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InvisibleMad Season
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Improve your sterile techniques! (a comprehensive guide to agar) * 33
    #21889950 - 07/02/15 08:38 PM (8 years, 11 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
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Edited by Mad Season (05/15/17 10:14 AM)

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OfflinePsilosoulful

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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season] * 2
    #21890391 - 07/02/15 10:18 PM (8 years, 11 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 10:18 PM)

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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: Psilosoulful] * 1
    #21890424 - 07/02/15 10:26 PM (8 years, 11 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
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Edited by Mad Season (07/02/15 10:39 PM)

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Offlinenooberst
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season] * 1
    #21890496 - 07/02/15 10:41 PM (8 years, 11 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] * 1
    #21890765 - 07/02/15 11:41 PM (8 years, 11 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
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Offlinenooberst
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season] * 1
    #21892057 - 07/03/15 10:43 AM (8 years, 11 months ago)

Will do:thumbup:


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OfflineTh3Issu3
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Re: Mad Season's sterile techniques: Inoculation [Re: nooberst] * 2
    #21892426 - 07/03/15 12:25 PM (8 years, 11 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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InvisibleFriedEggS
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Re: Mad Season's sterile techniques: Inoculation [Re: Th3Issu3] * 1
    #21894789 - 07/03/15 10:04 PM (8 years, 11 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] * 1
    #21894823 - 07/03/15 10:18 PM (8 years, 11 months ago)

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


--------------------





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Invisibleinsanemike

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Re: Mad Season's sterile techniques: Inoculation [Re: TravelAgency] * 1
    #21895365 - 07/04/15 01:55 AM (8 years, 11 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] * 1
    #21896854 - 07/04/15 02:00 PM (8 years, 11 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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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: firstTIMER420] * 1
    #21896900 - 07/04/15 02:14 PM (8 years, 11 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
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season] * 1
    #21909028 - 07/07/15 10:13 AM (8 years, 11 months ago)

Nice, bookmarked for sure


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OfflineFreeWorldOrder
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Re: Mad Season's sterile techniques: Inoculation [Re: bburnett227] * 1
    #21909933 - 07/07/15 02:23 PM (8 years, 11 months ago)

Very nice... :thumbup:


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OfflinefirstTIMER420
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season] * 1
    #21917010 - 07/08/15 09:01 PM (8 years, 11 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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InvisiblePastywhyteMDiscord
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Re: Mad Season's sterile techniques: Inoculation [Re: firstTIMER420] * 2
    #21917124 - 07/08/15 09:28 PM (8 years, 11 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] * 1
    #21918652 - 07/09/15 09:41 AM (8 years, 11 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 09:43 AM)

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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season] * 1
    #22006207 - 07/27/15 06:39 PM (8 years, 10 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-x
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Re: Mad Season's sterile techniques: Inoculation [Re: nooberst] * 1
    #22006250 - 07/27/15 06:48 PM (8 years, 10 months ago)

nice write-up mad :rockon:


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Re: Mad Season's sterile techniques: Inoculation [Re: cArcace-x] * 1
    #22006645 - 07/27/15 08:10 PM (8 years, 10 months ago)

:fuckinawesome:


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Re: Mad Season's sterile techniques: Inoculation [Re: taGyo]
    #22007082 - 07/27/15 09:53 PM (8 years, 10 months ago)

Good shit dude. Did my agar innoc from your EQ&Lipa Yai prints and saralove's P. mexicana CN using basically this exact sterile Tek today except from prints, very helpful. My transfers from the dirty old Chilean print looked clean last time around finally so hopefully this will be the last transfer for them today too, super fast growth& very rhizo :smile: shit basically covers a plate in 5 days  since every transfer after the second. Kept taking the fastest sector every time, now there is no stopping it. Can't wait to see what it does to some grain jars. Thanks for writing this up, I know a lot of people probaly have this stuff up else ware but this one was way easy to follow.


-MIM


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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: MagicInMichigan]
    #22007210 - 07/27/15 10:25 PM (8 years, 10 months ago)

:blush: you guys are too kind! I'm glad you find it easy to follow. I was trying to be as thorough as I could. There are a bunch of one's I've read. I just wanted to share what techniques I developed to get my success


--------------------
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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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22007671 - 07/28/15 12:47 AM (8 years, 10 months ago)

After I transfer mycelium to a new agar plate, is it necessary to wrap the dish in parafilm while still in the SAB to ensure that no contams get in once it is removed and stored?

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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: uninc4life2010]
    #22007710 - 07/28/15 01:03 AM (8 years, 10 months ago)

Yessir! Generally only with Petris. Not pasty plates, which should have filtered gas exchange. Lots of people use saran wrap instead. It's a way cheaper option.


--------------------
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 (07/28/15 01:22 AM)

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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season] * 1
    #22248188 - 09/16/15 09:00 PM (8 years, 9 months ago)

:bump2: Great post


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Invisiblenatedawgnow
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Re: Mad Season's sterile techniques: Inoculation [Re: cronicr]
    #22248799 - 09/16/15 11:07 PM (8 years, 9 months ago)

Good stuff, man!

Very informative!


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OfflinefirstTIMER420
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Re: Mad Season's sterile techniques: Inoculation [Re: uninc4life2010]
    #22248911 - 09/16/15 11:49 PM (8 years, 9 months ago)

Quote:

uninc4life2010 said:
After I transfer mycelium to a new agar plate, is it necessary to wrap the dish in parafilm while still in the SAB to ensure that no contams get in once it is removed and stored?





Glad I looked at mads answer, I usually open my SAB and wrap them outside. Havent gotten any contams I can think of because of it, but I guess ill have to be more careful.

Also mad, how long do you wait till you take a transfer? I usually wait until about half the plate is covered with Myc so I can generally see how its going to grow/group out


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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: firstTIMER420]
    #22250338 - 09/17/15 10:05 AM (8 years, 9 months ago)

Quote:

Mad Season said:
Transferring

First transfer (Agar2Agar)

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





I like to transfer away from contams. I just go for thicker places with fast growth normally. I'd choose the bottom growth anywhere from 7-11 o'clock.

Afterwards if I'm just going to transfer again I'd like it to get to around this size:
(I'd stay away from the 6 o'clock area of the bottom growth)

I generally like to let them get almost to the edge before putting into grain jars.


--------------------
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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Offlinebennylava
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22301188 - 09/28/15 01:39 AM (8 years, 8 months ago)

Can someone go into a little more depth, about the collars he said you could make, in that first video in the OP?

Right now, my SAB just has two holes. That's two holes for contams to drift right on in. I'd like to have some of these collars, that hold the gloves in place permanently. Is there some place that sells them? I'd like to avoid having to make them out of wood.

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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava]
    #22301564 - 09/28/15 07:15 AM (8 years, 8 months ago)

The pvc collars are so bleh to me. A still air box is much better than a glove box. Glove boxes are so 2003. You'll have a better success rate in a proper sab over a gb.

I mean make it with collars if you want. Just saying that it's not necessary.


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
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OfflinefirstTIMER420
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22305993 - 09/28/15 11:49 PM (8 years, 8 months ago)

Quote:

Mad Season said:
Quote:

Mad Season said:
Transferring

First transfer (Agar2Agar)

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





I like to transfer away from contams. I just go for thicker places with fast growth normally. I'd choose the bottom growth anywhere from 7-11 o'clock.

Afterwards if I'm just going to transfer again I'd like it to get to around this size:
(I'd stay away from the 6 o'clock area of the bottom growth)

I generally like to let them get almost to the edge before putting into grain jars.




Man, still cannot get my damn pan cyan to germinate...ehem.....

What the hell is a collar?

also, you place two transfers on 1 100mm plate?


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Re: Mad Season's sterile techniques: Inoculation [Re: firstTIMER420]
    #22306062 - 09/29/15 12:22 AM (8 years, 8 months ago)

give it some time, if the plate is clean just leave it mine took 16 days to germinate(9 year old print though)


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Re: Mad Season's sterile techniques: Inoculation [Re: cronicr]
    #22306547 - 09/29/15 04:36 AM (8 years, 8 months ago)

What causes it to just stall out, and never take off? What are the causes of it just failing to germinate?

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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava] * 1
    #22306752 - 09/29/15 06:14 AM (8 years, 8 months ago)

inviable spores (usually old) or media is not soft/watery enough.
if u see germination, then it dies back, do not use.


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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season] * 1
    #22313365 - 09/30/15 10:51 AM (8 years, 8 months ago)

Nice post man!

Love all the extra photos. Really makes it extra clear on what you're doing.

Also, why do you wet the inside of the SAB? Does it help keep it more bacteria-free?


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Re: Mad Season's sterile techniques: Inoculation [Re: LogicaL Chaos] * 1
    #22313448 - 09/30/15 11:14 AM (8 years, 8 months ago)

Yes. As air moves around in it the contams and other particles will get stuck to the walls :smile: it aids in keeping the box free of particles. Even moving big objects like jars can disturb the air enough for things to start spreading though. That's why I recommend the biggest clear tote you can find. More air to move means it takes more to move it


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Re: Mad Season's sterile techniques: Inoculation [Re: blindingleaf]
    #22313458 - 09/30/15 11:16 AM (8 years, 8 months ago)

Quote:

Mad Season said:
The pvc collars are so bleh to me. A still air box is much better than a glove box. Glove boxes are so 2003. You'll have a better success rate in a proper sab over a gb.

I mean make it with collars if you want. Just saying that it's not necessary.





So I'm fine just using a bleach solution on my bare bathroom, and then noc'ing my stuff inside a SAB? Doing that, I should be largely free of contamination?

Quote:

blindingleaf said:
inviable spores (usually old) or media is not soft/watery enough.
if u see germination, then it dies back, do not use.




What about bacteria (or something) beating your spores to the punch? What if your spores never germinate, but you never really physically see the contaminants? Is that something that could happen? Or do you always see the mold or bacteria buildup? Can it just be a bad smell?

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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava]
    #22313484 - 09/30/15 11:19 AM (8 years, 8 months ago)

It's possible for nothing, including contams to not be able to germinate for months. It happened with my tampanensis spores... :shrug: not sure why.

If the whole plate gets covered in bacteria it's pretty much garbage, but in the op, the 3rd and 2nd plate, both those plates had bacteria and other shit germinate before mycelium. I was still able to save it. Hope that helps.

As for the bathroom, if the room has fairly still air, and you're actually ok with breathing in bleach.... go for it. Personally I just use soap water lol.


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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22313507 - 09/30/15 11:25 AM (8 years, 8 months ago)

Quote:

Mad Season said:
It's possible for nothing, including contams to not be able to germinate for months. It happened with my tampanensis spores... :shrug: not sure why.

If the whole plate gets covered in bacteria it's pretty much garbage, but in the op, the 3rd and 2nd plate, both those plates had bacteria and other shit germinate before mycelium. I was still able to save it. Hope that helps.

As for the bathroom, if the room has fairly still air, and you're actually ok with breathing in bleach.... go for it. Personally I just use soap water lol.




Yeah I'll probably just go the soap and water route, now that you mention it.

But your reply raises another question for me. Inside the SAB, there has to be a very strong alcohol smell. Often times, alcohol is being used inside the box. Just from using it inside my SAB and nowhere else outside of the SAB, it made the whole bathroom smell pretty badly of alcohol fumes. So I can only imagine, what the air is like, inside my petri dishes, and anywhere else that the air gets a chance to get into. Could this potentially be bad for germination, or otherwise hinder mycelium growth?

Edited by bennylava (09/30/15 11:28 AM)

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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava]
    #22313517 - 09/30/15 11:27 AM (8 years, 8 months ago)

It'll be fine :smile: don't need to worry too much about fumes for them. I was just saying for your convenience lol


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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22313670 - 09/30/15 11:59 AM (8 years, 8 months ago)

When mycelium first appears, can it have an ever so slight, opaque look to it? I know its supposed to be sort of a bright white, and it looks like it sort of is, but if I look closely, its just a tiny bit... see through. Looks like if it were much larger, you just might be able to see through it, in the very center. But to be more clear, this piece of suspected mycelium, is only about 1/5 the size of a BB. And it is round.

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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava]
    #22313729 - 09/30/15 12:09 PM (8 years, 8 months ago)

Let it grow out some. Wait for any visible rizmorphs. Usually has to get to the size of a dime. Show some pics if you can.


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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22313968 - 09/30/15 12:57 PM (8 years, 8 months ago)

Quote:

Mad Season said:
Let it grow out some. Wait for any visible rizmorphs. Usually has to get to the size of a dime. Show some pics if you can.





Ok I'll post a pic or two, once its of a significant size that will be big enough for pics. Two more questions:

1. Is it ever ok to open the lids of your jars or petri dishes? Is this ever done?

2. Self healing silicon inoculation ports seem to be all the rage these days. Is this really necessary? Or is it more for people who suffer from frequent contamination? Some people don't practice very much sterile technique, and they don't seem to have too many problems. I mean they'll do the basics, but they don't go the extra mile. And other people, seem to want damn near a cleanroom or a filtered sterile lab to work in. So that makes me wonder, if these "self healing" ports are really even that helpful/important.

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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava]
    #22314003 - 09/30/15 01:02 PM (8 years, 8 months ago)

I don't have any ships. As you can see in the tek i just pop the lids and go to town haha.

As for if you can open the lid to take a pic.. I really don't recommend it. If anything. Take a pic after you've transfered.


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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22314038 - 09/30/15 01:09 PM (8 years, 8 months ago)

Quote:

Mad Season said:
I don't have any ships. As you can see in the tek i just pop the lids and go to town haha.

As for if you can open the lid to take a pic.. I really don't recommend it. If anything. Take a pic after you've transfered.




Ok, say say someone had a contamination, but they went ahead and grew everything to full fruition. Harvested the mushrooms, and ate them. In what manner, could the contaminants hurt the person? Is there mold on the mushroom itself, and the person eats the mold? Can someone break down how this goes down? Just trying to understand, I don't have any intention of eating shrooms grown from a contaminated jar.

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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava]
    #22314058 - 09/30/15 01:13 PM (8 years, 8 months ago)

The person who eats it will be fine. No one has gone to the hospital from eating a fruit from a contaminated substrate. Ever. Every mushroom picked in the wild has a substrate with some sort of contamination. Any visible molds on fruit bodies I'd stay away from tho.


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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22314107 - 09/30/15 01:25 PM (8 years, 8 months ago)

Quote:

Mad Season said:
The person who eats it will be fine. No one has gone to the hospital from eating a fruit from a contaminated substrate. Ever. Every mushroom picked in the wild has a substrate with some sort of contamination. Any visible molds on fruit bodies I'd stay away from tho.




So the fight against contamination, is just so that the mushrooms will ever grow. That sound about right?

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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava]
    #22314552 - 09/30/15 02:49 PM (8 years, 8 months ago)

I'm not sure. But it applies to every mushroom species. Including edibles. Mushrooms/molds will compete against each other, and most mushrooms will just contain contaminations to stop from growing. They don't draw up any toxins into the fruiting bodies tho


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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22317862 - 10/01/15 08:26 AM (8 years, 8 months ago)

Well it looks like my little white dots have gained some little friends. They're orange, or perhaps a bit of a brownish orange. Growing about a half inch away from the white dots. But only in a couple of my petri dishes, not all.

If the white dots turn out to actually be mycelium, and some other contaminants are growing in there with them, can I just cut some of the myc out of there, and just use that to colonize a different dish? Or even a grain jar?

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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava]
    #22318035 - 10/01/15 09:12 AM (8 years, 8 months ago)

A new dish. Never put it in a grain jar until the whole dish gets almost colonized completely


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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22322105 - 10/02/15 03:43 AM (8 years, 8 months ago)

And now a question that's a bit more broad and general.

Do you think that most people, once they get a good colony a mycelium going, just use that to colonize new stuff? Maybe they only use a spore syringe once. Sure they might make some spore prints just so they'll always have a source of spores in case anything happens, but they really don't use spore prints or syringes anymore. They just draw from their already growing colony of mycelium. After all, why wait for the mycelium to appear, when you already have some on hand, and you can just put a little of that, in your jar or whatever you're wanting to grow in.

Is this how its generally done, to save some time?

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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava]
    #22322135 - 10/02/15 04:34 AM (8 years, 8 months ago)

most people will save clones/isolates, and pull from that if its good genetics.
once u get used to germinating spores, its like anything else though, u just kinda assume its going to take a week or so to see growth.
i'd guess most regulars on here have several things going at once, so if one thing is taking longer, they might not even notice it because other things are occupying their time.


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Re: Mad Season's sterile techniques: Inoculation [Re: blindingleaf]
    #22322187 - 10/02/15 05:25 AM (8 years, 8 months ago)

Quote:

blindingleaf said:
most people will save clones/isolates, and pull from that if its good genetics.
once u get used to germinating spores, its like anything else though, u just kinda assume its going to take a week or so to see growth.
i'd guess most regulars on here have several things going at once, so if one thing is taking longer, they might not even notice it because other things are occupying their time.




What would make the genetics bad? I'm assuming that the people selling the spores, are high end "users". And that they spores they're selling, are from the best stock.

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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava]
    #22322210 - 10/02/15 05:42 AM (8 years, 8 months ago)

two harvard graduates can still have retard babies right?

u start over from spores, although some tendencies can carry over as you go further into print>grow>print>grow, etc. 

but as a general rule, as growers, we assume that when we do MS grows, we have same chance for awesome flushes and fruits as we do for not so good flushes and fruits because we can't be exactly sure what kinds of genetic expression will occur when hundreds of spore matings have taken place.


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Re: Mad Season's sterile techniques: Inoculation [Re: Psilosoulful]
    #22444506 - 10/28/15 11:39 AM (8 years, 7 months ago)

Very understandable and the tip you gave of NOT allowing your hand to go over the open container is a good one. Thank You


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Re: Mad Season's sterile techniques: Inoculation [Re: bennylava]
    #22444543 - 10/28/15 11:52 AM (8 years, 7 months ago)

I highly doubt You will ever get 100 % NO contams for 7 consecutive years and I bet if you tried anyone's tek for 7 years you will get a few  jars full of clean Mycelium, so what is possible lies somewhere in the middle.
Do all you can to eliminate contaminated air around your grow and You will do better.


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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22444601 - 10/28/15 12:06 PM (8 years, 7 months ago)

I was sure I'd already posted in this thread, great write up Mad! :hatsoff:

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Re: Mad Season's sterile techniques: Inoculation [Re: spacechildo]
    #22444722 - 10/28/15 12:42 PM (8 years, 7 months ago)

:takingnotes:

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Re: Improve your sterile techniques! (a comprehensive guide to agar) [Re: Mad Season]
    #22710722 - 12/28/15 08:42 PM (8 years, 5 months ago)

:snort::bump:


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Re: Improve your sterile techniques! (a comprehensive guide to agar) [Re: Mad Season]
    #22710886 - 12/28/15 09:32 PM (8 years, 5 months ago)

:tongue2:


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Re: Mad Season's sterile techniques: Inoculation [Re: taGyo]
    #22720249 - 12/31/15 09:48 AM (8 years, 5 months ago)

:popcorn:


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Re: Mad Season's sterile techniques: Inoculation [Re: taGyo]
    #22720347 - 12/31/15 10:14 AM (8 years, 5 months ago)

:lol: I really need to get around to updating this :facepalm:


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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22745172 - 01/06/16 06:01 PM (8 years, 5 months ago)

:nyan:


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Re: Mad Season's sterile techniques: Inoculation [Re: taGyo]
    #22870967 - 02/06/16 12:00 PM (8 years, 4 months ago)

Bumping this to help people improve their sterile techniques :smile:


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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22922818 - 02/19/16 11:47 AM (8 years, 4 months ago)

:bump: :thumbup:

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Re: Mad Season's sterile techniques: Inoculation [Re: Psilosoulful]
    #22925173 - 02/20/16 12:18 AM (8 years, 4 months ago)

Wow, it's crazy that 9 months ago I hadn't meddled in agar yet. This post helped me a lot, and now agar is the norm for me. Between Pasty and You, I have learned so much about agar and how to use it and it has changed how I do mycology forever. Newbies read this please.


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OfflinePsilosoulful
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Re: Mad Season's sterile techniques: Inoculation [Re: TravelAgency]
    #22925205 - 02/20/16 12:28 AM (8 years, 4 months ago)

:levitate:

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InvisiblePastywhyteMDiscord
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Re: Mad Season's sterile techniques: Inoculation [Re: Psilosoulful]
    #22925867 - 02/20/16 09:06 AM (8 years, 4 months ago)

Agar is killer stuff and fun as well. Glad yer on board :rockon:

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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: Pastywhyte]
    #22926059 - 02/20/16 10:27 AM (8 years, 4 months ago)

:whathesaid: :awedance:


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OfflinePsilocyBen17
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #22986961 - 03/08/16 07:47 PM (8 years, 3 months ago)

Thanks for the pictoral Mad Season. I found it helpful.


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OfflineWill7401
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Re: Mad Season's sterile techniques: Inoculation [Re: Pastywhyte]
    #23134117 - 04/19/16 01:08 PM (8 years, 2 months ago)

Quote:

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




My advice to anyone just staring out in this field...research and practice with agar before wasting any time or money with any other method. Agar IMNO (in my newbie opinion) is the best method regardless of how daunting it may appear at first. it really isn't that difficult and making mistakes are very cheap compared to, oh I don't know, say going through two whole 12 cc MS of Cambodia and eventually feeding the birds and squirrels 20 jars of rye grain. Pasty you have been a champ in my corner so far and Mad Season, you just helped me tremendously with a couple of things i see that I had been doing wrong. Namely, when I placed a drop or two of MS on the agar I tried to be as careful as possible to not move it around thinking this would help it germinate faster by keeping the spores all together. From here on out I will definitely try to disperse them now. Makes total sense too. I'm glad to know it's okay to use the side of the jar to get the wedge off my scalpel. I am having a rather difficult time getting them to come off whether it be agar to agar transfer or agar to grain. They're very sticky. A heated coffee can huh? Don't I feel like a dumbass. This is a kickass write up brother. Thank you.


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Invisibledankington
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Re: Mad Season's sterile techniques: Inoculation [Re: Will7401]
    #23659240 - 09/19/16 04:11 PM (7 years, 9 months ago)

:bumpthread:

A bunch of people need to see this. I read it every couple of months :lol:
Thanks for taking the time to put this together, Mad.

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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: dankington]
    #23659613 - 09/19/16 06:17 PM (7 years, 9 months ago)

Lol it's been 6 months since this was bumped? Wow time flies


--------------------
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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OfflineChips
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #23909203 - 12/09/16 05:52 AM (7 years, 6 months ago)

thank you for this tek, the tip about cutting into the receiving plate with the agar made my work so much cleaner, just a simple cut and drag instead of scraping the scalpel around everywhere trying to get the wedge off :lol:

i hope it's okay to bump this, i have a SAB technique question

after prepping grain, i store it on a shelf in a closet. i leave the foil covering the top

i wipe down the jars (including the foil) before bringing them in to the SAB

after removing the foil inside the SAB, do i need to wipe off the lid before opening for inoculation? or is the foil sufficient enough to keep it safe?

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OfflineTiamo
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Re: Mad Season's sterile techniques: Inoculation [Re: Chips]
    #23909325 - 12/09/16 07:20 AM (7 years, 6 months ago)

Just recently did my first agar plates... Inoculated 18 of them with various species and strains. I hope I was sterile enough, I think I might have been too impatient and rushed. We'll see. This is nerve wracking, lol.


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InvisibleMad Season
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Re: Mad Season's sterile techniques: Inoculation [Re: Chips]
    #23909381 - 12/09/16 07:51 AM (7 years, 6 months ago)

Quote:

Chips said:
thank you for this tek, the tip about cutting into the receiving plate with the agar made my work so much cleaner, just a simple cut and drag instead of scraping the scalpel around everywhere trying to get the wedge off :lol:

i hope it's okay to bump this, i have a SAB technique question

after prepping grain, i store it on a shelf in a closet. i leave the foil covering the top

i wipe down the jars (including the foil) before bringing them in to the SAB

after removing the foil inside the SAB, do i need to wipe off the lid before opening for inoculation? or is the foil sufficient enough to keep it safe?



Foil should be fine. Realistically if you did things right and only had the sterile lid bottoms on top of the open media, you don't even need to wipe things down. Just don't let anything but the underside of the lid go over it. Alcohol is more of a safety net than a necessity IMO.


--------------------
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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Invisiblevan hattonFacebookDiscord
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #23909702 - 12/09/16 10:27 AM (7 years, 6 months ago)

:takingnotes:


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If I ever give out misinformation please inform me so I can have the correct information. :cheers:

Tmethyl said:
Chuck Norris once roundhouse kicked a monotub that wasn't pinning fast enough. The force of the kick rearranged the genetics of the mushrooms, we now call them Penis Envy.

Caps McGee said:
:thumbsup:
Fun part is figuring out what works best for you

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OfflineChips
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #23911656 - 12/09/16 09:56 PM (7 years, 6 months ago)

Quote:

Mad Season said:
Quote:

Chips said:
thank you for this tek, the tip about cutting into the receiving plate with the agar made my work so much cleaner, just a simple cut and drag instead of scraping the scalpel around everywhere trying to get the wedge off :lol:

i hope it's okay to bump this, i have a SAB technique question

after prepping grain, i store it on a shelf in a closet. i leave the foil covering the top

i wipe down the jars (including the foil) before bringing them in to the SAB

after removing the foil inside the SAB, do i need to wipe off the lid before opening for inoculation? or is the foil sufficient enough to keep it safe?



Foil should be fine. Realistically if you did things right and only had the sterile lid bottoms on top of the open media, you don't even need to wipe things down. Just don't let anything but the underside of the lid go over it. Alcohol is more of a safety net than a necessity IMO.




:inlove3:

it took me 2 hours to noc 24 jars the other night because i was meticulously wiping every jar before it went in and after i removed the foil lol

this makes a lot more sense, ty!

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InvisibleNothingsChanged
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Re: Mad Season's sterile techniques: Inoculation [Re: Chips]
    #24224343 - 04/07/17 12:15 AM (7 years, 2 months ago)

Great tek. Helped tremendously. :toast:


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Offlinegermanauslander
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Re: Improve your sterile techniques! (a comprehensive guide to agar) [Re: Mad Season]
    #24224406 - 04/07/17 12:48 AM (7 years, 2 months ago)

:like:

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OfflineFenriss
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Re: Improve your sterile techniques! (a comprehensive guide to agar) [Re: Mad Season]
    #24550904 - 08/13/17 02:37 PM (6 years, 10 months ago)

Thank you for demystifying agar for me a little. I love the part about thinking carefully about your next move before you make it. That's helping me to generate less motion to stir up contaminants. Trying to treat it like a meditation :smile:

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Offlinemildfoodpoisoning
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Re: Mad Season's sterile techniques: Inoculation [Re: Fenriss]
    #24552719 - 08/14/17 08:33 AM (6 years, 10 months ago)

Wow, thx for sharing the knowledge,

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Offlineseekinglight
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season]
    #24812205 - 11/28/17 03:56 PM (6 years, 6 months ago)

Nice pictures of contaminants.  I'm just learning the basics and appreciate the pitures.

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Offlinecatnip40
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Re: Mad Season's sterile techniques: Inoculation [Re: seekinglight]
    #24812444 - 11/28/17 05:28 PM (6 years, 6 months ago)

:takingnotes::popcorn:

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OfflineTsteigs
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Re: Mad Season's sterile techniques: Inoculation [Re: catnip40]
    #24931546 - 01/21/18 02:21 PM (6 years, 5 months ago)

Sorry to bump an old thread.  Just wanted to say Thanks.

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OfflineBoatswainBob
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Re: Mad Season's sterile techniques: Inoculation [Re: Tsteigs]
    #25587520 - 11/02/18 06:08 PM (5 years, 7 months ago)

Bumping this again as it has been instrumental in helping me plan my agar/oat jar knocs & moves way easier. Cheers mate, thank you so very much!!


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"If we're willing to give up hope that insecurity and pain can be exterminated, then we can have the courage to relax with the groundlessness of our situation." -Pema Chรถdrรถn

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Re: Mad Season's sterile techniques: Inoculation [Re: BoatswainBob]
    #26362785 - 12/05/19 11:07 AM (4 years, 6 months ago)

Nice write up eh!  :gocanada:


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Tek's I use

LAGM2020

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OfflineStainless2
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Re: Mad Season's sterile techniques: Inoculation [Re: Mad Season] * 2
    #28137737 - 01/13/23 09:58 AM (1 year, 5 months ago)

Thank you for this write-up.  Hopefully, it brings it to the top so other noobs like me see and learn from it.

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OfflineDERRAYLD
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Re: Mad Season's sterile techniques: Inoculation [Re: Stainless2] * 1
    #28137741 - 01/13/23 10:01 AM (1 year, 5 months ago)

I'm glad you resurrected it to be honest, some solid information in here presented in a simple, visual manner.

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Offline7anja7od_OG
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Re: Improve your sterile techniques! (a comprehensive guide to agar) [Re: Mad Season]
    #28263326 - 04/05/23 09:43 AM (1 year, 2 months ago)

Peace. Thanks for the great info. I have a SAB and I have been looking into being as sterile as possible. I have never heard of the size of the holes making a difference and I have 4in holes. I have had the box built for yrs and have only recently gotten into agar work so I have been reading up on how to be as clean as possible without having a flow hood. U are also the first I have seen not use gloves and such. U have done a great service in helping to demystify the process and I thank U for that. ๐Ÿ‘๐Ÿพ๐Ÿค™๐ŸพโœŒ๐Ÿพ
Peace.

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InvisibleWay
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Re: Improve your sterile techniques! (a comprehensive guide to agar) [Re: 7anja7od_OG]
    #28263397 - 04/05/23 10:34 AM (1 year, 2 months ago)

Quote:

7anja7od_OG said:
Peace. Thanks for the great info. I have a SAB and I have been looking into being as sterile as possible. I have never heard of the size of the holes making a difference and I have 4in holes. I have had the box built for yrs and have only recently gotten into agar work so I have been reading up on how to be as clean as possible without having a flow hood. U are also the first I have seen not use gloves and such. U have done a great service in helping to demystify the process and I thank U for that. ๐Ÿ‘๐Ÿพ๐Ÿค™๐ŸพโœŒ๐Ÿพ
Peace.




Thanks for bumping this thread. I had never seen it before and was always curious about hole size and air movement.  Seeing that someone tested it makes me even more happy with my decision to go 8 inch diameter arm holes. So much room for activities and I never bump my SAB any more. Highly recommend.


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That's the way she goes, boys. Sometimes she goes, sometimes she doesn't, cause that's the fuckin way she goes.

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