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Offlinefiggusfiddus
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A newb's reflections on sterility... shortcuts work, if you're smart. * 1
    #6620357 - 02/28/07 03:12 PM (14 years, 2 months ago)

Okay, so I admit it--I really enjoy reading up on and talking about methodology. I can tell that other people do too, hence our nice big forum. But contamination is essentially the word of the day in every single tek and post I read, and I am honestly a little confused as to why it is the scourge of so many first-time attempts (and tenth-time attempts). This is intended not as information from an expert, but from a more-or-less newbie to those even less experienced. Take it as you will.

Here's a little caveat: I don't know why contams aren't a problem in my cakes. I have so far seen contamination only in my experimental cakes (e.g. whole rice using uncertain spore prints, et cetera). I have avoided contamination altogether in all five of five PF-style cakes (from mail spores), and seven of seven tissue culture cakes (also PF-Tek formula). Now, here's the funny thing:

I use a pot, not a pressure cooker. I line my terrarium with verm for moisture (sterility no-no, in theory). I spray my cakes with tap water. I essentially ignore the notion of airborne contaminants, save when working with very sensitive material. My apartment is filthy. Really, filthy. I work in an atmosphere that simply must have tons of mold spores. I am in theory a terrible mushroom cultivator.

Then why does it work? I think it has to do with some of my shortcuts.

* Shortcut number one--ALCOHOL.

91% isopropyl alcohol, from your local supermarket. It evaporates quickly, doesn't seem to harm the mushrooms (in reasonable amounts), and leaves behind sterile water.

Why are you boiling that syringe? Why are you boiling anything unless it has substrate in it? The only things you should be boiling are things you cannot bake (like wet substrate) and cannot sterilize with alcohol. Put a reasonably large amount in a little spray bottle and you're golden. Just be sure you get some on the bottle's exterior, too, if you're doing anything sensitive.

For instance: you're sucking a fresh print's worth of spores into your syringe, or maybe some tissue slurry. Your syringe has been sitting around a while maybe, or maybe you sucked in some air by accident. No biggie. Squeeze it all out, and fill it up with alcohol. You can use a tiny little vial or glass and simply recycle alcohol between inoculations if you want--your alcohol doesn't get contaminated. It's ALCOHOL! You don't need to heat up the tip of your syringe as long as you let it sit in the alcohol for a second or two. If you have a contaminant that is surviving that alcohol bath, it's going to survive the fucking nuclear holocaust, and getting your syringe all heated up and corroded isn't going to do a damn thing to it. EDIT: Just be sure to flush the syringe with sterile water after clearing it with alcohol. I don't know how quickly alcohol kills spores, but it will likely kill tissue in a water suspension very quickly. I like to keep a big glass full of sterile water with seran wrap for this very purpose--you don't even need to let it cool to room temp, since it's just for flushing.

Handling your cakes? Spray some alcohol on your gloves. (Do use gloves, though--your hands aren't as easily sterilized, even by alcohol, as a flat, latex surface). Putting that tinfoil back over a freshly-inoculated jar? Spray some alcohol on it and let it evaporate. It's easy, man. Give all jars/glasses a little spray and a rinse before you fill them with substrate, too.

Don't boil your food processor when making a slurry. Spray it down with alcohol. You will kill MORE contaminants with a simple alcohol spray than you will with 212F water. If you're really OCD, do both I guess.

When dunking, you don't really need to sterilize your water. Tap is relatively clean, as long as you don't have a well-water source (which is sometimes common in the extreme boonies). What you do need to do is sterilize your container. Spray it down good, and rinse with tap, then refill it and dunk as usual. Make sure you do rinse, because I don't think the cakes should sit in even a weak isopropyl solution overnight.

* Shortcut number two--the oven.

Most of your contaminants will not come from the air, or (unless you're stupid) from contact, but from the ingredients. Whole grains are full of them, and so is your verm. You can get your verm to 300 or 400F in maybe twenty minutes without ever touching a pressure cooker, and you should. Do the same with your grains or flour, but don't get them over 300 for long or they'll probably start to get toasty (and that will probably reduce the accessibility of their nutrients to the cakes).

If you're a whole-rice person, it's also nice to rinse the rice before you cook it, and to dry it during sterilization in an oven or convection oven. It removes some of the tasty starches, but it keeps things from clumping in your jars. Personally, I don't see the appeal of whole rice, but it's a nice trick.

Ovens are great for utensils, as well. Why would you boil that fork you're going to use to break up and case your cakes? Boiling gets it to 212F, which is probably fine, but it's not like there's some temperature limitation on the fork itself. Bring it up to 300F and let it cool. Or, if you're me, spray it down with alcohol and cut out the wait.

* Third shortcut--conditions.

Now, this isn't really a shortcut, but it will save you a hell of a lot of problems if you aren't already doing it: INCUBATE AT IDEAL TEMPERATURES.

Look at it this way: You have a nice, rather clean little home for mushrooms in your jar. You also have a big wad of spores or tissue, something probably larger than most wild mushrooms ever use to get their foothold. So what excuse in the world is there for some random opportunistic bacterium or mold to win in a fight with your mushroom when you have thousands and thousands of spores on your side? Sure, shit happens sometimes, and you're bound to have the odd contaminated cake, but it shouldn't be a common thing. If it is, you are probably handicapping your culture.

How? Keeping it at room temp is one way. You know your culture's ideal colonizing temperature, 85F, and you should use that knowledge to your advantage. The faster your colony spreads, the less likely its enemies are to get a foothold first. Because that's what it's really about--beating bacteria and mold to the punch. You're going to have SOME contaminants in your cake no matter what you do, the key is to make sure your cake colonizes all the substrate before they can colonize any of it. If you make sure your colony is spreading quickly by maintaining the right temperatures, you keep its enemies from ever having a chance. The only way they should win is if they get introduced to the substrate at a level comparable to your spores or tissue, and unless you're sprinkling bits of moldy cantaloupe over your substrate that shouldn't be happening very often.

Finally, sterilize your incubation chamber regularly. When you pop in to check on your babies, spray the inside of the incubator down with some alcohol (or Lysol, if you prefer, though it doesn't evaporate as cleanly or as quickly). Let it air out for thirty seconds or so, fanning if you want, and close it back up. Usually you don't want to fan an incubator, but I am of the strong opinion that a few airborne contams are a much smaller problem than the loads and loads of contact contaminants that you get growing inside a plastic incubator. I haven't had a problem with alcohol fumes harming my cakes, but I recognize it's probably not good for them, hence the fanning.


Okay, that's my rant. Hopefully it will help someone. I am amazed that the benefits of alcohol are not more often included in teks. Heat is great, but you can't heat everything all the time. If you have a lot of contamination problems, sure, invest in a pressure cooker--but the difference you get between the 212F of the boiling pot and the temperatures in the pressure cooker probably won't solve your problems. Some spot sterilization, on the other hand, requires a very small effort and negligible expense, and it can go a disproportionately long way toward keeping your jars clean and healthy until they're big enough to fend for themselves.


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FGSFDSFGSFDSFGSFDSFGSFDSFGSFDS
FGSFDSFGSFDSFGSFDSFGSFDSFGSFDS
FGSFDSFGSFDSFGSFDSFGSFDSFGSFDS


Edited by figgusfiddus (03/01/07 12:20 PM)


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Invisiblegeko
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: figgusfiddus]
    #6620815 - 02/28/07 05:01 PM (14 years, 2 months ago)

:thumbup: Very nice post! I have always been thinking along the same lines and so far in my brief mushroom cultivating experience I have seen very little contamination.


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InvisibleHipsterDoofus
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: geko]
    #6621742 - 02/28/07 09:20 PM (14 years, 2 months ago)

Like you,I don't know why contams aren't a problem in my cakes. I work out in the open on a table. No gloves, box or flowhood. My sterile procedure is only to wipe the table, spray the air and wash my hands. I only use alcohol to swab the needle.

From my point of view your "shortcuts" are just a lot more work and trouble.


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OfflineTobolam
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: figgusfiddus]
    #6621848 - 02/28/07 09:52 PM (14 years, 2 months ago)

I'd have to argue here.
1) Shortcut alcohol - Alcohol is great for quick sterilization, but you need something more than a quick spray. After using 91% alcohol I found diluted 70% Alcohol (Making it about 50) more effective. Boiling when you need to boil saves the trouble. If you put some alcohol in a syringe you will need to get rid of that alcohol by something other than squirting it out, as microparticles still stay in there (try this - fill up a syringe with alcohol, squirt it all out, full it with 1 mL of water, squirt the water out on your eye, I bet it burns). You will need to rinse it out with water....boiled water... So while you are setting up a pot of boiling water, wouldn't it be easier to throw the syringe in there?

2) The oven would reduce contaminants. A pressure cooker would OWN the oven at reducing contaminants. Only PF worked IME without a PC, and therefore if you are using anything other than PFtek you need to use a PC, rendering the oven obsolete and heartbroken. PF works fine without the oven. It's just redundant and unnecessary to use the oven. Boiling or PCing FTW!.

3) You are neglecting the fact that bacteria grows faster in incubated conditions...Much faster. So If you incubate to help mycellium to outgrow the bacteria, you are actually helping the bacteria to outgrow the mycellium (assuming your shit is contaminated)


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Offlineyading
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: Tobolam]
    #6623698 - 03/01/07 10:55 AM (14 years, 2 months ago)

I think ulterlize alcohol would be a quite useful shortcut...


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Offlinefiggusfiddus
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: Tobolam]
    #6623946 - 03/01/07 12:31 PM (14 years, 2 months ago)

Quote:

Tobolam said:
I'd have to argue here.
1) Shortcut alcohol - Alcohol is great for quick sterilization, but you need something more than a quick spray. After using 91% alcohol I found diluted 70% Alcohol (Making it about 50) more effective. Boiling when you need to boil saves the trouble. If you put some alcohol in a syringe you will need to get rid of that alcohol by something other than squirting it out, as microparticles still stay in there (try this - fill up a syringe with alcohol, squirt it all out, full it with 1 mL of water, squirt the water out on your eye, I bet it burns). You will need to rinse it out with water....boiled water... So while you are setting up a pot of boiling water, wouldn't it be easier to throw the syringe in there?

2) The oven would reduce contaminants. A pressure cooker would OWN the oven at reducing contaminants. Only PF worked IME without a PC, and therefore if you are using anything other than PFtek you need to use a PC, rendering the oven obsolete and heartbroken. PF works fine without the oven. It's just redundant and unnecessary to use the oven. Boiling or PCing FTW!.

3) You are neglecting the fact that bacteria grows faster in incubated conditions...Much faster. So If you incubate to help mycellium to outgrow the bacteria, you are actually helping the bacteria to outgrow the mycellium (assuming your shit is contaminated)




1) You're right, and I added the bit about the rinse. That was just an omission. Boiling a glass of water is easier than making tea, so I don't consider that a difficulty, but boiling objects and ensuring their sterility on removal, etc, is a monstrous pain. I don't think boiling certain objects, such as syringes, does all that much, honestly--surface contaminants get right back on them as soon as you're done boiling, and they're -wet- to boot, meaning everything sticks. I would rather see someone spraying regularly, not assuming anything is clean, than someone thinking their boiling actually did the deed in any permanent way. Shortcuts are great, but they should be real shortcuts, not just the mental kind.

2) Ovens will reduce contaminants in dry material much, much better than boiling, and just as well as a pressure cooker (or better, depending on its max temp). If you want, you can crank your verm up to 500F in the oven--and why not? If you're not using a PC, you SHOULD use the oven. If you are, I can see why you would say it is redundant.

3) Of course incubation helps bacterial growth too, but it's a question of relative rate. Generally speaking, the "ideal temperature" we'd be using if we were trying to grow contaminants would be a little higher than the ideal for cubensis. I would suspect it's in the low nineties, though I'm not going to pretend any solid knowledge of this--anyone who does is welcome to post. At any rate, the improvement in the growth rate of your mycelium from raising your incubation to 85F should be greater than the growth rate boost given to the bacteria by the same, if only because this is the native operating temperature of cubensis. Yeah, it's theory, not certainty, but I think it's founded in reason even if it is shooting from the hip a bit. There's no reason not to incubate at ideal temps, though--even if you're speeding up contamination, you'll at least know your cakes are toast a few days sooner! None of this "colonizing for a month" crap for me, thanks.

EDIT: Okay, I've rethought this, and I think there's a better problem with your statement that the bacteria will outgrow the cubensis at incubated temperatures. Simply put: Look at the quantity of cubensis material you have introduced. If it's a PF-style syringe, not a lot probably, but if it's a homemade spore syringe or a tissue culture we're talking massive amounts of material. The contaminants enter the substrate with the tiniest number of cells, even in the worst conditions. No matter what your relative growth rate, the amount of time it takes the contaminants to recover from their initially low numbers is about enough time for you to colonize the whole cake. The mycelium is actually growing very quickly compared to the bacteria because its initial numbers are so huge, and while incubation will speed up the growth rate of both it will benefit your culture disproportionately well so long as it's got a good foothold.

Your point has possibly made me reconsider the value of incubating a low-spore PF-style syringe, though, all the same, but there's no way it has me reconsidering the incubation of a tissue culture. Good points, though.


Also, saying that because only PF didn't tell you to use a PC, you need a PC with anything but PF is not strictly logical. You shouldn't engage in high-risk growing techniques without some alternate form of sterilization, sure, but in some cases the sterilization of bulk substrate can be achieved in oven conditions. Your oven gets HOTTER than your pressure cooker! And as long as the material is dry, you can cool it down before it really cooks. Simple.


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FGSFDSFGSFDSFGSFDSFGSFDSFGSFDS
FGSFDSFGSFDSFGSFDSFGSFDSFGSFDS
FGSFDSFGSFDSFGSFDSFGSFDSFGSFDS


Edited by figgusfiddus (03/01/07 12:41 PM)


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Offlinemikemushroom
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: figgusfiddus]
    #6624063 - 03/01/07 01:10 PM (14 years, 2 months ago)

OK- alcohol will kill bacteria, but it takes an amount of time for it to kill viruses. Alcohol is not very effective at killing contaminating spores. This is why some people use 10% bleach solution. A glovebox keeps airflow to a minimum, and you can make a poor man's GB with about $3.
I think it is lazy to not be safe (safer). And considering alcohol and bleach cost so little, a say why not (but personally I only use 91% alcohol. 70% is better according to the vet. that was in here last night, because it takes water to penitrate cells.
"An ounce of prevention is worth a pound of cure." So I respectively disagree. Perhaps I am a little OCD, but thats fine with me.


--------------------
Want to be of the shaman mind? Carlos Castaneda is the godfather of the New Age Movement which is based off the peyote & shroom eating natives in Mexico.

Read his library of books to expand your mind.


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Offlineminesstudent
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: mikemushroom]
    #6624117 - 03/01/07 01:27 PM (14 years, 2 months ago)

I don't think we need to worry about viruses in mushroom cultivation.


--------------------
"The universe is the way it is because if it wasn't we wouldn't be here to talk about it"


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Offlinemikemushroom
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: minesstudent]
    #6624133 - 03/01/07 01:31 PM (14 years, 2 months ago)

The point-alcohol is not the most effective solution for sterility when used alone. But once again, thats the only chemical I use.


--------------------
Want to be of the shaman mind? Carlos Castaneda is the godfather of the New Age Movement which is based off the peyote & shroom eating natives in Mexico.

Read his library of books to expand your mind.


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Offlinexhooliganx
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: mikemushroom]
    #6625001 - 03/01/07 06:26 PM (14 years, 2 months ago)

Quote:

Here's a little caveat: I don't know why contams aren't a problem in my cakes. I have so far seen contamination only in my experimental cakes (e.g. whole rice using uncertain spore prints, et cetera). I have avoided contamination altogether in all five of five PF-style cakes (from mail spores), and seven of seven tissue culture cakes (also PF-Tek formula). Now, here's the funny thing:

I use a pot, not a pressure cooker. I line my terrarium with verm for moisture (sterility no-no, in theory). I spray my cakes with tap water. I essentially ignore the notion of airborne contaminants, save when working with very sensitive material. My apartment is filthy. Really, filthy. I work in an atmosphere that simply must have tons of mold spores. I am in theory a terrible mushroom cultivator.



the main focus on sterility is when you're inoculating your jars. Once you have your cakes colonized you shouldn't really have to worry about the verm for moisture or tap water or airborne contaminants.

The majority of what was said in this post was utter BS. The 86*F as ideal temp is false. I know that's what it says in TMC, but it's been repetitively tested by RR who is friends with Staments that you have no increase in colonization above 82*F. You've just been fortunate with not having contam problems.

I mean seriously the pftek was developed so that you could make all of those shortcuts and it not matter. Try doing any agar or G2G with alcohol and an oven.


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


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Offlinefiggusfiddus
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: xhooliganx]
    #6627925 - 03/02/07 02:30 PM (14 years, 2 months ago)

No, I agree, I'm talking about simple-simple cultivation, PF-tek level if not strictly PF-tek. I don't think your average tissue clone is far beyond PF level though, in terms of complexity or the likelihood of contamination. Using a food processor cleaned only with alcohol and rinsed with boiling water, I've produced many healthy cakes now with no contamination whatsoever. Method? Take a cross-section of stalk, spray it down with alcohol to kill off surface contams and rinse with room temp water. I did this after Lysoling the air and holding the plastic lid more or less over the work area, since it only takes about 20 seconds. Pop into clean processor, blend with boiled room temp water, and inoculate. Failure rate 0%, at least so far.

I know I'm not an expert, and tried to make it clear, but I thought it should be noted that for beginning procedures like PF and the above tissue clone, first-timers can probably hold back on a few of the standard sterile procedures if they're careful with the routine stuff. I am inclined at this point to think that most contamination at the basic level comes from human error, that is outright exposure to obvious contaminants, not from insufficient sterilization.

I'm really glad you cleared up the matter on the temp though, if the ideal temp is about 82F then exceeding it is just encouraging bacterial growth. I wish that information were more readily visible though--I, like most (probably), have been going by the PF (or was it MMGG?) standard of 85-86. I guess that's what I get for taking 1995 info as gospel.

Anyway, I warned people not to take me seriously. I admit here and now that I may be full of it! But I think some of the tips might still be useful to the new grower, because they've worked flawlessly for me.


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Offlinefromthemoon
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: figgusfiddus]
    #6629345 - 03/02/07 09:25 PM (14 years, 2 months ago)

I bet once one gets a bunch of contaminated jars/casings and are set back 1 or 2 months, you won't want to short-cut on sterility.

Frankly, I think this is a noobie mistake that is common.  First grow or two w/ no contaims and one think contaims are a myth or something so one gets lazier and lazier until total contamination and loss of time and effort - that's when the noobie gets back to sterility.

I enjoy being safe, carefull, and sterial through the entire process.

Just my 2 cents :wink:


--------------------
Look at the atoms in the air and allow your mind to see the other planes that coexists in the same physical space where you sit. There are vibrational strings in each and every atom containing infinite realities, universes, and dimensions. Multidimensional beings and entities are RIGHT IN FRONT OF YOU!!! Only gravity and mental energy can pass through these different atomic branes and mushrooms can break the mental/spiritual membranes separating one reality from the other. TAKE A LOOK!


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Invisibleralphroks
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Re: A newb's reflections on sterility... shortcuts work, if you're smart. [Re: fromthemoon] * 1
    #6719011 - 03/28/07 04:51 AM (14 years, 1 month ago)

One time in biology class, we washed our hands w/ antibacterial soap
to prove it doesnt sterilize your hands by colonizing a thumbprint
on agar. I didnt wash my hands and rubbed my armpits, butt and my sack,
and rubbed my hands all over the plate. O man the colors where amazing..
chiks totally dig you if they know your that dirty


--------------------
"Please read and learn and relay knowledge not misinformation! Thank you"-hyphae


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Mushrooms, Mycology and Psychedelics >> Mushroom Cultivation

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