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Omnicracker
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Strain Isolation from Syringe: check my procedure.
#5908980 - 07/28/06 01:33 PM (17 years, 6 months ago) |
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I finally got my perti dishes and today i am ready to delve into the world of strain isolation. here is my plan:
AGAR: -Boil up 250ml of MEA solution and fill 10 petris. -Allow to cool in a stack to avoid condensation. -Wrap the stack in foil and PC for 30min at 15psi. -Put in glove box to cool
Spores: -While PCing the petri, also PC 1 liter of water in a Qt. jar fitted with a aspiratible lid. -After it cools, Inject 1cc of spore solution into the sterile water. -Fill new syringe with diluted spore solution.
Then i'll inoculate the p.dishes with 1/4cc Dil.Spores each, inside of the glovebox.
then i will watch for the most agressive/rhizomorohic substrain and isolate it into a fresh petri, and into a liquid culture master jar.
sound good?
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creamcorn
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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
#5908989 - 07/28/06 01:40 PM (17 years, 6 months ago) |
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On the prep, you want hot/boiling water and to slowly add your media to it while constantly stirring so it dissolves nice.
From there you put it in a flask/jar/something and PC it. Don't let it cool first. Letting it cool, then heating it up a second time, can destroy the protiens that cause it to gel up.
After the PC run, allow it to cool some, but still pretty warm to the touch, then pour it into plates. Don't PC plates with agar already in them. Often the plastic disposable ones will melt. They aren't meant to be autoclaved, that's why the come pre-sterile and are called disposable. If they're glass ones, wrap them in foil and PC them to sterilize them, but do so with them empty, and pour your agar after.
I see where you're coming from with the contam situation, but you'll ruin the agar and/or the plates doing it the way you describe - and this is why you need to pour agar in a glovebox or in front of a hood.
And even though your spore solution is dilute, it only takes about a drop... 1/4 cc is maybe overdoing it. You'll see how much a drop of liquid is even when compared to your plate.
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Omnicracker
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Re: Strain Isolation from Syringe: check my procedure. [Re: creamcorn]
#5909034 - 07/28/06 02:08 PM (17 years, 6 months ago) |
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yeah, there seems to be mixed information about pouring before or after. i can pour it after, thats fine with me. i probably would have made a mess my way.
1/4cc IS probably too much, i was just going to eyeball the amount. just a wee squirt.
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creamcorn
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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
#5909128 - 07/28/06 02:38 PM (17 years, 6 months ago) |
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there shouldn't be mixed information. anyone suggesting PC'ing plates already poured was... just plain wrong. 
you definitely absolutely certainly should pour after.
even taking the melting out of the picture, PC'ing plates full of agar means it boils and sputters all over the place, getting your media all over the walls and the top plate, rather than leaving you with a nice flat clean surface and clear window on top to view through - and potentially giving contams a nutritious 'path' to find their way inside the dish. just because you COULD get away with it and it COULD work, doesn't mean its the right way to do it.
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fastfred
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Re: Strain Isolation from Syringe: check my procedure. [Re: creamcorn]
#5909463 - 07/28/06 04:18 PM (17 years, 6 months ago) |
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An easier and safer way to do media is to fill a rack of test tubes with the media you'll need for 1 or 2 petris in each tube. Then you can just pour each plate from one tube. It makes things a little easier and less chance of contams.
-FF
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Omnicracker
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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
#5909514 - 07/28/06 04:47 PM (17 years, 6 months ago) |
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with foil over the top of the test tubes?
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RogerRabbit
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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
#5909680 - 07/28/06 06:00 PM (17 years, 6 months ago) |
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I use a liquor bottle for agar. You definitely want to pour the dishes after the agar has been sterilized, not before. You can stuff the top of the liquor bottle with polyfill, then cover with foil to prevent contaminants from entering your media while cooling. Personally, I modify a synthetic filter disk to fit inside the original liquor bottle lid, after drilling a 1/4" hole in the lid, so it can be screwed on tight, then covered with foil. You want to let the agar cool in the bottle until you can handle it without heat protection for your hands. Of course, you still need to wear surgical gloves when you pour. The reason for letting the agar cool is to avoid the excess condensation that will form in your stack of petri dishes.
Mix the MEA powder with cold water. If you try to mix into warm or hot water, it clumps up just like flour does when making gravy. Mixing with cold water eliminates that problem.
When the agar has solidified, just one or two drops from a spore syringe or a swipe of spores from a print on each dish, or each section if you use divided petri dishes like I do.
Don't allow the weight to rattle on your PC while the agar cooks. If you do, the agar will boil over and make a mess in your PC. This requires attention to the heat setting on your stove. As the PC nears pressure, stay ahead of the curve and turn down the setting so you can reach pressure, but not vent excess steam. I use 45 minutes at 15 lbs, not 20 for MEA. After the cycle, make sure the PC cools very slowly to also prevent the agar from boiling over. I use three or four times as much water in the PC when sterilizing agar as I do with jars for this reason. The extra thermal mass from using more water holds the heat longer, slowing down the cooling cycle.
One last thing. Be sure to keep your petri dishes in the plastic sleeve they came in right up until you're ready to pour. I wipe the plastic sleeve down with alcohol before opening. This is also a good time to wipe your latex gloves with alcohol as well. Good luck. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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Omnicracker
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Re: Strain Isolation from Syringe: check my procedure. [Re: RogerRabbit]
#5909768 - 07/28/06 06:44 PM (17 years, 6 months ago) |
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great advice rabbit! but ive already fired up this first batch, its cooling right now. i realize now im going to have a hard time pouring these dishes from a Qt. mason jar...
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fastfred
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Re: Strain Isolation from Syringe: check my procedure. [Re: RogerRabbit]
#5909772 - 07/28/06 06:47 PM (17 years, 6 months ago) |
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I think you're overcomplicating things a bit. I've never had any problems with agar boiling over using a regular PC cycle.
If you let the media cool down in the PC you don't have to worry so much about contams.
I also always use hot water to dissolve malt extract, it works fine you just have to add it slowly to prevent clumping.
> with foil over the top of the test tubes?
I use tube caps or foil. Both with good success.
-FF
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Omnicracker
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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
#5909813 - 07/28/06 07:13 PM (17 years, 6 months ago) |
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i mixed it in simmering water, and it clumped a little bit, but i was able to whisk it away.
how about the spore dilution idea? no one has said anything about that.
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RogerRabbit
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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
#5909955 - 07/28/06 08:18 PM (17 years, 6 months ago) |
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That would be too much spore dilution unless you're wanting to breed monokaryons. Also, 1/4 cc is too much to use in a petri dish. Only add a drop or two. A spore syringe will already be diluted enough for isolation.
Don't look for rhizomorphic sectors because they rarely show up right away. You will want to transfer all the sectors you find to new dishes. It will take two or three transfers before you begin to see rhizomorphic growth. In fact, you may not even see sectoring on the first dish if lots of spores have germinated. The reason is that so many substrains will be growing that it will look like a mass of cottony mycelium. If that's the case, just take ten or so pieces the size of a grain of rice from around the leading edge of the growth.
You'll see sectoring when they begin to grow out. Be sure to grow out several of the sectors on grains, then to fruiting, because some are sure to be bunk, but one or two will be awesome.
I hope you have several sleeves of petri dishes. I usually go through two or three sleeves of the three section petri dishes doing isolations, so if you're using single dishes, you may need more. However, if you're going to transfer every two or three days, you can transfer three or four pieces of mycelium to each dish on the second and third rounds even if you're using non-divided petri dishes. Good luck. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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Omnicracker
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Re: Strain Isolation from Syringe: check my procedure. [Re: RogerRabbit]
#5910000 - 07/28/06 08:40 PM (17 years, 6 months ago) |
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sounds good. i making 10 dishes this batch, i have 50 dishes total. can i inoculate only 3 and leave the other 7 for the next round of transfers if i keep them undisturbed in the glovebox?
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fastfred
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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
#5910031 - 07/28/06 08:51 PM (17 years, 6 months ago) |
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Sure. It's a good way to check your sterile technique and weed out any contaminated ones.
> how about the spore dilution idea? no one has said anything about that.
The problem with that is that a lot of vendors cut costs by sending out spore solution that's already highly diluted. So the dilution has probably already been done for you.
It mostly depends on the volume that the vendor has to do that week or day. If they get 30 orders and have enough spore prints to make only 10 syringes what do you think they do? The aren't going to refund money or hold up orders and get bad word-of-mouth.
Sometimes the problem is so bad that people don't get any germination at all. Some people here have even scoped solution and been unable to find ANY spores.
So YMMV with diluting an unknown concentration.
-FF
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Omnicracker
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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
#5910060 - 07/28/06 09:00 PM (17 years, 6 months ago) |
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they are juicy ralphster spores, but i wont dilute them either way.
UPDATE: i simmered together a MEA solution (5g LME, 4.5g agar, 250mil water.) after it was entirely dissolved, i added a little water to make up for what had boild off and bump it back up to 250ml. i then PCed for 30 minutes @15psi. (in a Qt. jar) the PC slowly cooled to touch over 3 hours. i got everything ready in the glovebox and then opened the PC to get the jar and it HAD SOLIDIFIED ALREADY. too cool i guess. or did i go wrong somewhere else?
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RogerRabbit
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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
#5910095 - 07/28/06 09:09 PM (17 years, 6 months ago) |
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No. You don't want to leave petri dishes in the glovebox for any length of time. Just wrap them with parafilm or slip back into the sleeve they came out of and seal it up. Remember, petri dishes don't seal at all so that gas exchange can take place. They need to be wrapped or packaged to keep contaminants out.
Did you make provisions on the quart jar you prepared the agar in to prevent contaminants entering as it cooled? Remember, as the PC cools, the contents naturally shrink, so that air from outside is drawn in. The same thing happens in your jars, so the contaminants have a sneaky way of finding their way into them unless some sort of filtering is provided for. A filter disk or polyfil/tyvek will usually do the trick. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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fastfred
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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
#5910098 - 07/28/06 09:10 PM (17 years, 6 months ago) |
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For 3% MEA (a good choice) you would want 7.5g malt extract and 3.75 to 5g agar per 250 ml.
You cooked it a little too long, but your agar concentration seems fine. You must have let it cool too long. Take it out of the PC when it is just bearable to touch for about 5 secs with your hand. That should be close to right. The agar shouldn't totally solidify until it's fairly cool.
Just remelt your agar by placing your jar in some boiling water for awhile. It should still be OK.
-FF
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RogerRabbit
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Re: Strain Isolation from Syringe: check my procedure. [Re: RogerRabbit]
#5910112 - 07/28/06 09:13 PM (17 years, 6 months ago) |
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You can reheat the agar to liquify it again. You don't want it to be 'cool' but just barely to the point where you don't need a pot holder or paper towel or anything of the like for heat protection.
I wouldn't use that first boil. Just mix your dry ingredients in cool water, then PC for the recommended time. I use 45 minutes at 15psi. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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fastfred
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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
#5910126 - 07/28/06 09:17 PM (17 years, 6 months ago) |
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Petri dishes should keep about a week at room temp. After that they tend to dry out a little, but that should be your only problem storing them as long as you keep them out of drafty air. They should be fine in your glovebox until they start to dry out. Sometimes they are overly wet to begin with and will keep for awhile longer.
A PC is at pressure when it's heated so there's not a whole lot of air intake as it cools. After it reaches 0 psi it might take in a small amount of air, but it's nothing to worry about. As long as you have a tinfoil lid covering your media you'll be fine.
-FF
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fastfred
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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
#5910132 - 07/28/06 09:20 PM (17 years, 6 months ago) |
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As for boiling to dissolve the ingredients... that's as per FDA specs (FDA M93). So if you want to make non-standard media feel free, but it's usually best to go with standardized methods.
Malt Extract Agar (3%)(MEA)(General Microbiology)(FDA M93) ---------------------------------------------------------- 30 g Malt extract 20 g Agar 1 L Distilled water
Boil to dissolve ingredients. Avoid overheating, which causes softening of agar and darkening of medium color. Autoclave 15 min at 121°C. Dispense 20-25 ml into sterile 15 x 100 mm petri dishes. Final pH, 5.5 ± 0.2.
This medium is recommended as a general maintenance medium. [1]
[1] Bacteriological Analytical Manual, 8th Edition, Revision A, 1998.
-FF
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Omnicracker
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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
#5910158 - 07/28/06 09:28 PM (17 years, 6 months ago) |
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alright. im going to reheat the first jar (fitted with a filter lid), along with a bottle with polyfill (for RR) and a rack of TestTubes (for FF) about 2/3 filled with MEA. i hope the testtubes dont boil over....
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fastfred
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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
#5910200 - 07/28/06 09:38 PM (17 years, 6 months ago) |
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They shouldn't. Just make sure you let the PC cool normally.
I should have pointed out that when boiling to dissolve ingredients you have to be careful because the media will quite easily foam up and boil over. Be ready to remove it from the heat if it suddenly starts to boil over. And it does happen quite suddenly sometimes. It's best to use a very gentle boil with stirring.
There's nothing wrong with using polyfill or foam plugs in your media vessels, it's just not required IME. 98% of the items in a lab just get a simple tinfoil cover to prevent contamination. The foam plugs are kind of handy, but I only bother to use them if the tinfoil isn't next to the autoclave where it should be.
Be sure to post if you like my test tube idea. I have a lot of tubes laying around, so it works pretty well for me. It also prevents any contamination from ruining a whole batch of media.
-FF
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RogerRabbit
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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
#5910426 - 07/28/06 10:37 PM (17 years, 6 months ago) |
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Quote:
fastfred said: 98% of the items in a lab just get a simple tinfoil cover to prevent contamination.
Fred, you're nothing but a goddamn highschool moron. In fact, I probably beat up your daddy or grandpa when I was in high school. You follow me around just to troll my posts, and then say the opposite no matter what, and I'm sick and fucking tired of it. I've asked you privately to wait until you've grown your first mushroom crop before passing out your bad advice, but you simply don't listen.
Just two weeks ago, you didn't even know what mushroom mycelium was, telling a guy with an obviously contaminated jar that it was fine, for no other reason than you saw that I had posted and told him that a jar that colonizes fully from spores in two days is colonized with mold. Even after he posted pictures of the jar, http://www.shroomery.org/forums/showflat.php/Number/5866649#Post5866649 you told him it was ok, because you have no idea what a jar of mushroom mycelium looks like.
To even suggest using tin foil to prevent contamination proves you're an absolute moron. Perhaps in a multi-million dollar sterile lab, such will work, but nobody here has a sterile lab. We have kitchens with moldy fruit and bedrooms with carpet, curtains, bedding, dogs and cats, etc.
I'm trying to help people here out of a love for the art, and you're nothing but a post whoring kid trying to make a name for yourself at the expense of others with your keyboard.
The problem with idiots like you is the new folks who are trying to learn the hobby take bad advice and then ruin their projects with it and lose their hard earned money all for nothing.
You need to sit back, study for a while, then attempt to grow a crop yourself before telling others how to do it. A high school biology class doesn't make you a mycologist. The advice I gave on agar preparation is straight from 20+ years of experience doing isolations of hundreds of species and from my work with internationally known mycologists, from whom I've learned the ropes. I will never teach the easiest, lazy-ass way of doing anything. I will explain what I know works, and will only post teks that I have used and proved for years.
Now, go take a flying fuck at a rolling doughnut, and stay off my back.
I sincerely apologize to anyone offended by the above. I've tried using the nice approach. I've tried gently pm'ing the guy to ask him to stop trolling. Other growers have tried both nicely and not so nice to get the guy to grow up and act like an adult. He even spouted off to a moderator earlier that tried to prod him in the right direction. He simply refuses to act like an adult. I'm over it with the idiot. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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fastfred
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Re: Strain Isolation from Syringe: check my procedure. [Re: RogerRabbit]
#5910493 - 07/28/06 10:55 PM (17 years, 6 months ago) |
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Quote:
RogerRabbit said:
Quote:
fastfred said: 98% of the items in a lab just get a simple tinfoil cover to prevent contamination.
Fred, you're nothing but a goddamn highschool moron. In fact, I probably beat up your daddy or grandpa when I was in high school. You follow me around just to troll my posts, and then say the opposite no matter what, and I'm sick and fucking tired of it. I've asked you privately to wait until you've grown your first mushroom crop before passing out your bad advice, but you simply don't listen.
Just two weeks ago, you didn't even know what mushroom mycelium was, telling a guy with an obviously contaminated jar that it was fine, for no other reason than you saw that I had posted and told him that a jar that colonizes fully from spores in two days is colonized with mold. Even after he posted pictures of the jar, http://www.shroomery.org/forums/showflat.php/Number/5866649#Post5866649 you told him it was ok, because you have no idea what a jar of mushroom mycelium looks like.
To even suggest using tin foil to prevent contamination proves you're an absolute moron. Perhaps in a multi-million dollar sterile lab, such will work, but nobody here has a sterile lab. We have kitchens with moldy fruit and bedrooms with carpet, curtains, bedding, dogs and cats, etc.
I'm trying to help people here out of a love for the art, and you're nothing but a post whoring kid trying to make a name for yourself at the expense of others with your keyboard.
The problem with idiots like you is the new folks who are trying to learn the hobby take bad advice and then ruin their projects with it and lose their hard earned money all for nothing.
You need to sit back, study for a while, then attempt to grow a crop yourself before telling others how to do it. A high school biology class doesn't make you a mycologist. The advice I gave on agar preparation is straight from 20+ years of experience doing isolations of hundreds of species and from my work with internationally known mycologists, from whom I've learned the ropes. I will never teach the easiest, lazy-ass way of doing anything. I will explain what I know works, and will only post teks that I have used and proved for years.
Now, go take a flying fuck at a rolling doughnut, and stay off my back.
I sincerely apologize to anyone offended by the above. I've tried using the nice approach. I've tried gently pm'ing the guy to ask him to stop trolling. Other growers have tried both nicely and not so nice to get the guy to grow up and act like an adult. He even spouted off to a moderator earlier that tried to prod him in the right direction. He simply refuses to act like an adult. I'm over it with the idiot. RR
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Roadkill
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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
#5910496 - 07/28/06 10:56 PM (17 years, 6 months ago) |
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This thread has been closed.
Reason: enough!~
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