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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Edited by figgusfiddus (03/01/07 10:20 AM)
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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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