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Strain Isolation from Syringe: check my procedure.
    #5908980 -

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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xxx..Learn Something..xxx

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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
    #5908989 -

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. :smile:


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Cloning the easy way with LC - newbie friendly pictorial tek

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Re: Strain Isolation from Syringe: check my procedure. [Re: creamcorn]
    #5909034 -

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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xxx..Learn Something..xxx

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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
    #5909128 -

there shouldn't be mixed information.  anyone suggesting PC'ing plates already poured was... just plain wrong. :smile:

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. :smile:


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Re: Strain Isolation from Syringe: check my procedure. [Re: creamcorn]
    #5909463 -

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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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
    #5909514 -

with foil over the top of the test tubes?


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xxx..Learn Something..xxx

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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
    #5909680 -

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


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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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Re: Strain Isolation from Syringe: check my procedure. [Re: RogerRabbit]
    #5909768 -

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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Re: Strain Isolation from Syringe: check my procedure. [Re: RogerRabbit]
    #5909772 -

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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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
    #5909813 -

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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xxx..Learn Something..xxx

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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
    #5909955 -

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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Re: Strain Isolation from Syringe: check my procedure. [Re: RogerRabbit]
    #5910000 -

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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xxx..Learn Something..xxx

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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
    #5910031 -

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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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
    #5910060 -

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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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
    #5910095 -

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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Re: Strain Isolation from Syringe: check my procedure. [Re: Omnicracker]
    #5910098 -

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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Re: Strain Isolation from Syringe: check my procedure. [Re: RogerRabbit]
    #5910112 -

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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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
    #5910126 -

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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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
    #5910132 -

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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Re: Strain Isolation from Syringe: check my procedure. [Re: fastfred]
    #5910158 -

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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xxx..Learn Something..xxx

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