|By Sanchez (Sanchez) on Sunday, December 23, 2001 - 11:01 am: The Nook|
I am not exactly sure what I am doing wrong here... Here's the deal.
I tried, in a shittily made glovebox to clone a mushroom, dropped a little piece of a mushy into some sterilized honey water.... and several days later I had a hideous white cloud of bacteria. Hooray!
So, I built another, much cooler glove box, the one in the archives that uses the computer fan with the filter over it. Actually, I'm still stoked about the box it's so damn cool.
So I cloned another mushy in my new box, and this time it turned out exactly like it was supposed to. Whitish/amber little strand like particles sitting on the bottom of the jar.
Now this is what I don't get. Inside the new, cool glove box, I injected a little bit of the good honey water into 3 other jars of honey water, hoping I would have 4 jars of honey water to shoot a grip of jars with. I also shot two 1/2 pint jars of BRF substrate with the good honey water just to see what happens since I haven't seen the honey tek in action yet.
It's 6 days later, and I finally had a chance to check on things - and here's what I find.
3 Contam'd honey water jars. I guess lack of cleanliness would have to explain it although I don't understand how - and I know my Glovebox works. The original honey water jar still looks totally fine, although it's been in the fridge since the day I shot the others.
The two jars of substrate I shot with the GOOD honey water are what's really weird. They don't look infected, but they sure as hell aren't growing any mycelium. They just look like jars full of substrate with nothing going on in them.
Does anyone have any idea where I screwed up? Does this sound like I'm not being sterile enough, or what? I'd pretty much know what to think if the BRF jars had some funk in them now, but they don't, why would they have absolutely nothing going in them?
On another note, this has nothing to do with this situation, but does the BRF formula work decent with pint jars? What kind of colonization times are we talking? And are grain teks in pint jars less likely to contam that the BRF pints or something? Or faster?
Any help/suggestions is very much appreciated!
|By quote: (Quote) on Sunday, December 23, 2001 - 04:16 pm: The Nook|
never start liquid cultures from a liquid culture, that's my rule.
the original jar prolly has low levels of bacteria, which you transfered and multiplied in your secondary jars.
brf flour works ok in pints.
|By Nan (Nanook) on Sunday, December 23, 2001 - 07:02 pm: The Nook|
It's a good rule too. It can be done, but you have to have your sterile technique down solid.
It sounds like you have the equipment together, but it takes a few practice sessions (and yes you can expect some failures) before you knock off the rough edges and develop a smooth sterile technique for working with these new toys.
Never let the syringes contact the walls or floor of the box, even though they are clean, treat them as non-sterile surfaces. I load in some PC sterilized foil and some sterile saucers when I am preparing for glove box work... The foil can be unfolded to make little sterile trays for holding syringes, jar lids, foil covers... Are you spraying all of the items you are loading into the glovebox with lysol? Contams will rest on the foil covers of liquid culture jars, I spray everything down, place them in the box, turn on the fan(s) and wait 20 minutes for the sanitizer to work and dry before putting on gloves (and rinse & rub gloves with strong sanitizer) to start opening/unwrapping things in the box.
Flame needles before you contact liquid culture.
Contamed inoculum will not show growth in jars. Another problem with inoculum that does not work is that people forget to thrash or draw up and eject the liquid culture prior to drawing up for shooting jars. It is easily possible to draw up a syringe containing nothing but sterile sugar solution unless you somehow fragment the filiments of mycelia in the culture prior to drawing up syringes for use.
Hope this helps - Nan
|By Sanchez (Sanchez) on Tuesday, December 25, 2001 - 06:54 am: The Nook|
Thanks guys, I guess I just have to work on being CLEANER. What a pain in the ass to be that clean. Oh well, it'll be worth it in the end.