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agar
old hand
Registered: 11/21/04
Posts: 9,056
Loc: Somewhere Else
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CONTAMINATE VECTORS
#6609421 - 02/25/07 05:07 PM (17 years, 1 month ago) |
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Spore syringes from well established reputable vendors. Are what most new hands start with. And, are usually clean of contaminates.
Prints from reputable vendors are also reliable. But, not always foolproof. Because of the very nature of how prints are made.
SporeWorks & Ralphster; Has the most reliable syringes & prints, I am aware of.
Prints from FreeSporeRing are certainly dubious; as are prints from most trades. Simply because most are often made in not very sanitary conditions. By no means am I bad mouthing either source. But, you get what you pay or trade for.
If you wish to insure yourself from contaminates. It is a mistake to make syringes from a print. Unless the print came from a VERY reliable source. The best thing to do is; go to agar/petri dish's, with spores from a print.
A 50 pack sleeve of sterile plastic disposable petri's. Is not very expensive. Nor, is a pound of agar & PDA nutrients. Glad Cling Wrap will work in place of Parafilm. Not all cling wraps are the same. GLAD CLING WRAP is gas permeable. Some others are not.
An inexpensive set of stainless steel dental picks. Are good tools for agar petri culture transfer isolation. You can buy a beginner agar/perti kit for $30 range (+s/h). From several vendors here: For example, SporeWorks has a a nice one, at that price. http://sporeworks.com/store/catalog/Basic-Agar-Culturing-Kit-p-16211.html
Going to a few perti's to insure a culture is clean. In reality saves you lots of time/labor/materials/aggravation; (in the long run.) As a dirty print, or syringe will get you contaminates, every-time. That show up later in grain spawn jars.
It is also wise to do agar/perti work in a STILL AIR glovebox. Simply because, if a culture is not clean. In still air, you can transfer clean mycelium to another plate. In moving air, contaminate spores from a dirty culture get blown into clean plates. When you attempt to make transfers.
Once you have a clean culture isolated on perti's. It is simple do still air glove box transfers. Of a cleanly colonized wedge. To PC'ed grain jars, or a properly prepared LC.
Assuming you PC spawn grain jars long enough & at proper pressure. And, the lids have proper filtered gas exchange provisions. And, some sort of injection port, if needle inoculated.
You can then propagate colonized jars by G2G. One to the other, a heaping tablespoon or two per jar. Or, once an LC is colonized thickly, inoculate with that.
The next contaminate vector is in fact syringe inoculation. Even in front of a flow hood, or in a glovebox. ALWAYS keep the needle wrapped in a alcohol soaked gauze pad.
It takes a little practice to get used to. But, once you learn the technique. It will cut grain spawn jar contamination, to near zero.
If you G2G, it must be done in still air, very sanitary conditions. (preferably, in a glovebox).
Once you have cleanly colonized grain spawn jars.
The next contaminate vector is the substrate, you spawn to.
You must insure it is properly pasteurized. And, the moisture content is within an optimal range Otherwise, all is most often lost.
Improper pasteurization leads to contaminated substrate. To wet, often gets you slow colonization & bacterial bloom. To dry leads to slow poor colonization, that often stalls.
The next contaminate vector is the actual spawning. You want to do that under the most sanitary conditions. You can muster.
Meaning, in a very small still air sanitized room. Or, in front of a flow hood, or in a glovebox. Whatever the case, wear clean cloths. Exam gloves, face mask & hair covering. (these are not expensive things & are easy to find)
The next contaminate entry point is during the spawning run. To alleviate that & still allow for ample gas exchange. I cover my trays with Glad Cling wrap. Held in place with simple masking tape.
Then, slit the Cling wrap & cover the slits with Micropore tape.
If you don't have WIDE Micropore tape.
You can cover wide slits; with sanitized polyester non-woven stabilizer backing material. Used in drape & craft making, available at most fabric stores. It is the same as polyfill, but made into a non-woven fabric. Fabric weights differ & you want as stiff/heavy as you can get.
(above are 8X8 inch sheets, you can cut to any size)) Held in place with masking tape.
The object being, to minimize entry of contaminate spores; During spawn run.
Once a bulk substrate is 101% colonized.
The next contaminate vector is the "casing" layer. Which should have a light airy non-nutritive composition: At an optimal Ph of 7.5 to 8. And, properly pasteurized.
Once "cased" and mycelium surfaces on the casing layer. You initiated pinning with a 12/12 off/on light cycle. And, give significant fresh air exchange & high Rh.
At this point, sterility no longer matters. But, don't neglect good sanitation.
Edited by agar (02/25/07 11:53 PM)
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2FiNiTe
ConsideratlyKilling Me
Registered: 06/12/06
Posts: 1,635
Loc: New England
Last seen: 7 years, 6 months
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Re: CONTAMINATE VECTORS [Re: agar]
#6609438 - 02/25/07 05:11 PM (17 years, 1 month ago) |
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**Bookmarked**
Good writing, clear, and informative. I don't think anyone can ask for more. For someone like me who is a new hand I've often wondered about these types of techniques and you just told me everything I needed to know. Thanx for the time and the effort you put into this, strictly for the progression of the hobby.
-------------------- "Ours is a world of nuclear giants and ethical infants. We know more about war that we know about peace, more about killing that we know about living." General Omar N. Bradley
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mycocurious
Mike O. Kuerias
Registered: 02/09/07
Posts: 1,265
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Re: CONTAMINATE VECTORS [Re: agar]
#6609463 - 02/25/07 05:17 PM (17 years, 1 month ago) |
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perfect dude, looks like an awesome rough draft for a rather definitive tek on contaminates and generalities of the process.
-------------------- Don't mistake my tone for a "matter-of-fact" attitude. I'm just presenting what I believe to be correct, until I'm corrected... - How Myco-Curious Prepares Coir & Compost Substrates - How Myco-Curious Builds A Bulk Humidifier - How Myco-Curious Builds An Automated Greenhouse ------------------------------------ figgusfiddus said: Keep in mind that inoculating or whatever in front of a flow hood won't help your bad substrate, your bad inoculant, your bad sterile procedure, etc. etc. etc. It's not a +3 flowhood of magic, it's just a tool.
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agar
old hand
Registered: 11/21/04
Posts: 9,056
Loc: Somewhere Else
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This little blurb evolved from a PM, I sent to someone; plagued with contaminates. After I wrote the pm, I thought some parts of it were worth posting.
The general points being, where contaminate entry usually occurs.
ALL mushroom cultures start from spores. A syringe, or print is just the beginning. Followed by numerous steps. Any of which, if done haphazardly, or wrong. Lead to contamination, or poor / stalled growth.
You have to get each step RIGHT. (or, very close to right) Otherwise, you negate all you have invested.
Each step requires some rudimentary knowledge. Once you have the simple basics down pat.
Then, getting better at mycology, becomes an art form. You don't need to be an ARTIST. But, using their techniques, help greatly in succeeding.
Sadly, most beginners do some initial steps well. Then neglect one or more of the other critical steps. Along the way.
All it takes is one small blunder, anywhere in the chain of steps. And, the whole process goes wrong.
The point being, learn to crawl before you attempt walking. Learn to walk, before you attempt running. If you don't, you will almost certainly stumble, along the way.
--------------------
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Cerius
Space Cowboy
Registered: 04/27/05
Posts: 297
Last seen: 7 years, 2 months
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Re: CONTAMINATE VECTORS [Re: agar]
#6610426 - 02/25/07 09:00 PM (17 years, 1 month ago) |
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just two replys for all the writing? tsk tsk. kudos, agar!
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Vibrate
with me
Registered: 11/18/06
Posts: 256
Last seen: 15 years, 2 months
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Re: CONTAMINATE VECTORS [Re: Cerius]
#6610547 - 02/25/07 09:20 PM (17 years, 1 month ago) |
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Great info Agar. Thank you.
-------------------- I love you
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KaptKid
Spaced Pirate
Registered: 12/11/03
Posts: 6,252
Loc: Bright Side of the Sun
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Re: CONTAMINATE VECTORS [Re: agar]
#6610608 - 02/25/07 09:28 PM (17 years, 1 month ago) |
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Great write up.
So many places for contam's to come from.
-------------------- Child of the 60's, Tripping ever since.
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Legend9123
Registered: 09/24/06
Posts: 2,590
Last seen: 10 months, 10 days
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Re: CONTAMINATE VECTORS [Re: KaptKid]
#6610835 - 02/25/07 10:14 PM (17 years, 1 month ago) |
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Thank you once again Agar you are a great helping hand.
-------------------- Those who would give up a little freedom to get a little security shall soon have neither. -Benjamin Franklin
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VampireSlayer
killing ghosts,zombies andvampires forlife
Registered: 08/26/06
Posts: 2,529
Last seen: 16 years, 6 months
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Re: CONTAMINATE VECTORS [Re: KaptKid]
#6610857 - 02/25/07 10:20 PM (17 years, 1 month ago) |
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cool agar! this is one of if not the best write ups on sterile procedures out there. I myself have always been very cautious about sterility, just wish I had a glove box. I've seen glove boxes that I could buy online but most are really large, which is good, but not good for someone living in a small apartment. I know I'm missing out a ton without a glovebox because I can't do agar or grain to grain properly.
-------------------- I Don't come to fight flesh and blood but spiritual wickedness in high and low places
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etam
it's a hobby...
Registered: 04/24/06
Posts: 1,126
Loc: East Coast
Last seen: 15 years, 5 months
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Quote:
Pikashroom said: cool agar! this is one of if not the best write ups on sterile procedures out there. I myself have always been very cautious about sterility, just wish I had a glove box. I've seen glove boxes that I could buy online but most are really large, which is good, but not good for someone living in a small apartment. I know I'm missing out a ton without a glovebox because I can't do agar or grain to grain properly.
Take a wide mouth lid, heat it up on your stove, and use it to melt armholes in a clear rubbermaid bin. Bam, glove box.
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RogerRabbit
Bans for Pleasure
Registered: 03/26/03
Posts: 42,214
Loc: Seattle
Last seen: 1 year, 1 month
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Re: CONTAMINATE VECTORS [Re: Legend9123]
#6610888 - 02/25/07 10:31 PM (17 years, 1 month ago) |
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And, bear in mind the point of all those precautions and sterile procedures is primarily to protect our crop from US. We the cultivators are the number one source of contaminants. We stir up mold spores when we walk across the floor. Mold spores are on our clothing if we haven't put on freshly washed clothes just before beginning work. Bacteria flows from our mouths when we breathe, and fills the air with live bacteria if our armpits are sweating. If we sneeze, up to 200,000 thousand bacteria can be released. These attach to dust particles in the air and float for a long time.
That's why the most successful, long term growers shower, wash hair, brush teeth and use mouthwash, put on freshly washed clothes, wear gloves that are washed with alcohol, masks, hairnets, etc. every time they do sterile work. Sometimes we can get away with less, but one failed batch of jars, or petri dishes swiped with rare spores can pay for a lot of gloves and toothpaste. 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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2FiNiTe
ConsideratlyKilling Me
Registered: 06/12/06
Posts: 1,635
Loc: New England
Last seen: 7 years, 6 months
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Quote:
RogerRabbit said:
That's why the most successful, long term growers shower, wash hair, brush teeth and use mouthwash, put on freshly washed clothes, wear gloves that are washed with alcohol, masks, hairnets, etc. every time they do sterile work. Sometimes we can get away with less, but one failed batch of jars, or petri dishes swiped with rare spores can pay for a lot of gloves and toothpaste. RR
Being a new hand at this hobby I havn't had a whole lot of experience with doing certain procedures or techniques but this bit of advice like that I quoted from the Rabbit is probably the single most important part of my equation for success. I've had some good success at this hobby so far, I love doing it and reaping the benefits is just a bonus for me. I owe it all to that golden bit of advice, anyone that is new at this hobby should know that this is one of the biggest things in all mushroom cultivation, you can apply this advice to all species and methods. Keep an open mind to others ideas, especially the ones that exceed you in skill and always be able to pick those people out.
This stuff helped me a lot, and plenty of others. We should be thanking these guys.
-------------------- "Ours is a world of nuclear giants and ethical infants. We know more about war that we know about peace, more about killing that we know about living." General Omar N. Bradley
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Helltick
Player
Registered: 10/30/05
Posts: 684
Loc: Deep...
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Re: CONTAMINATE VECTORS [Re: agar]
#6610986 - 02/25/07 10:55 PM (17 years, 1 month ago) |
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Excellent read.
-------------------- My Agar Tek. My Greenhouse Tek. "It's not good when it's done, it's done when it's good"
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VampireSlayer
killing ghosts,zombies andvampires forlife
Registered: 08/26/06
Posts: 2,529
Last seen: 16 years, 6 months
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Re: CONTAMINATE VECTORS [Re: etam]
#6611047 - 02/25/07 11:22 PM (17 years, 1 month ago) |
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Quote:
etam said:
Quote:
Pikashroom said: cool agar! this is one of if not the best write ups on sterile procedures out there. I myself have always been very cautious about sterility, just wish I had a glove box. I've seen glove boxes that I could buy online but most are really large, which is good, but not good for someone living in a small apartment. I know I'm missing out a ton without a glovebox because I can't do agar or grain to grain properly.
sounds great but how do i do this without burning my hands?
Take a wide mouth lid, heat it up on your stove, and use it to melt armholes in a clear rubbermaid bin. Bam, glove box.
-------------------- I Don't come to fight flesh and blood but spiritual wickedness in high and low places
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cpw1971
Mr
Registered: 10/07/06
Posts: 5,615
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kickass thread btw, anyone see that mythbusters episode where they tested the toothbrushes for feces??? they found the fecal spores were everywhere
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agar
old hand
Registered: 11/21/04
Posts: 9,056
Loc: Somewhere Else
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Quote:
Pikashroom said: cool agar! this is one of if not the best write ups on sterile procedures out there. I myself have always been very cautious about sterility, just wish I had a glove box. I've seen glove boxes that I could buy online but most are really large, which is good, but not good for someone living in a small apartment. I know I'm missing out a ton without a glovebox because I can't do agar or grain to grain properly.
With a bit of design ingenuity. You could build a G/B, with hinges, pins or screws. That could/would quickly disassemble & fold flat for storage. Just a thought.
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Blutjager
Inhuman
Registered: 06/11/06
Posts: 9,220
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Re: CONTAMINATE VECTORS [Re: agar]
#6611383 - 02/26/07 01:51 AM (17 years, 1 month ago) |
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Another thing that is good to use to keep the needle sterile are those little make up sponges soaked in alcohol
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getfasterer
Stranger
Registered: 03/05/10
Posts: 115
Loc: lalaland
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Re: CONTAMINATE VECTORS [Re: agar]
#12350461 - 04/07/10 05:59 PM (13 years, 11 months ago) |
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perfect
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afrosheen
9Lives the cat
Registered: 03/06/10
Posts: 1,878
Last seen: 2 years, 5 months
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Re: CONTAMINATE VECTORS [Re: agar]
#12350606 - 04/07/10 06:19 PM (13 years, 11 months ago) |
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I love agar's wacky formatting, everything he writes looks like poetry.
--------------------
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OxyProx
Deoxy.org
Registered: 03/09/10
Posts: 81
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Re: CONTAMINATE VECTORS [Re: afrosheen]
#12351048 - 04/07/10 07:21 PM (13 years, 11 months ago) |
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I don't have anything useful to add. I just wanted to say thank you for posting this...
Thanks.
-------------------- Remember remember the fifth of November Gunpowder, treason and plot. I see no reason why gunpowder, treason Should ever be forgot...
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