|Isolating on Agar||-|
|Cleaning up agar|
|Treating dirty cultures||-|
|By Purge (Purge) on Tuesday, October 02, 2001 - 03:28 pm:|
i recently recieved a spore print from a local friend, but i have reason to think it might be contaminated (if you knew my friend you would understand). is there any way to sterilize the print without damaging the spores?
|By monkeyod (Monkeyod) on Tuesday, October 02, 2001 - 06:03 pm:|
You'll have to run it on agar and take the clean mycelium and transfer it to a new agar, grow that out and repeat untill you get no contaims.
|By A Drunk Bastard (Adb) on Monday, October 22, 2001 - 07:58 am:|
doh! my goal of just leaching off this wonderful site failed. was a member of the origional n temp site n (lol) everybody mispelled my name (no biggie letters do look alike) n had a bout of the uncalled for paranoia that reinforced the idea that if ya gotta throw stuff away, don't put it in your garbage, that's what gas stations are for. anyways i'm still just chillin countin my blessings n thinking of the future n had a question of sorts.
was wondering how everybody's experiences have been with the FSR prints, granted the 1$ or whatever isn't a financial risk at all.
specifically i'm wondering generally how steril they are, if i went that direction would i have to go the agar route to ensure success? i only have limited experience dealing with prints, 1 from afoaf n a couple from another guy n all were contaminated, least the ones the other guy did i can kinda think of where the weak link was in taking the prints. i'm trying to put off gettin into agar for a bit, i've used it b4 in academic situations n such, but just don't want to deal with it at home right now.
i'd like to give pan cyans a whirl as i hear nuttin but supurb about the potency n ralphster44's pics are beautiful, where does the Nook pan cyan peeps recommend getting spores from? granted i haven't done casings yet, but from what i've read they're not that much more maintenance than casings with cubes. was thinkin of goin that tek where ya mix worm castings brf verm n stuff into 1/2pints, pc n then pretty much flat cake tek it, maybee case with verm.
so i'm plannin that next time i'll build a cheapy glove box n try my hand at tissue cloning, sounds like fun to me!! what i was wondering bout that is how long do liquid cultures remain viable? i've done the honey tek for mycelium production b4 but i didn't store it for any legnth of time. was wondering how long those jars will stay viable lets say with refridgeration a week or so after inoculation & any difference between honey n dextrose with that? (think i'll go dextrose next time just to try it)
sorry bout all the questions here in 1 post. you all have done well at maintaining the vibe of the origional site, hopefully we can get OT postin more frequently as that superbrick is/was the coolest thing i've ever seen! talk about an innovator.
|By SYDYSTYK (Addict) on Monday, October 22, 2001 - 08:35 am:|
are you sure it was the prints and not something you did(no offense)
|By A Drunk Bastard (Adb) on Monday, October 22, 2001 - 07:02 pm:|
no offense taken, we're all here for the same reason
lol yea i'm pretty sure it was the prints, with the print from afoaf, i'm almost 100% positive it was contaminated as there was some white stuff on part of the print, but just for kicks i loaded a syringe using a section of the print that _visually_ appeared to be clean(pc'd 1/2 pinter with water in it n worked on the oven rack), but no luck. the prints from 'another guy' were his 1st ever taken, no glove box or anything n he later told me that he could think of a couple weak links in the sterility of his procedure. so that's my story with prints so far.
|By Nan (Nanook) on Monday, October 22, 2001 - 09:24 pm:|
Agar work is called for then if the print is rare or valuable. Go to the archives and look in the cloning folder. Archives->Cloning
|By SYDYSTYK (Addict) on Tuesday, October 23, 2001 - 09:58 am:|
im kinda talking out my ass here but i was curious about using agar to save a valuable(to me) print, what if the print had bacteria(spores?) can anything be done?
also i have bob's "growing wild mushrooms" when he is describing to construction of his glove box he mentions "a germicidal UV (ultra-violet) fluorescent lamp...The UV lamp only works on bacteria, not fungi.(the DNA structure in bacteria is sensitive to UV light and is destroyed by it, while fungi are not.)"
from "growing wild mushrooms" by bob harris
any experience anybody?
|By Nan (Nanook) on Tuesday, October 23, 2001 - 03:29 pm:|
Dirty Prints... You innoculate PDA or MEA Plates and hope that rhysome mycelia break out and away from the germinating contams.
A section with a clean strand, or a small cube of agar with the cleanest mycelia fanning away from contams is removed to a second plate of Peroxidated Agar where it is placed upside down on the solid media to trap contam spores... Spores have a hard time germinating on Peroxidated Agar, which is why you have to use PDA or MEA for step one. See: H202
Rhysomes should grow out from between the agar sandwich and on to the Peroxidated Agar. A clear section of rhysome containing agar is then removed and placed upside down on the third plate of Peroxidated Agar which is hopefully then clean. It is tedious work.
If Peroxidated Agar fails (bacterial contam), make a trip to the pet store and make up a batch of MEA with some fish tank antibiotic: Tetracycline is good. Follow the dilution directions and use the anitbiotic solution for the liquid portion of your agar and try more spores on these plates.
UV lights are effective against Bacteria, it's only an edge, and is used more often on Flow Hoods and large commercial boxes. Not required for the stuff we are doing here.
See: Cleaning up Agar Cultures
|By SYDYSTYK (Addict) on Tuesday, October 23, 2001 - 07:46 pm:|
|By Fishy1 (Fishy1) on Thursday, October 25, 2001 - 12:45 am:|
Ralphs Pan prints RULE! Both are recommended, and should be clean prints.
I have some Ps. Samuiensis prints coming, and these will be done on agar, as they are straight from the field, and seem to be a rarity.
I have 2 prints coming....hopefully I will have them (prints) avail. sooner or later.....
Good luck w/ the prints.......fishy1
|By Nan (Nanook) on Thursday, October 25, 2001 - 04:47 am:|
as to taming a wild spore print.
this is nothing new.
the steps are quite basic and you're only going to run into problems if you stray too far off the beaten path.
first, you assume the spore sample is contam'd.
of course you make every effort to get a clean a print as possible, but under field coinditions it's pretty safe to assume some contams will get in.
so the first thing you must do is germinate it on a solid media.
folks use agar for a very good reason, namely it's not very appetizing to micro-organisms, and they are much slower growing on agar than otherwise.
one can use half pint jars as an acceptable substitute for petris but there is no good substitute for agar.
once the spores germinate on the agar, all work being done in a sterile environment from this point, one snags a tiny bit of mycellia from the cleanest best looking part of the petri and transfers that to some antibiotic/peroxidated agar.
the antibiotic/peroxide will help to further clean up the colony as it grows out.
then, you repeat the process, again snagging a good looking piece of rhizo mycellia for transfer, you want it as far away from any contams that might be present on the petri as possible.
the process of growing it out, and transfering, is repeated until you get some that grow out 100% clean.
yopu now have your basic culture from which you can procede to domesticate the strain.
to further select for better fruiting/growing substrains, begin growing out your new cultivar on various substrates and select the best examples to clone and propagate further until you get where you like what you see.
Rare Spores : Shroom Glossary