I am actually trying to do with this method is to lessen the amount of bacteria in the substrate in question before introducing their new neighbours who will be taking over.
The rest is just climate control afterwards to keep other competitors such as "cobweb" down. Air circulation, humidity, and amount of nitrogen seem to be big triggers for mushroom competitors.

Mookjong said:

A bit of topic, but I used the oven door trick. Turn it on low and leave the door open and do all the work on the door with rubber gloves, n95 respirator, and haste. I didn't have any contamination issues. I'm not exactly sure why this worked for me or if it was just a placebo effect. I just thought it might be worth sharing.

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I've heard of this, the core concept being that the rising heat floats any particles up away from your substrate.
Preheating the oven to 350F for 10 minutes to kill any bacteria in the oven then reducing the heat so you can work around above the open door.
Good idea, but I might rather a glove box than inoculating 10 jars over the oven.

SnakeByte said:

Well there are ways we can trick the endospores like "Bacillus sp." in certain substrates. Grain for instance can be soaked 12 to 24 hrs before sterilizing. This allows them to germinate and become vulnerable. The other bacteria I've looked at are just too weak to withstand the chemical sanitizing of alcohol and peroxide.
These steps I've described are just merely off the top of my head and can easily be amended if you like. For one: The strength of peroxide was never determined. Would it be best to use 5% or 35% food grade hydrogen peroxide?


But what else are you doing when we sterilize with the pressure cooker? We're humans, that's what we do - It's our thing. We change our environment to meet our needs.
What I am actually trying to do with this method is to lessen the amount of bacteria in the substrate in question before introducing their new neighbours who will be taking over.
The rest is just climate control afterwards to keep other competitors such as "cobweb" down. Air circulation, humidity, and amount of nitrogen seem to be big triggers for mushroom competitors.



I've heard of this, the core concept being that the rising heat floats any particles up away from your substrate.
Preheating the oven to 350F for 10 minutes to kill any bacteria in the oven then reducing the heat so you can work around above the open door.
Good idea, but I might rather a glove box than inoculating 10 jars over the oven.

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I think you are failing to differenciate between stages of sterililty. We look for 100 percent sterility when we are seeking to isolate a single organism - we don't want any competition at all. That is where we autoclave and in most instances there isn't much to autoclave - a few liters of agar at most - and I don't believe chemical sterilization works at this level at all.

Next there is the production of spawn - this as well needs to be fully sterilized as we are working with pure and highly nutritous substrates - possibly creating mother sources from which grain to grain samples will be taken - this as well lends itself poorly to chemical sterilization and this as well is - realtively low in bulk. The stage that is inhibited is of course the bulk stage, the straw or compost or what haveyou - now this substrate does not lend itself to sterilization, in fact sterilization is usually not a good idea because of what I have said, creating a situation where all competetitiors have an equal chance. We don't want that, we want to give the mycelium in preference a jump start that is hardly possible with complete sterilization.

Now farms are aware of the cost and inconvenience of full sterilization of the substrate. They will use the natural temperature rise of the nature of compost to kill the bad things and leave the good ones. In the case of artificial substrates or non composted substrates they will use steam boxes with steam genies produceing there required 160 - 180 degree temperatures for as long as required - no autoclave necessary.

All this does is give you desired mycelium a window of opportunity - of a week or two after which the substrate returns to a more normal balance ofgood microbes and fungus and bad. No chemical method I am aware of will handle this sort of bulk and still allow the normal progression of life on the substrate - life that in the case of some mushrooms is essential.

You're right, that I'm not really aware of all the "steps of sterilization", I admit I am very much a noob at this but I think the core concept is there.
The idea is not to eradicate but to rid the environment of just enough bacteria to give your culture the head-start. While keeping the incubating chamber safe from any possible outside contaminants (constantly cleaning tools and such).
For this to happen, we need our change on that environment to be naturally reversible so that the proper bacterias can do their thing. It also needs moisture and air pockets (lots of surface area).
The terrarium also needs to be clean, humid, and have proper air circulation.

But I do know that both sterilization and pasteurization are achieved by denaturation of the tertiary structure of proteins and/or disruption of nucleic acids hydrogen bonds in the bacteria.
That is actually what the autoclave is accomplishing with the steam.

Denaturation
1. a change in the usual nature of a substance, as by the addition of methanol or acetone to alcohol to render it unfit for drinking.
2. in proteins and nucleic acids produced by heat or certain chemicals, usually results in loss of function. In proteins, various non-covalent bonds are disrupted resulting in unfolding of the polypeptide chain; in nucleic acids hydrogen bonds between nucleotides are disrupted converting double-stranded molecules or parts of them into single-stranded forms.

Heat Killing of Bacillus subtilis Spores in Water Is Not Due to Oxidative Damage
Researching a little bit on "Wet Spot; Sour Rot" - Bacillus sp. I found out that the bacteria killing in autoclave heat happens through denaturing of proteins.

"Certainly, the preponderance of evidence presently available supports the hypothesis that inactivation of a key protein or proteins is the major mechanism whereby heat kills bacterial spores. The challenges now are to prove this hypothesis conclusively and to identify the protein(s) that is the target in this process."

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Denaturation of Proteins
Looking into it further I found that Ethanol is used often in denaturing all sorts of proteins when only 70% ethanol is used - but here is something interesting, that if higher concentrations of alcohol are used, the proteins coagulate.


Degradation of oxidatively denatured proteins in Escherichia coli
Here is another study but about the effects of oxidative stress on E. Coli.
It indicates that while H2O2 will not necessarily kill the bacteria, it absolutely slows the growth. This happens through protein degradation by disrupting the nucleic acids hydrogen bonds.

Found this interesting article. Possibly points to the use of black light to fight bacteria?
It also confirms that Silver is bad for bacteria and fungus.
Physical Methods Of Microbial Control:


Both of these (Ethanol and H2O2) do something different to the bacteria. I'm re-thinking the "solution" idea, as it would be more effective if one was added to the substrate, and THEN the other.
Or
If using grain, soaked in one, THEN the other.
Though IF successful, one serious downside is very possibly not being able to use BRF with this "dry" process.

So IF the denaturation of the bacteria can be achieved AND reversed, than I can say with reasonable confidence that we might be onto something...

now i like where your going, and looking into tissue culture may help you with questions you seem to be asking, in tissue culture you wash the plant material in several solutions before it is considered sterile enough for the culture container.

Here is yet another study. This one about that "solution" I was thinking of.
Oxidation of ethanol with hydrogen peroxide:

I'm still re-thinking the whole solution idea and making it step-by-step though.
Interesting information just the same

thought i would also throw the idea up of radiation sterilization. dont know if its possible but there are some pretty powerful Uv-C lamps out there, and then again there is always the microwave... just a thought.

Yes, I was also thinking of microwaves and read a little about the UV method but think I need to know more about the latter.
Though microwaves do kill bacteria (quite effectively), there is one thing that using a microwave on substrate that bothers me. I've noticed it when using the microwave for food. As I'm sure many of you will be able to relate.

When I put things in the microwave, they tend to stick together quite a bit. Not only that but the food in question is always fast to dehydrate when I take it out.
To fight this, I've been wondering if it's not better to:
-Microwave food in containers with lids, then wait a moment before opening it.
-Adding a little water in the container to whatever I'm cooking in there
-Cooking it for longer periods of time, though intermittently so to shake my container of food

You might laugh but this seems to have solved my problems of the welded dry food.
Something similar might work on non-flour based substrates. Like grain or bird seed... I've never put bird seed in the microwave before though. lol

when drying my weed in the micro i use the freezer to cool it quickly to prevent it from becoming crispy this allows me to come close to a very normal bud consistency. if that helps but with food i use closed containers... and follow the directions, as most say to leave the food in the microwave for a few minutes before removing.

I'm also trying to find a viable replacement for vermiculite. Any thoughts on that?