Home | Community | Message Board

Sporeworks
This site includes paid links. Please support our sponsors.


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!

Shop: Bridgetown Botanicals Bridgetown Botanicals   PhytoExtractum Buy Bali Kratom Powder   Original Sensible Seeds Bulk Cannabis Seeds   North Spore Injection Grain Bag   Kraken Kratom Red Vein Kratom   Left Coast Kratom Buy Kratom Extract   Mushroom-Hut Liquid Cultures

Jump to first unread post Pages: 1 | 2 | 3 | 4 | Next >  [ show all ]
Some of these posts are very old and might contain outdated information. You may wish to search for newer posts instead.
InvisibleFrankHorrigan
The Inquisition
Male


Registered: 01/04/11
Posts: 10,573
Trusted Cultivator
Making slants with Frank * 27
    #18182209 - 04/28/13 04:49 PM (10 years, 8 months ago)

This is part of my series on how I get things done. I hope it helps!



There's not a lot said about slants around here and I'm not sure why that is.

A slant is basically just a culture on agar in a test tube. The agar is cooled in such a way that it provides a nice sloping surface for the mycelium to grow on.

I use two kinds of slants: master slants (with wood) and regular slants (just agar).
By adding a small slice of wood to each tube along with the agar, the mycelium can be stored safely long-term.

I love slants because they let me keep cultures without having to deal with a fridge full of bulky petri dishes.
It also makes condensation an issue of the past.
And master slants will keep cultures viable for many years :awesomenod:

They're very simple to make and if you can work with petris using good sterile technique, you'll have no problem with slants.

This tek is based off of Mephisto's fantastic master slant tek.
RR also has a great video about making master slants on his DVD series.



You'll need a few things to start:
Test tubes (17x100mm size is pictured, but larger sizes are easier to work with) with caps
Agar
Syringe + needle
Wide mouth pint jars
Lids with GE filter (grain lids)
Pressure cooker
Parafilm
OPTIONAL- Thin wooden sticks of some sort (craft sticks, coffee stirrers, etc)


Preparation:

For tubes I use 17x100mm CulTubes for this write up. These hold 12ml of liquid. The cap is excellent and allows either GE or an airtight seal. The only issue is that 17x100mm is very small and somewhat difficult to work with. I would honestly recommend larger tubes.



Regarding Vacutainers:

You can also use vacutainers but I don't like the rubber stoppers that much. I'm clumsy with those caps.

A little prep work is required next so our slants cool at the right angle. I use a wire shelf wrapped in foil but you can wrap just about anything for the same purpose. Whatever you settle on, make sure it fits inside your still air box or in front of your hood.

One side needs to be propped up an inch or two. Metal jar lids or craft sticks work well for this. Just keep stacking them one at a time under the shelf until you have a good angle. Here is a small wooden cutting board, wrapped in foil for easy cleaning and propped up at a good angle using two stacks of seven metal jar lids :thumbup:


Fill a tube up with water to the 5-6ml line and lay it on your propped up shelf. There should be a nice slope of liquid, slanting towards the cap. It should taper off before hitting the top.


Adjust the slope until you find an angle you like. Don't fret about specifics too much! You'll find a sweet spot after a couple tries :wink:


Wood or no wood?

Do you want to make master slants or omit the wood?  If you decide not to use wood, RR suggests transferring your cultures to new slants twice a year:


Quote:

RogerRabbit said:
When making transfers from culture slants, cut a small piece of mycelium and transfer it to a Petri dish. Allow it to grow out approximately 1 cm(unless contamination appears sooner), and then transfer from the leading edge of the growth to a new Petri dish. Once the mycelium on the second dish grows out 1cm, if no contamination has become evident, place a tiny piece of mycelium from the leading edge into a fresh test tube slant for further storage. The remaining mycelium on the Petri dish can then be used to inoculate grains for mushroom growing.




If you want to use wood for master culture slants, pick up a package of craft sticks or coffee stirrers. Mycelium will stay viable in cold storage for many years once they colonize the agar-soaked wood.


I cut these craft sticks in half, then heat some water on the stove. Take it off the heat when it boils and toss in your craft sticks. Cover it and let them soak for one hour.



Filling the tubes:

Do a little math and figure out how much agar you are going to need.
For this pictorial I am making 125ml of agar for 20 tubes or so.
I'm using light MEA powder for my agar, it calls for a ratio of 25g of powder to 500ml of water.
Each culture tube will need 5-6ml of agar.
So for 20 cultures at 6ml each I need to make around 125ml of agar.
That means I'll need 6.25g of MEA powder to 125ml of water.

Measure out the agar and mix it together in a glass or bowl.


Suck it up into a syringe (I use 60cc syringes) with a needle. Make sure it is mixed well every time you aspirate the solution into your syringe.


Cap the needle and wait until your hour is up with the sticks or continue on if you are not using them.

After an hour you'll notice all of your fully hydrated sticks sitting on the bottom of the pan. They are ready to go!


Take them out and add one to each tube. They should fit comfortably inside and not touch the top.


Get your pint jars opened up and ready. I lay mine on the side, propped up a bit. It's easier to load the test tubes that way. I'll explain why we're using pint jars shortly :thumbup:


Shake your syringe well and make sure the agar is mixed before filling each tube. Carefully fill each tube to the 5-6ml mark. Second picture is a tube with wood, third is a tube with none.


After you fill each one, push the cap on but only across the first snap. It should jiggle loosely but not come off easily- this will allow GE during sterilization.

This is how the caps for the vacutainers need to be attached before PCing:
Quote:

Mephistophelian said:

For Vac-Tainers see attached picture.








Loading the jars

Place each tube upright inside a pint jar. You can fit 8 cultubes of this size into one wide mouth pint jar but you should only load 7 in each for now.


After your pint jars have 7 tubes in them, fill each with a little water and add the eighth tube if you are going to. Make sure the water in the jar only goes up to the 3-4ml mark on the tubes, otherwise you may end up getting water inside of them while they are sterilizing. Place a grain lid with GE filter on each jar.


The reason for using these pint jars and filtered lids is so we can move the tubes from the PC into a still-air box or in front of a hood without risking contamination. It is important that the cultubes be shut completely before they are exposed to open air.

It is also because the water inside the jars provides some extra insulation during cooling. It is very common to have a few tubes boil over, this helps minimize that.

I recommend having at least six tubes per jar. You don't want them to lay on their side and boil over while they are being PC'd. If you need to, just use empty tubes as place holders :thumbup:

Once you're finished with the jars, place them in your PC and add water until about 1/4-1/2 way up the jars. PC for 25-40 minutes at 15psi.


Cooling the slants

Let the PC cool for 1-2 hours, until it is no longer too hot to touch.

Put on gloves, tyvek sleeves, and prepare your still-air box. Wipe down the foil with an isopropyl-soaked paper towel and wipe your gloves down as well.


Open the PC, remove the jars and tighten each lid. Place them in your still-air box or in front of your hood.

Wipe your gloves down one more time and wipe down each jar. Open the first jar and carefully snap each cap down so it is airtight. After each cap is snapped, remove them from the jar and place them on your shelf.


Make sure the angle of your slants is appropriate :thumbup:


Let them cool for an hour or so before inoculating. That's all there is to it.



A few notes about using these:

Transfer clean mycelium from a petri dish into your slants. Make sure to flame sterilize the scalpel, work using good sterile technique, and do not leave the culture tube open for any longer than needed.

After sterilizing my scalpel, I open the receiving tube and place the cap face-up on the sanitized foil surface. While holding the scalpel in one hand, I use the other to hold the receiving tube and lift the lid of the petri dish. It takes a bit of manipulation to hold a tube and the lid of a petri dish with one hand while taking a transfer with a scalpel in the other.  Practice a few times using blank dishes and tubes before attempting to inoculate them, it helps a lot :awesomenod:

Make sure to snap the cap down one notch after inoculation for GE. Wrap the top of the tube in parafilm to ensure no contams can get through.

During colonization, I store my tubes in Ziploc bags. When they are ready to go into storage, I snap the cap down all the way (air-tighT) and put them into plastic baggies again. These bags go into insulated lunchboxes and sit at 36F in my culture fridge.

When you are ready to use your cultures, transfer a small piece of mycelium to a petri dish and let it grow out. You can do this with either an inoculation loop or a scalpel. Make sure to only insert the sterile part of your scalpel blade into the tube if you use one.



:cheers:


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

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


Edited by FrankHorrigan (10/01/13 04:21 PM)


Extras: Filter Print Post Top
InvisibleKhii Khwaay
black tooth grin

Registered: 04/16/12
Posts: 2,277
Re: Making slants with Frank [Re: FrankHorrigan]
    #18182438 - 04/28/13 05:48 PM (10 years, 8 months ago)

Wow, this is a really awesome tek. Very well thought out. Just a couple of questions:

1. How far do you normally let the myc colonise the agar before you put it into storage?

2. Where do you get those culture tubes?


Extras: Filter Print Post Top
OfflineSaint Stephen


Registered: 03/05/13
Posts: 152
Loc: In and out of the garden
Last seen: 10 years, 7 months
Re: Making slants with Frank [Re: Khii Khwaay] * 1
    #18182453 - 04/28/13 05:50 PM (10 years, 8 months ago)

Will this work with BD vacutainers? Ive got a few around and i was wondering if this is an option or stick storing spores?


great tek either way thanks man


Extras: Filter Print Post Top
OfflineSagescruffy
CH
Male


Registered: 10/30/09
Posts: 2,011
Loc: PNW
Last seen: 6 months, 11 days
Re: Making slants with Frank [Re: Saint Stephen]
    #18182455 - 04/28/13 05:51 PM (10 years, 8 months ago)

nice write up!


--------------------
Love.


Extras: Filter Print Post Top
OfflineAngry_Goomba
Stranger Danger


Registered: 03/25/13
Posts: 116
Last seen: 9 years, 4 months
Re: Making slants with Frank [Re: FrankHorrigan]
    #18182534 - 04/28/13 06:12 PM (10 years, 8 months ago)

Awesome man. I always enjoy your posts. I'm going  to do some cloning on agar shortly, and my next step is doing some slants. Thank  you and bookmarked. :awesomenod:


Extras: Filter Print Post Top
OfflineFungiFractals
Sacred Geometry
Male User Gallery


Registered: 04/06/09
Posts: 224
Last seen: 2 months, 11 days
Re: Making slants with Frank [Re: Angry_Goomba]
    #18182619 - 04/28/13 06:35 PM (10 years, 8 months ago)

everything looks great, as expected from frank. i know that i for one will refer to this thread in the future.

Thanks for being such a positive influence in the community frank.


Extras: Filter Print Post Top
InvisiblezMan
zMan


Registered: 09/08/12
Posts: 2,191
Re: Making slants with Frank [Re: Angry_Goomba]
    #18182638 - 04/28/13 06:39 PM (10 years, 8 months ago)

Frank have you tried letting them cool right in the jar?

Also what's the deal with how much slant you want? Is more slant better because the myc can take more water from the agar or less slant so the myc can take water longer?


--------------------
Information for everyone: http://www.mushroomvideos.com
Are your grains or spawn clean? CLICK THIS CHECKLIST


Extras: Filter Print Post Top
OfflineCoreyRubble
Just Getting Started
Male User Gallery


Registered: 04/03/10
Posts: 1,134
Loc: Right now???? streets :(
Last seen: 8 years, 2 months
Re: Making slants with Frank [Re: FungiFractals]
    #18182646 - 04/28/13 06:41 PM (10 years, 8 months ago)

Thank frank! Your my idol buddy.

I love that you help and aren't condescending.

Rare quality around these parts Sometimes.

Maybe you just haven't been on the boards long enough, Lol.

Hats off to you sir!

I was just looking for how to do slants yesterday, and I like your style.


Extras: Filter Print Post Top
OfflineBuddyGreen
tripping

Registered: 07/20/04
Posts: 264
Last seen: 8 years, 1 month
Re: Making slants with Frank [Re: zMan]
    #18182650 - 04/28/13 06:42 PM (10 years, 8 months ago)

Frank, you sexy bitch. :datass:

this is an awesome write up.  i'm going to have enough money to build a flow hood by the end of the may, and have been reading a bit about isolations and clones with agar.  I've seen a lot of references to master slants, but nothing quite written up with this.  kudos, and thanks for being the man. :thumbup:


Extras: Filter Print Post Top
InvisibleFrankHorrigan
The Inquisition
Male


Registered: 01/04/11
Posts: 10,573
Trusted Cultivator
Re: Making slants with Frank [Re: CoreyRubble] * 1
    #18182728 - 04/28/13 07:01 PM (10 years, 8 months ago)

Quote:

Khii Khwaay said:
Wow, this is a really awesome tek. Very well thought out. Just a couple of questions:

1. How far do you normally let the myc colonise the agar before you put it into storage?

2. Where do you get those culture tubes?




I let the myc colonize the whole thing usually. At least to the end of the agar on the slant. It never hurts to know the entire tube is colonized and it's much easier to get mycelium transferred out. I ordered the culture tubes online from a medical supply store. I would search around online for a good price on a case of 500.

Quote:

Saint Stephen said:
Will this work with BD vacutainers? Ive got a few around and i was wondering if this is an option or stick storing spores?




Yes, you certainly can use vacutainers. I expanded the OP to include mephisto's information on how to attach the vacutainer cap for PCing.  I just always bump those caps off :facepalm:

Quote:

zMan said:
Frank have you tried letting them cool right in the jar?

Also what's the deal with how much slant you want? Is more slant better because the myc can take more water from the agar or less slant so the myc can take water longer?




You can let them cool in jar but you can't lay it down to make the slant properly because there is water in the jar that will leak in. It's easier to just take em out after snapping the cap down fully.
You want enough slant to be able to reach in and take some myc off the agar without compromising the sterility of the tube.  It's difficult to cleanly pull myc out of a tiny test tube if it only has a 5mL pool of solid agar on the bottom.  The slant gives it more surface area to colonize as well.

Edit: RR also says this, I found:
Quote:

RogerRabbit said:
The extra surface area of a petri dish causes them to completely dry out after six to eight months in the refrigerator.  Test tubes have more agar with less surface area for much slower gas exchange, thus slowing down evaporation.
RR




There may be other benefits I'm not aware of. Perhaps someone who has more experience with master slants can chime in.

To everyone else...
:thanx: :thanx: :thanx:

I hope it makes slants an easy job :mushdance:


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

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


Edited by FrankHorrigan (04/28/13 07:59 PM)


Extras: Filter Print Post Top
InvisiblezMan
zMan


Registered: 09/08/12
Posts: 2,191
Re: Making slants with Frank [Re: FrankHorrigan]
    #18182747 - 04/28/13 07:05 PM (10 years, 8 months ago)

There has to be other benefits other then removing mycelium. Start chiming in!


--------------------
Information for everyone: http://www.mushroomvideos.com
Are your grains or spawn clean? CLICK THIS CHECKLIST


Extras: Filter Print Post Top
OfflineiOmniphobia
Not all who wander are lost
Male User Gallery

Registered: 03/29/13
Posts: 148
Last seen: 10 months, 28 days
Re: Making slants with Frank [Re: zMan]
    #18182812 - 04/28/13 07:19 PM (10 years, 8 months ago)

Yet another great guide by Frank. Thanks man!


Extras: Filter Print Post Top
InvisibleFrankHorrigan
The Inquisition
Male


Registered: 01/04/11
Posts: 10,573
Trusted Cultivator
Re: Making slants with Frank [Re: iOmniphobia]
    #18183016 - 04/28/13 07:58 PM (10 years, 8 months ago)

Quote:

RogerRabbit said:
The extra surface area of a petri dish causes them to completely dry out after six to eight months in the refrigerator.  Test tubes have more agar with less surface area for much slower gas exchange, thus slowing down evaporation.
RR




:themoreyouknow:


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

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


Extras: Filter Print Post Top
Offlinetripdawg420
low life with no life
Male


Registered: 02/02/09
Posts: 7,071
Loc: illinois
Last seen: 3 hours, 32 minutes
Re: Making slants with Frank [Re: FrankHorrigan]
    #18183392 - 04/28/13 08:43 PM (10 years, 8 months ago)

:congrats: nice :thumbup:


--------------------
HUSTLER
How U Survive This Life Everyday Resourcefully
epic GT mono tub
http://www.shroomery.org/forums/showflat.php/Number/17277772

wbs tek
http://www.shroomery.org/forums/showflat.php/Number/11525679
coir tek
http://www.shroomery.org/forums/showflat.php/Number/11917410
results :thumbup:


Extras: Filter Print Post Top
InvisiblezMan
zMan


Registered: 09/08/12
Posts: 2,191
Re: Making slants with Frank [Re: FrankHorrigan]
    #18187580 - 04/29/13 04:35 PM (10 years, 8 months ago)

Quote:

FrankHorrigan said:
Quote:

RogerRabbit said:
The extra surface area of a petri dish causes them to completely dry out after six to eight months in the refrigerator.  Test tubes have more agar with less surface area for much slower gas exchange, thus slowing down evaporation.
RR




:themoreyouknow:





So in theory and according to RR you shouldn't make your slant as big as possible but not small either. Make it big enough that you wont have much trouble later to move it over to a petri and it will last the max allowable time.


--------------------
Information for everyone: http://www.mushroomvideos.com
Are your grains or spawn clean? CLICK THIS CHECKLIST


Extras: Filter Print Post Top
InvisibleFrankHorrigan
The Inquisition
Male


Registered: 01/04/11
Posts: 10,573
Trusted Cultivator
Re: Making slants with Frank [Re: zMan]
    #18187664 - 04/29/13 04:52 PM (10 years, 8 months ago)

5-6mL per slant is perfect :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


Extras: Filter Print Post Top
InvisibleEvilMushroom666
Heretic
Male User Gallery


Folding@home Statistics
Registered: 11/18/09
Posts: 10,289
Loc: Canada Flag
Trusted Cultivator
Re: Making slants with Frank [Re: FrankHorrigan] * 2
    #18187699 - 04/29/13 04:58 PM (10 years, 8 months ago)

Nice write up, nothing like a bunch of freshly poured slants to get the blood moving:



Extras: Filter Print Post Top
Invisible36fuckin5
Alchemycologist


Registered: 08/11/03
Posts: 12,079
Loc: Diving into Mystical Territori...
Trusted Cultivator
Re: Making slants with Frank [Re: EvilMushroom666]
    #18382783 - 06/07/13 11:48 AM (10 years, 7 months ago)

y u no Parafilm?!


--------------------
Redd Foxx said:
If you're offended I don't give a shit and don't come see me no more.

Pat The Bunny said:
A punk rock song won't ever change the world, but I can tell you about a couple that changed me.

bodhisatta said:
i recommend common sense and figuring it out.

These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.


Extras: Filter Print Post Top
InvisibleFrankHorrigan
The Inquisition
Male


Registered: 01/04/11
Posts: 10,573
Trusted Cultivator
Re: Making slants with Frank [Re: EvilMushroom666]
    #18382794 - 06/07/13 11:51 AM (10 years, 7 months ago)

Quote:

EvilMushroom666 said:
Nice write up, nothing like a bunch of freshly poured slants to get the blood moving:






Sexy slants ya got there, EM :awesomenod:


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

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


Extras: Filter Print Post Top
Invisiblelessismore
Registered: 02/10/13
Posts: 6,268
Re: Making slants with Frank [Re: FrankHorrigan]
    #19435759 - 01/17/14 11:52 PM (10 years, 12 days ago)

Nice tek :-)

ice cream sticks probably work good, and a good excuse to eat icecream
got a few I saved for hobby purpose/slant use :wink:

also works wonders for raising the bridge on a guitar if glued to the old bridge


sounds like a good idea to put the test tubes in a jar like in the tek.... else I would have to loosen the screw lid a bit for them not to burn/melt likely
which would introduce contams maybe... if unlucky
jar ftw


Extras: Filter Print Post Top
Jump to top Pages: 1 | 2 | 3 | 4 | Next >  [ show all ]

Shop: Bridgetown Botanicals Bridgetown Botanicals   PhytoExtractum Buy Bali Kratom Powder   Original Sensible Seeds Bulk Cannabis Seeds   North Spore Injection Grain Bag   Kraken Kratom Red Vein Kratom   Left Coast Kratom Buy Kratom Extract   Mushroom-Hut Liquid Cultures


Similar ThreadsPosterViewsRepliesLast post
* Calcium Chloride (drying questions) bjkroll 2,025 5 12/12/02 04:03 PM
by resin
* Potassium Chloride (is it just as good??) bjkroll 679 2 01/25/03 07:23 PM
by bjkroll
* Desiccant question (anhydrous calcium chloride) Canisdormit 2,084 7 01/27/02 10:12 PM
by jalfonso
* Calcium Chloride, any dessicating properties? InjectTruth 1,328 7 04/23/04 03:00 AM
by Joshua
* Calcium Chloride - Can it be reused? CaptBeefheart 3,056 3 02/17/03 09:42 AM
by dog
* Calcium Chloride Poisoning? DigiAngel 1,092 2 02/14/03 09:03 PM
by Rastafari
* perlite & calcium chloride question..... DNICEONER 867 2 05/03/04 08:56 PM
by DNICEONER
* Calcium Chloride question Hammerloaf 940 1 10/28/02 09:30 PM
by mickey_rourke

Extra information
You cannot start new topics / You cannot reply to topics
HTML is disabled / BBCode is enabled
Moderator: Shroomism, george castanza, RogerRabbit, veggie, mushboy, fahtster, LogicaL Chaos, 13shrooms, Stipe-n Cap, Pastywhyte, bodhisatta, Tormato, Land Trout, A.k.a
54,758 topic views. 32 members, 268 guests and 52 web crawlers are browsing this forum.
[ Show Images Only | Sort by Score | Print Topic ]
Search this thread:

Copyright 1997-2024 Mind Media. Some rights reserved.

Generated in 0.023 seconds spending 0.007 seconds on 14 queries.