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firstTIMER420
Born the son of a sharecropper..



Registered: 05/08/15
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Loc: US, maybe?
Last seen: 6 years, 4 days
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MS agar/Streaking
#22116420 - 08/20/15 12:05 PM (8 years, 5 months ago) |
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Been reading about streaking and different methods of inoculating agar with a MS syringe. Ive also talked to a few people, and have been told a few methods. Ive had problems with my last round of MS innoculations, ended up with 6 plates of bacterial messes. My technique has been improving.
Now, methods of MS inoculation I am aware of:
1. Flaming needle and placing drop in middle of plate, doesn't need to be flamed between every plate. --the problem im having is that the drop doesn't stay in the middle of the plate and ends up just sliding around the plate and creating a mess. my first ms inoculation even though that happened still turned out fine. This got me thinking about streaking the drop either with a sterile cotton swab or an inoculation loop. I am leaning towards using the loop, but am curious about the technique in doing so.
Flame the loop, wait a few seconds, then streak the drop. Repeat for every plate. I have read that if it is a bigger plate, you can zig zag, if its smaller, better to make a straight line, or maybe even a half circle. That's basically what im assuming the process of using one is, if im missing anything please let me know. I am also wondering if using the hot loop would cause the agar to melt or damage the spores in the drop in any way.
2. Another way of MS inoculation I read was to dip the tip into the agar and then squirt the drop in the divot of the plate as you are making the hole.(or you can make a slice with a scalpel) Havent really been able to find much experience reports on this, and was wondering if it would work very well without causing contams. Any experience/opinions?
3. Sandwiching - Pouring about half of the plate, just enough to cover the bottom with a little more, inoculating with a drop, then pouring the other half of the plate. Before inoculating, must wait until the first layer hardens, which only takes a minute or so if poured at the right temp.
--haven't attempted this, but thinking about doing it since the first pan syringe I have is so contaminated, the spores didn't have a chance to even germinate. The syringe was on its second use, and was stored in the fridge in between uses( is this necessary?)
I am looking to colonize pan cyan myceliym from MS syringes. I also have antibiotic powder and was thinking about making antibiotic agar, but still need a viable technique for MS inoculation.
If you read this far, thank you. I have read a lot on these techniques and people say so many different things its hard to actually say what will work on a regular basis.
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natedawgnow
Rocky mountain hood rat



Registered: 02/09/15
Posts: 8,939
Loc: ation
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Only read the first half but you can just put a drop on the plate and then gently tilt it to get the drop to disperse across the plate.
It works the same as streaking but with 1 less contam vector. It also seperates the spores making it easier to isolate smaller colonies of strains instead of having 1 big blob with a million genetic pools.
Good luck!
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Edited by natedawgnow (08/20/15 12:12 PM)
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Mad Season
hookers and blackjack



Registered: 09/16/12
Posts: 12,666
Loc: Canada
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don't let it touch the walls of the dish. Definitely much better to have it spread out, fuck those contams!
Transfer after a few days of initial growth. Like at this point:

When it's just perfect enough to discern it's cubensis and not some mold, but not too big either.
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natedawgnow
Rocky mountain hood rat



Registered: 02/09/15
Posts: 8,939
Loc: ation
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Forgot that part, thanks mad!
Ya don't let the drop touch the sides of the dish when you tilt it.
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Mdahmer
Aloysius devadander abercrombie



Registered: 04/05/14
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Last seen: 6 years, 6 months
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i have never gone syringe to agar personally but i would go with what nate said, the less steps the better.
as far as your 3rd option i've read about sandwiching but not doing it for the initial inoculation. like since you know you have a contam syringe you would pour a thin plate. inoc. wait for the myc and bacteria to grow then pour another thin layer on top of that. the idea being the heat will subdue the bacteria hopefully long enough for the myc to grow through to the top layer where you can snatch it off and transfer.
i am not an expert in this area though so im just throwin stuff out there
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firstTIMER420
Born the son of a sharecropper..



Registered: 05/08/15
Posts: 1,025
Loc: US, maybe?
Last seen: 6 years, 4 days
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Re: MS agar/Streaking [Re: Mdahmer]
#22116563 - 08/20/15 12:38 PM (8 years, 5 months ago) |
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thanks for the quick responses guys, im cooking the agar now so I will be trying that shortly. I really want to grow some pans cyans but cant seem to get my PF pan jars to colonize, and the grain I recently inoculated doesn't show any signs yet either. Like I said the first try with Pan cyan agar failed horrible because of the syringe, and I have a new one now.
Will try tilting the plate and see what happens.
Have any of you tried streaking though? If so with a loop or swab?
Also MAD, I have a couple plates maybe a month old, from MS, no contams at all, but they are really overgrown. How long can plates sit and still be used?
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Edited by firstTIMER420 (08/20/15 12:41 PM)
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Mad Season
hookers and blackjack



Registered: 09/16/12
Posts: 12,666
Loc: Canada
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I've streaked both loops and swabs in a zigzag fashion. 1 single swipe. All you need is a drop on it. Very easy actually. It all comes down to personal preference.
Until they dry out . I love overgrown plates. You might as well wait for a pin, and transfer it. It'll be sterile, and you have a very young clone specimen
Edited by Mad Season (08/20/15 12:51 PM)
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tahoe
Noob Slayer



Registered: 11/26/03
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I press the syringe plunger just enough to get a lil drop to form on the end of the needle. Then I drag it across without touching the needle to agar. It's usually a drop the size of a grain of salt. I never flame the needle because I never let the needle touch substrate.
-------------------- Stop experimenting half way through your first grow. Grow it to maturity, watch it, learn from it. Do this a few times then experiment with different ideas and figure out what works best for you.
My Legacy https://www.shroomery.org/forums/showflat.php/Number/22140987#22140987 Teh=The I need to proofread
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36fuckin5
Alchemycologist


Registered: 08/11/03
Posts: 12,079
Loc: Diving into Mystical Territori...
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Re: MS agar/Streaking [Re: tahoe]
#22117588 - 08/20/15 04:53 PM (8 years, 5 months ago) |
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If you want to streak a plate, flame your syringe and inoculation loop. In your sterile area, put one drop of solution on the loop to cool it and the syringe. Then one more drop to actually pick up spores. Streak the plate with that.
But we really want growth starting from the middle of the plate so we can see the sectors and so that contams will be confined to one spot. Streaking is more for bacteria.
-------------------- Redd Foxx said: If you're offended I don't give a shit and don't come see me no more. Pat The Bunny said: A punk rock song won't ever change the world, but I can tell you about a couple that changed me. bodhisatta said: i recommend common sense and figuring it out. These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.
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firstTIMER420
Born the son of a sharecropper..



Registered: 05/08/15
Posts: 1,025
Loc: US, maybe?
Last seen: 6 years, 4 days
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Re: MS agar/Streaking [Re: 36fuckin5]
#22119216 - 08/20/15 10:25 PM (8 years, 5 months ago) |
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reading what you guys wrote about using a loop, im probably going to end up with a mess of contamination in each plate lol.
I kind of just went where the drop was and made circles from the middle outwards on 4 different points to disperse the liquid enough so it wouldn't run.
This leads me to my next question: What do you guys do immediately after innocing with the MS syringe, if you just tilt it to make it run evenly, can you let the plate sit in the SAB or whatever for 12 hours so it has enough time to dry? Because when I try to wrap the plates, the water ends up running and I get fucked. What do you guys do?
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kmetric



Registered: 08/23/14
Posts: 140
Last seen: 7 years, 10 months
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You may be over thinking this. Just put a drop in the center of a bunch of plates, sit back and be patient. Eventually something will start growing in one of the plates. Then continue to transfer wedges until you get some clean looking mycelium.
Agar work will seem like a piece of cake after a few runs once your technique and eye for myc growth improves.
Quote:
What do you guys do immediately after innocing with the MS syringe
I put them away in a corner without doing anything special and don't worry about them until I see growth.
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firstTIMER420
Born the son of a sharecropper..



Registered: 05/08/15
Posts: 1,025
Loc: US, maybe?
Last seen: 6 years, 4 days
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Re: MS agar/Streaking [Re: kmetric]
#22119424 - 08/20/15 11:25 PM (8 years, 5 months ago) |
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I have found the need to wrap them, which is where my problem is, after I place the drop, when I try and wrap it runs most of the time ruining the plate. this is why i was asking if its ok to let them sit for say 12 hours after inoculating before wrapping, giving it time to soak in.
I cant believe I only had 2 contams out of 20 plates my first time doing agar, i didn't even wrap half of them and the other half i wrapped with tape.
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Edited by firstTIMER420 (08/20/15 11:27 PM)
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36fuckin5
Alchemycologist


Registered: 08/11/03
Posts: 12,079
Loc: Diving into Mystical Territori...
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Spore syringes were never intended for agar. You're forcing it to happen, you're gonna have issues.
Spore prints don't have this problem, and you can get them free or almost free.
-------------------- Redd Foxx said: If you're offended I don't give a shit and don't come see me no more. Pat The Bunny said: A punk rock song won't ever change the world, but I can tell you about a couple that changed me. bodhisatta said: i recommend common sense and figuring it out. These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Re: MS agar/Streaking [Re: 36fuckin5] 1
#22120874 - 08/21/15 06:54 AM (8 years, 5 months ago) |
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Streaking is best for any spores. It spreads them out so if you have really strong growth from some spores it won't all be jumbled in the middle. It helps you pick out isolated fast performance mycelium.
Also helps you notice contamination faster
Also it's a great way to get the right amount of spores. Squirt onto the loop so you only have a single drop
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inked4life
Fungi finesse


Registered: 06/28/15
Posts: 555
Last seen: 4 years, 9 months
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do u use a sab for agar work or open air. what i have done in the past is lay down your wrap pre cut to the lenght u want, put ur dish on top of that , flame your needle , remove the lid and place a drop onto it and place lid, and then you simply wrap the top without picking the dish up, i used a deep baking pan like a 2 in sides, pre cutting your pieces of wrap is essentially the most important pre planned step if u dont want them to run. besides the most important steps , sterilization and hand technique.
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tetherface
get in where you fit in



Registered: 10/05/14
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Loc: wild
Last seen: 6 years, 6 months
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Is it really recommended to pour hot agar over a plate with mycelli growth for the "sandwitching" method doesn't agar cool at 45c so we pour at 47c/116f which is gonna kill every bit of myc on that plate from what I understand at least myc dies at about 106f can someone elaborate on this?
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Time and heat kill. 106 may kill but it takes a long time. 122f kills in way less time. 140f kills on contact.
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inked4life
Fungi finesse


Registered: 06/28/15
Posts: 555
Last seen: 4 years, 9 months
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i wouldnt sandwich agar with growth on it, that makes no sense to me, cuts air off, therefore cutting of gas exchange, stalling , killing any type of growth, especially at that temp. myceluim slows down past 90 ○f so i imagine any higher would destroy your work.
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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It cools down In under 5 minutes you're pouring 10ml over 10ml of hardened agar. Don't worry about GE its a plate not a substrate. The whole point is the myc grows thru anyway
Edited by Trusted cuItivator (08/21/15 11:32 AM)
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tetherface
get in where you fit in



Registered: 10/05/14
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Loc: wild
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Bodhi so it is a legit method? wouldn't the short exposure to the extreme heat at best weaken the myc and make it easier for the contams to thrive? I recall reading a tek that recommended placing a clean solidified cake on top of a contam plate to achieve the sandwich technique or does this fall into the "more than one way to skin a cat" category?
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Bacteria can't grow thru the agar. Mycelium will.
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Mad Season
hookers and blackjack



Registered: 09/16/12
Posts: 12,666
Loc: Canada
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An extremely bacterial plate can be saved with the sandwich. I know I've tried. It was amazing
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tetherface
get in where you fit in



Registered: 10/05/14
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right on thanks guys
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36fuckin5
Alchemycologist


Registered: 08/11/03
Posts: 12,079
Loc: Diving into Mystical Territori...
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Quote:
tetherface said: from what I understand at least myc dies at about 106f can someone elaborate on this?
They naturally grow in FL, and abundantly. How many 110 F days do you think they have each year?
-------------------- Redd Foxx said: If you're offended I don't give a shit and don't come see me no more. Pat The Bunny said: A punk rock song won't ever change the world, but I can tell you about a couple that changed me. bodhisatta said: i recommend common sense and figuring it out. These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.
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firstTIMER420
Born the son of a sharecropper..



Registered: 05/08/15
Posts: 1,025
Loc: US, maybe?
Last seen: 6 years, 4 days
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Quote:
bodhisatta said: Bacteria can't grow thru the agar. Mycelium will.
THIS^
which is the reason I wanted to do it, clean a dirty syringe after I get it to grow some myc on the first plate.
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