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InvisibleMad Season
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Improve your sterile techniques! (a comprehensive guide to agar) * 24
    #21889950 - 07/02/15 09:38 PM (5 years, 3 months ago)

This is a bit of a guide for those who are having contamination problems. This might not get rid of your all contamination problems, but it's ALWAYS the first place to start working on. I think logically. When working with contaminations, I always start at the inoculation stage/starting point of it all, then the spawn stage, then the pasteurization stage and so on.

Let's get started!

Things you'll need:
-Still air box
-Squirt bottle with soap and water
-Scalpel or x-acto with #11 blade
-Blow torch/clean blue flame (I use propane, but butane or alcohol lamp works too.)






Things to look out for

This thread has pictures of bacteria, and other contams on agar. Quite a few of those are pretty much garbage, and can't be saved.

This is hidden contams on agar. I made this thread to show how important organisation, and being thorough is for agar inoculant.

Some contaminations you can save by cutting away from the contamination. Like this one. The bacteria is the bright murky yellow all around the edges, so I went in the middle where I can see it's growing into a clean area


This one has yellow bacteria on the outside, a couple white bacterial growths in the middle, and a white mold that is clearly not mycelium. That's why I transfered from the clean place with visible rizmorphs:


This one has 5 different contaminations. Aspergillus, some really fuzzy white mold, a grey cobweb looking mold, and 2 types of bacteria. I was able to save it by going from the place with rizmorphs:







Making the still air box (SAB)

This is a picture of my 110 qt clear still air box:

Notice it's wet/condensated inside. That's what it's supposed to look like the whole time you're working with it.

I made it mimicking this video, except I SERIOUSLY recommend you get a 100 qt+ tub.



Here's another SAB I made without a coffee can:
https://www.shroomery.org/forums/showflat.php/Number/23897456

I took the cut outs of the holes and taped them to make little doors. This is to keep the air still if I'm waiting for things to cool, or if I want a smoke break.

My SAB holes are 6 inches. Honestly this is the MINIMUM diameter. I took incense and lit it in my box (kudos to kizzle for the idea) and found the arm holes moved the most air (smoke) when moving my arms around, and in and out. By increasing the size of the arm holes, and working in a fairly still room with the vents turned off .. I've been finding that moving your arms in and out will cause no air movement. And thus very rare contams.





Prep work!

For all things agar try out these links:
http://www.shroomery.org/11332/Agar-and-Culture-Storage
Pastywhyte's Easy Agar TEK
Pouring Dishes TEK.

I could type out how to get prepped in a wall of text, or show you a 40 second video..



This is so simple, yet a really good way to prep. I personally am not that sanitary, but if you're fighting contamination, it really wouldn't hurt. I use a spray bottle of soap and water to cover the walls of the still air box.

For this demonstration, the towel is the still air box area, and my bare hands are actually gloved hands :wink:. Everything in the towel is inside the still air box. Notice the blowtorch is outside the towel.(SAB)






Inoculation

As usual, always flame the syringe.


Open the dish a cm or so like:


and slip the needle in. Squirt a drop or 2 of solution into the dish.

You don't want to melt the sides of the dish or the lid when you do.

Close er up and rock the dish around slowly to distribute spore solution all over the dish. You want it to cover as much surface area as you can. This will really help in cutting away from contaminations because it'll germinate all around the dish, rather than everything germinating in one spot.





Transferring

First transfer (Agar2Agar)

You want to transfer basically a few days after you see growth. Like:


In my demonstration, I use an overgrown dish. I strongly advise you don't let it get overgrown on the first transfer.


Flame scalpel


While flaming the scalpel, you want to simultaneously pop and slide the lid off like this. I find sliding it really helps in keeping the air still:


Cut a wedge in the agar. Make sure not to let your gloved hand hover over the media!


Stab at it at a 45 degree angle and lift it straight out. Again with your hand not over any open media.


pop and slide the lid off the receiving plate you wish to transfer to using the same method as above, while simultaneously taking your transfer. It should only require 1 hand anyway to take the lid off.

Don't put the receiving lid down. Hold it. This next part shouldn't take longer than 5 Seconds and only requires 1 hand

Place it onto the receiving plate and cut into the agar of the receiving plate. Slide/cut a line into the agar. The wedge should stick to the receiving plate and come off. This way you aren't jostling the plate trying to knock the wedge in, AND NOT HAVING A GLOVED HAND GO OVER THE PLATE have I said not to let anything go over open media enough times yet? Lol..


Didn't get that? Here's closer pics!


Put the lid on the exact opposite of how you took it off. Slide it over and pop it back on (slide and pop)






Grain and LC inoculation with agar wedges (grain wedges are generally much bigger than this)



Take off the screw on part of the mason jar and put it off to the side/far corner


Flame scalpel, and simultaneously Pop and Slide the lid off the agar dish and cut out a wedge, like seen in the first transfer. For LC a small wedge works, for grains aim for a bigger wedge. Even multiple wedges can get stabbed and transferred at the same time into a grain jar. Realistically, a small wedge works too. It would just increase colonization times.

Now while you've got the wedge on the blade, slide the jar lid off until your fingers are no longer over the jar.

if you have the rubber attached I highly recommend taking it off. This will make life much easier.



Using the rim of the jar, take it off the scalpel and let it drop into the jar. Sometimes it sticks to the jar so give it a good shake/stir AFTER THE LID IS BACK ON to get it to go down.


Now slide the lid back on


If you did a grain jar, give it a good shake to rub the mycelium off onto grains. Also to get it under the surface. Shake again at 20-30% as you normally would.





Conclusion

Using this tek, you should have very little air movement when opening lids. So little in fact I've been having amazing success without using ANY sanitizers. Your hands should never be over open media, and you shouldn't have lids open for longer than 5 seconds. I hope this really reduces contamination rates for you, or even gives you the motivation to try agar. This right here is what made me get rid of ALMOST all my contaminations. Hope this helps!





Quick tips to always keep in mind

-Make sure to always work at the back of the still air box
-Always flame tool between transfers
-Slow gentle movements are key. The less air you stir, the better
-Always think about what you're going to do next before you do it. You don't want to be making any mistakes. Especially with open media
-Always keep flames outside of the still air box.
-NEVER LET ANYTHING BUT YOUR SCALPEL OR NEEDLE GO OVER OPEN MEDIA


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


Edited by Mad Season (05/15/17 11:14 AM)


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OfflinePsilosoulful

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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season] * 1
    #21890391 - 07/02/15 11:18 PM (5 years, 3 months ago)

Nice write-up!!! Very easy to follow and great for noobs as well.  :headbang: :awethumb:

I was looking to make a SAB soon. So far, I've been using a cheap, cardboard glovebox with pretty decent success. It is definitely time for an upgrade, since I'll be doing a lot of agar work/G2G transfers in the near future. :smile:

Should the holes in the tub be that big, or can I scale them down to just fit my arms through. I figured it would cut down on contamination rates by reducing drafts made by moving in and out of the tub. Also, did you flame the scalpel inside or outside the tub? If you aren't using alcohol to sanitize the inside the tub, there wouldn't be a risk of a fire, and it would be more sterile than exposing it to contaminates outside the tub.


Edited by Psilosoulful (07/02/15 11:18 PM)


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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: Psilosoulful]
    #21890424 - 07/02/15 11:26 PM (5 years, 3 months ago)

I edited the op a little. The blow torch is outside the still air box. A flame needs fresh air to burn, so if it was inside it'd definitely have a draft caused by circulating air. You don't need to have big 6 inch holes. I did it for convenience sakes, also only coffee can I had lol.

Thanks brotha! It's really nice to know I can at least help 1 person out :smile:. I wanted to put all the research I've done, and my own technique into a tek. I figured since it EXTREMELY raised my success rate, it would help others, and it should also be the first place to start with common advice :smile:.


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


Edited by Mad Season (07/02/15 11:39 PM)


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Offlinenooberst
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #21890496 - 07/02/15 11:41 PM (5 years, 3 months ago)

:takingnotes: this makes everything much simpler. Nice sterile guide Mad :thumbup:


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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: nooberst]
    #21890765 - 07/03/15 12:41 AM (5 years, 3 months ago)

Quote:

nooberst said:
:takingnotes: this makes everything much simpler. Nice sterile guide Mad :thumbup:



Thanks man :smile: :thumbup: you gotta report back in a couple months and tell me if you're noticing any results!


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


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Offlinenooberst
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #21892057 - 07/03/15 11:43 AM (5 years, 3 months ago)

Will do:thumbup:


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OfflineTh3Issu3
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Re: Mad Season's sterile techniques: Inoculation [Re: nooberst] * 1
    #21892426 - 07/03/15 01:25 PM (5 years, 3 months ago)

Nice write up, being overboard on photos really helped me when I first came here and I'm more than sure this will help more than a few people. +5 from me!


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InvisibleFriedEgg
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Re: Mad Season's sterile techniques: Inoculation [Re: Th3Issu3]
    #21894789 - 07/03/15 11:04 PM (5 years, 3 months ago)

:kaneclap:


my only real suggestion is to add a section for screw-on PP5 lids (the kind that are used in pasty's easy agar tek)


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OfflineTravelAgency
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #21894823 - 07/03/15 11:18 PM (5 years, 3 months ago)

Bookmarked! Thanks for the great write up! Can't wait to start agar


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Invisibleinsanemike

Registered: 02/24/14
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Re: Mad Season's sterile techniques: Inoculation [Re: TravelAgency]
    #21895365 - 07/04/15 02:55 AM (5 years, 3 months ago)

Very easy to follow, well written and well structured thread. :vibin:


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OfflinefirstTIMER420
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #21896854 - 07/04/15 03:00 PM (5 years, 3 months ago)

just convinced me to make the jump into agar, although I will be using petris, that will be the only variation.  Great tek, I am also someone who also needs LOTS of pics, as you may have noticed from my grow log lol. thanks.

what recipe do you use?


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12 34 http://www.shroomery.org/forums/showflat.php/Number/22386794[/url]
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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: firstTIMER420]
    #21896900 - 07/04/15 03:14 PM (5 years, 3 months ago)

Thanks everyone! <3

I'm glad you're getting into agar first timer :smile: it's really not hard. Trust me. Pf tek is harder.

For this one I did 2 grams agar and 2 grams malt extract for every 100 ml (2 dishes)


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


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Offlinebburnett227
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #21909028 - 07/07/15 11:13 AM (5 years, 3 months ago)

Nice, bookmarked for sure


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OfflineFreeWorldOrder
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Re: Mad Season's sterile techniques: Inoculation [Re: bburnett227]
    #21909933 - 07/07/15 03:23 PM (5 years, 3 months ago)

Very nice... :thumbup:


--------------------
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PastyWhyte's Easy Agar TEK
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OfflinefirstTIMER420
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #21917010 - 07/08/15 10:01 PM (5 years, 3 months ago)

I received my dishes, LME and agar, just waiting on my inoculation loops and I should be good to go!


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12 34 http://www.shroomery.org/forums/showflat.php/Number/22386794[/url]
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InvisiblePastywhyteM
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Re: Mad Season's sterile techniques: Inoculation [Re: firstTIMER420] * 1
    #21917124 - 07/08/15 10:28 PM (5 years, 3 months ago)

Nice writeup Mad! Agar is not hard at all is it :grin:


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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: Pastywhyte]
    #21918652 - 07/09/15 10:41 AM (5 years, 3 months ago)

:lol: no way pasty. Easy stuff! Glad you like it man :bow2:

I'm planning on updating this with a print work section, screw on lids section, and redoing the scalpel part to be in a jar or foil because I always pc it and feel it should be on as sterile a surface you can get.

But I've been so busy lately :frown:


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


Edited by Mad Season (07/09/15 10:43 AM)


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Offlinenooberst
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22006207 - 07/27/15 07:39 PM (5 years, 2 months ago)

Did some colonized brf paste transfer to 2 jars half filled with pf tek they both fully colonized without contam issues thanks :cheers:


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InvisiblecArcace-xS
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Re: Mad Season's sterile techniques: Inoculation [Re: nooberst]
    #22006250 - 07/27/15 07:48 PM (5 years, 2 months ago)

nice write-up mad :rockon:


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OfflinetaGyo
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Re: Mad Season's sterile techniques: Inoculation [Re: cArcace-x]
    #22006645 - 07/27/15 09:10 PM (5 years, 2 months ago)

:fuckinawesome:


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