Home | Community | Message Board


Crestline Sales - MycoPath
Please support our sponsors.

Mushrooms, Mycology and Psychedelics >> Mushroom Cultivation

Welcome to the Shroomery Message Board! You are experiencing a small sample of what the site has to offer. Please login or register to post messages and view our exclusive members-only content. You'll gain access to additional forums, file attachments, board customizations, encrypted private messages, and much more!

Amazon Shop for: Agar, Malt Extract, Parafilm

Jump to first unread post. Pages: 1 | 2 | 3 | 4 | 5 | Next >  [ show all ]
InvisibleStropharis
 User Gallery


Registered: 04/02/07
Posts: 1,364
Stro's Cleaning and Isolating On Agar * 14
    #18430998 - 06/17/13 09:17 AM (1 year, 5 months ago)

Stro's Cleaning and Isolating On Agar




I recommend putting all your spores and clones past agar, you want to know you are working with a clean culture before you introduce it to grains, and we also want to take out the guess work out of spore reliability.

So once you have a culture on agar, you will want to make sure that it is clean and free of contams. How do you get there? Well for starters you need a few things, pictured below is the majority of them.



You will need a Still Air Box to do this, don't have one, super easy using Stro's Still Air Box.

Items aside from that include paper towels, isopropyl alcohol, lighter, alcohol lamp or similar, gloves, scalpel, your cultures, new dishes, and parafilm (I prefer Glad Cling Wrap)

To demonstrate how I clean up my cultures if need be, I will be using a few clone cultures that I took using different levels of care so they would have different levels of  contamination for the purpose of this write up. A, B, and C. 

A


B


C



I don't think I have to tell you which clone was taken carelessly.

When taking A, I swung my un sterilized scalpel like a battle axe dismembering the pin and watched it fall on the substrate, then I stabbed it while doing my best Braveheart scream and gently placed it on the plate. I figured this was an a fairly unclimactic ending so I shook the dish and watched the nasty little pin roll around contaminating the entire thing.

You would always want to do a transfer from a dish like as soon as you can to avoid spreading any contaminant during transfers. The less there is growing on your dish before transfer, the better.

So now we need to clean these up and to do that we will be transfer contam free healthy mycelium from them to a new dish. Lets use A for an example, on a culture this nasty you might want to look at it and decide where you will be taking your transfer from before you are working inside you SAB and have a limited view.

Taking another look at A; there are limited options. Using a clock position, as people often use discussing agar sectors, the best option I see here is at about 4:30 but even this area is pretty close to bacteria which I have circled in red. Care must be take to avoid those areas so we can do this in one transfer. In addition, it is a good idea to get a good look from the back as well, sometimes bacteria can be underneath the mycelium and then you would be transferring contaminant laden mycelium. The green area is that actual wedge that I transferred from this plate to a new one.



In B you can see that there is only a small culture of bacteria that is by coincident also at the 4:30 and that all other area are suitable for transfer.


And C, does it even need a transfer? No it doesn't, you can see it is already clean culture and was ready for grains as is.  I did it anyways to keep all my clones running together and it will also further isolate the strain. 


So lets cover the transfer now;

First the working surface is wiped down with lysol and an air sanitizer sprayed into the air, and all necessary equipment wiped down with isopropyl alcohol and placed in an appropriate location relative to your work area. The bag of fresh plates was brought inside my SAB, opened, removed plates, closed, then removed from SAB. I don't normally do this but I did it to provide for more room and to facilitate this write up.

Here you can see I am just about ready to go.


All the wrap is removed from the dishes and they are set up. The dishes closest to me are ready for transfer and the ones furthest are "on deck".

Sterilize your scalpel over your alcohol flame (outside your SAB of course) and the bring it inside. Open your new dish and cool the blade on the outer perimeter of your agar. If you look, you will notice these marks on a lot of the dishes you see photos of. Here it is highlighted in blue. The mark from cooling the scalpel.


I will let the photos do most of the talking for the transfer.





A few things to note here is that my new dish is on on my left side, I do this because I do not want to be reaching over it for any reason. I am right handed and will be using my right hand to do the transfers, if you were left handed you would want to reverse this. I also do my best to have the clock position that I want easily accessible so I am not spending any more time than necessary with top off. When I transfer it to the new dish, I do my best to only reach over the agar with the scalpel and to not have my hand over the dish at any point. Normally I would complete all transfers before I wrap and mark my dishes but for the purpose of this write up, I then wrapped the new dish with the cling wrap.



Alright, that covers that for the most part. Lets talk sectoring, I will go ahead and use the images that have up to discuss this.

Although C is a clone, it isn't a true monoculture. Sectors aren't always easy to see but most of the time you can get a general idea of sectors by the outer perimeter of the mycelia growth. I will use a bit of imagery to show you what I mean, how to select them, and explain why it is important that you don't get too wrapped up in "sectors"

I honestly don't need to isolate C any further, it is going to produce and perform well and in fact there is a possibly that isolating it further can be counter productive. I actually only took one transfer from this culture as previously discussed as I currently can't facilitate very many projects at the moment.

Moving on, if we generalize here and at a glance establish sectors based on the outer permitter of the culture, we get something like this. You can see the fan like colonies spreading themselves out and growing at different rates and with different characteristics, sometimes separated with less dense areas refereed to as "lines of isolation". At first glance there are roughly six sectors there.



At a closer look though, there are more than that. For an example I will use only the 11:00 to 2:00 on the same image only cropped and inverted so I don't sit here all night drawing lines.

Highlighted in pink you can see even more lines of isolation and segregated individual sectors highlighted in green. Where I said we have two, well now I can see six.



I guess my point is, it isn't important to split hairs when sectoring, as long as you are isolated away from contaminates and you are taking small transfers from the outer edge of the healthiest, strongest, and fastest rhizomorphic mycelium. If you take a small peace from the very outer edge of the area where there is one distinct sector, when there are actually three, congrats you just snatched up the fasted monoculture in the area.

Another point I would like to make while I am in the process here. Some things outside genetics can have an effect on the characteristic of your culture, whether it be rhizomorphic (strandy) and tomentose (cottony).  Things like the nutritional value of your growth medium and temperature. These plates were transferred to a different batch of agar although the measurements and method of preparation were kept the same; using Sto's Agar Prep

500ml distilled water (1/2 liter)
10g Agar-Agar
10g Malt Extract



They were however moved to a higher temperature for colonization.  There appearance speak for themselves. The colonization times were faster and characteristics are different. If I don't keep medium and conditions consistant, the mycelium tricks me up. The most important thing here is we now have three clone cultures that are clean and nearly ready for grains.

Updated with a few photos, to keep things simple I will reorganize.


Original clone cultures



Seven days after first transfer from clone culture


Eleven days after first transfer from clone culture


As you can see the cultures are looking healthy and the strains ar showing their individual characteristics in A and B and as suspected C is a true monoculture.

As a learning tool, I will go over one or two things here. When I first posted the photo that were taken seven days after transfer someone was asking me a few questions and I said;

Quote:

Stropharis said:
C is now true monoculture, yes. As for A I believe there are two strains but it is hard to tell




What made me come to the conclusion that A had two strains when I posted that photo? Again it was primarily the outer paramater of the culture, although it was young and tomentose, take a look at the 12 o'Clock and you will see a very small sector with different behavior than the rest of the uniform culture.



Now just four days later, after it has matured quite a bit and has also developed  rhizomorphic hyphea, you can see the contrast in uniformity between that sector at 12 o'Clock, it has fanned out and distinguished itself even greater. I was happy to see C look so clean and that sector on A rep so hard after saying that.





- Stro


Edited by Stropharis (06/21/13 02:58 PM)


Post Extras: Print Post  Remind Me! Notify Moderator
OfflineMidnight Cyclone
StrangerDanger

Registered: 05/29/13
Posts: 399
Loc: oo ess aye oo ess aye Flag
Last seen: 10 months, 22 days
Re: Stro's Cleaning and Isolating On Agar [Re: Stropharis]
    #18431055 - 06/17/13 09:49 AM (1 year, 5 months ago)

Very nice write-ups man!

I just read through most or all of them. SO much good information!

Thanks for sharing the knowledge :mushroom2:


Post Extras: Print Post  Remind Me! Notify Moderator
OfflineForrester
medicine man
Male User Gallery

Registered: 02/05/13
Posts: 3,804
Loc: Northeast, USA Flag
Last seen: 8 hours, 16 minutes
Re: Stro's Cleaning and Isolating On Agar [Re: Midnight Cyclone]
    #18431096 - 06/17/13 10:14 AM (1 year, 5 months ago)

Very nice write up, this will be extremely helpful (along with some of your other write-ups) as I'm just starting to work with agar.  Thanks for taking the time!


--------------------
Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleFrankHorrigan
The Inquisition
Male


Registered: 01/05/11
Posts: 10,437
Trusted Cultivator
Re: Stro's Cleaning and Isolating On Agar [Re: Forrester]
    #18431128 - 06/17/13 10:38 AM (1 year, 5 months ago)

:congrats:

I love your write ups on agar Stro.

I've been meaning to get mine done but it's not going to happen too soon it seems.

So I'm going to point people over here til then :wink: :thumbup:


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

Yes, you can bump my old threads with a question.
Here is how I get things done.
You should take a look. :hehehe:


Frank's tips and tricks. Updated on 3/21/14
AMU- Get an answer here -AMU


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleStropharis
 User Gallery


Registered: 04/02/07
Posts: 1,364
Re: Stro's Cleaning and Isolating On Agar [Re: Forrester]
    #18431143 - 06/17/13 10:45 AM (1 year, 5 months ago)

Forrester, nice to see you around! I really enjoy your gourmet and medicinal work, inspirational to say the least.

Thanks Frank! It was a PITA for sure. It's really is hard to discuss without stepping on toes or surpassing the scope of purpose for the write up. Hopefully it will help a few folks who don't know where or how to start.


--------------------
Stro's Write Ups


Post Extras: Print Post  Remind Me! Notify Moderator
Invisiblebillb
The Wolf I Feed


Registered: 10/09/11
Posts: 983
Re: Stro's Cleaning and Isolating On Agar [Re: FrankHorrigan]
    #18431147 - 06/17/13 10:46 AM (1 year, 5 months ago)

Nice write up!!!


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleTonydanzawillpay
Registered: 11/11/12
Posts: 1,764
Re: Stro's Cleaning and Isolating On Agar [Re: billb]
    #18431166 - 06/17/13 10:57 AM (1 year, 5 months ago)

Great write up!


Post Extras: Print Post  Remind Me! Notify Moderator
OfflineForrester
medicine man
Male User Gallery

Registered: 02/05/13
Posts: 3,804
Loc: Northeast, USA Flag
Last seen: 8 hours, 16 minutes
Re: Stro's Cleaning and Isolating On Agar [Re: Stropharis]
    #18431210 - 06/17/13 11:21 AM (1 year, 5 months ago)

Thanks for the compliment :smile:

Quote:

Stropharis said:
Hopefully it will help a few folks who don't know where or how to start.




It certainly will, agar can be daunting to get into, and I've been putting it off forever.  Having really detailed information and pictures like these helps a ton. 

Especially going to the trouble to cause infection just for demonstration, that's awesome!  I just tried my first clone of a wild reishi a couple days ago and it will really help me on cleaning it up.


--------------------
Have some medicinal mushrooms and want to get the most out of them?  Try this double extraction method.


Post Extras: Print Post  Remind Me! Notify Moderator
Invisiblegopener
lil_dude
Male User Gallery

Registered: 06/16/13
Posts: 432
Re: Stro's Cleaning and Isolating On Agar [Re: Stropharis]
    #18431261 - 06/17/13 11:36 AM (1 year, 5 months ago)

Very very helpful!!:smile:


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


Post Extras: Print Post  Remind Me! Notify Moderator
Offlinespacechildo
Flower fuckin Power Child
Male User Gallery


Registered: 01/24/13
Posts: 5,287
Loc: Don't live here no more
Last seen: 9 hours, 5 minutes
Re: Stro's Cleaning and Isolating On Agar [Re: Stropharis]
    #18431318 - 06/17/13 11:56 AM (1 year, 5 months ago)

Great write-up, cleared up a lot of things that I couldn't figure out.
I'd be truly greatful if you could elaborate this a bit for me:

Quote:

Stropharis said:
If you take a small peace from the very outer edge of the area where there is one distinct sector, when there are actually three, congrats you just snatched up the fasted monoculture in the area.

- Stro




If there's actually 3 sectors just looking like 1 for the naked eye,
is that still a monoculture?
Or did you possibly mean that it's the best place to transfer from further?

Also, your absolute last pic of Orissa C, is that a monoculture?
If not, where would you choose to transfer from/sector next?

Sorry if I'm not making sense, I think this still is a bit confusing,
and my great breakfast probably didn't make my post as accurate as I wanted it to be

:pipesmoke:


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






1: Prep your grain, Rye Berries & Jar lids or WBS and SFD lids
2: Build a Simple SAB or a more advanced one
3: Inoculate and how to use a SAB
4: Build a monotub

5: Prepare Coir+verm with Bucket Tek
6: Dunk your grains right before Spawning them in your monotub!
7: When fully colonized Dial it in like a boss
8: Harvest with scissors and dry in dehydrator!


Post Extras: Print Post  Remind Me! Notify Moderator
OfflinecronicrM
Shhhhh
 User Gallery


Registered: 08/07/11
Posts: 32,296
Loc: Van Isle Flag
Last seen: 1 day, 8 hours
Trusted Cultivator
Re: Stro's Cleaning and Isolating On Agar [Re: spacechildo]
    #18431461 - 06/17/13 12:47 PM (1 year, 5 months ago)

:woot:
just want in on this one:thumbup:


--------------------
:evil:AMU Q AND A THREAD!:evil:
JUST DO WHAT HE DOES!

PRINT LOTTO ROUND 5

'It doesn't matter what I think of you..all that matters is clean spawn'


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleStropharis
 User Gallery


Registered: 04/02/07
Posts: 1,364
Re: Stro's Cleaning and Isolating On Agar [Re: spacechildo]
    #18431474 - 06/17/13 12:53 PM (1 year, 5 months ago)

It is only when you narrow a culture down to a smaller number of strains that sectoring becomes visible. Usually after cloning you can make out sectors, these sectors are nothing more than individual strains (monocultures/isolates) that are disdiqushing themselves by showing different growth characteristics relative to its neighboring strain creating lines of isolation between one another. When I say;

Quote:

Stropharis said:
If you take a small peace from the very outer edge of the area where there is one distinct sector, when there are actually three, congrats you just snatched up the fasted monoculture in the area.





I don't mean that these sectors are not visible to the naked but they are only noticed if you take a closer look and start "splitting hairs" or small sectors lol. If you do not need a lot of different strains from one dish then it isn't necessary, because that small peace of mycelium that you transfer will more than likely not carry all three genetics but only one. 

I want to make sure that I answer your question.

Quote:

spacechildo said:
If there's actually 3 sectors just looking like 1 for the naked eye,
is that still a monoculture?




No, a true mono-culture, isolate, or strain, should not show sectoring. When working with clones tissue on agar you have already narrowed it down and will most likely see sectors on the first culture, and although they are not true monoculture, the desired intent is met, a performing culture, therefore the reasoning behind reducing it further are limited.

Quote:

spacechildo said:
Also, your absolute last pic of Orissa C, is that a monoculture?
If not, where would you choose to transfer from/sector next?




C is now true monoculture, yes.


As for A I believe there are two strains but it is hard to tell, not relevant as I would have no reason to reduce it further.

B is not an isolate.



And you can tell why by looking where it came from.


There are so many strains in this culture that it is hard to make out sectors.

I will actually be adding to this writing up using the second dish of B to demonstrate further isolation of a strain.

I hope your answer is somewhere in my rambling.

I am late for a date but I will stop by later.


--------------------
Stro's Write Ups


Post Extras: Print Post  Remind Me! Notify Moderator
Offlinevan der griegen
Really Nice Guy


Registered: 08/26/12
Posts: 3,898
Last seen: 6 days, 8 hours
Re: Stro's Cleaning and Isolating On Agar [Re: Stropharis]
    #18449350 - 06/20/13 11:00 PM (1 year, 5 months ago)

I can't believe it's been three days and there aren't at least five pages of people telling you what an excellent resource this is.  :thumbup:  Thank you for this amazing pictorial write-up.


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleStropharis
 User Gallery


Registered: 04/02/07
Posts: 1,364
Re: Stro's Cleaning and Isolating On Agar [Re: van der griegen]
    #18450207 - 06/21/13 03:00 AM (1 year, 5 months ago)

Thanks Bro! There should be some updates coming soon.


--------------------
Stro's Write Ups


Post Extras: Print Post  Remind Me! Notify Moderator
Offlinenewbielife5
Enthusiast
 User Gallery


Registered: 09/04/12
Posts: 121
Last seen: 6 days, 10 hours
Re: Stro's Cleaning and Isolating On Agar [Re: Stropharis]
    #18450349 - 06/21/13 04:06 AM (1 year, 5 months ago)

This is an amazing write up! Props to you man:thumbup:


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

Mazatapec        PESH              Cambodian


Post Extras: Print Post  Remind Me! Notify Moderator
OfflinePussyFartM
Happy Bird Day!!!!
Male


Registered: 04/08/12
Posts: 18,914
Loc: Quahog, Rhode Island
Last seen: 20 hours, 40 seconds
Trusted Cultivator
Re: Stro's Cleaning and Isolating On Agar [Re: newbielife5]
    #18450351 - 06/21/13 04:07 AM (1 year, 5 months ago)

:whathesaid:  Very nice bro....cant't wait to see your updates.


--------------------
                 
THIS HOBBY IS NOT FOR THE IMPATIENT! PLEASE BE PATIENT, DON'T BE A PATIENT!
A Tale of 10 Isolates, GT Cluster Clone Monotubs, RR's Let's Grow Mushrooms DVD,
SGFC(Shotgun Fruiting Chamber), Monotub Tek, Damion5050's Coir Tek, TL's Tek List, Frank's Tek List,
EvilMushroom666's Pasteurization Tek, How It Should & Shouldn't Look - NEW CULTIVATORS GUIDE
  ^*~.,,.~*^*~.,_,.~*^*~.,_SHIT FOR NOOBS_,.~*^*~.,_,.~*^*~.,_,.~* 


Post Extras: Print Post  Remind Me! Notify Moderator
Offlinecubenpete
Aminita good excuse
Male User Gallery


Registered: 03/14/12
Posts: 809
Loc: The Carboniferous
Last seen: 1 day, 6 hours
Re: Stro's Cleaning and Isolating On Agar [Re: PussyFart]
    #18451645 - 06/21/13 02:05 PM (1 year, 5 months ago)

Wow! Agar 101-04. Great read.  :takingnotes:


Edited by cubenpete (06/22/13 04:06 AM)


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleStropharis
 User Gallery


Registered: 04/02/07
Posts: 1,364
Re: Stro's Cleaning and Isolating On Agar [Re: cubenpete]
    #18451862 - 06/21/13 02:59 PM (1 year, 5 months ago)

Updated.


--------------------
Stro's Write Ups


Post Extras: Print Post  Remind Me! Notify Moderator
OfflineDeadPhan
Male User Gallery

Registered: 05/06/04
Posts: 4,761
Last seen: 28 days, 9 hours
Re: Stro's Cleaning and Isolating On Agar [Re: Stropharis]
    #18630037 - 07/29/13 06:41 PM (1 year, 4 months ago)

Awesome dude.  Thanks for this!  :cheers:


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

Big Gulps!  Alright!  Well,  See ya later!
And if i claim to be a wise man, well, it surely means that i dont know!


Post Extras: Print Post  Remind Me! Notify Moderator
InvisibleStropharis
 User Gallery


Registered: 04/02/07
Posts: 1,364
Re: Stro's Cleaning and Isolating On Agar [Re: DeadPhan]
    #18632212 - 07/30/13 03:00 AM (1 year, 4 months ago)

Glad I could help.


--------------------
Stro's Write Ups


Post Extras: Print Post  Remind Me! Notify Moderator
Jump to top. Pages: 1 | 2 | 3 | 4 | 5 | Next >  [ show all ]

Amazon Shop for: Agar, Malt Extract, Parafilm

Mushrooms, Mycology and Psychedelics >> Mushroom Cultivation

Similar ThreadsPosterViewsRepliesLast post
* How do you clean your agar bottles? PussyFartM 718 11 12/22/12 11:37 PM
by Fischer
* Ps. Cyan without agar work? TheShroomHermit 484 9 04/09/03 04:16 AM
by deanofmean
* Need help with isolation. Agar pics included dmonkey1 590 16 07/02/11 02:02 AM
by afrosheen
* Is this strain isolated??? [Agar] - (PICS) CDS 1,172 9 10/25/08 02:17 AM
by The shroomy 1
* Is there a good way to isolate agar cultures after cobweb mold has sporulated in it? DoSedLiving 315 5 10/29/13 11:33 PM
by DoSedLiving
* Cleaning / reusing Agar jars Voodoo486 307 4 07/03/09 03:41 AM
by FractalXplora
* Isolated agar plate? Icon 192 6 06/26/14 04:54 PM
by Johnny Dont
* How clean is a clean colonized agar dish? Guaranteed bac free? sansa 321 6 07/13/13 11:17 PM
by sansa

Inbound Links
How many agar transfers/time does it normally take you guys to get an isolate, mono, or rhizomorphic growth? : shroomers
TEK Web links
LC has failed on 4 separate attempts : shroomers
Helpful links to start your own culture!
How to: Make Spore Prints - Milkwood - Skills for real lifeMilkwood – Skills for real life
I've got the itch again. Some basic tips from a retired grower : shroomers

Extra information
You cannot start new topics / You cannot reply to topics
HTML is disabled / BBCode is enabled
Moderator: Magash, Shroomism, george castanza, Prisoner#1, RogerRabbit, Citric, total, FooMan, 13shrooms, stonesun, EvilMushroom666, Javadog, cronicr, PussyFart
6,369 topic views. 16 members, 77 guests and 1 web crawlers are browsing this forum.
[ Toggle Favorite | Print Topic | Stats ]
Search this thread:
Myco Supply
Please support our sponsors.

Copyright 1997-2014 Mind Media. Some rights reserved.

Generated in 0.176 seconds spending 0.053 seconds on 41 queries.