Home | Community | Message Board


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: Kraken Kratom Red Vein Kratom   MagicBag.co All-In-One Bags That Don't Suck   North Spore Bulk Substrate   Bridgetown Botanicals CBD Concentrates   Left Coast Kratom Buy Kratom Extract   Original Sensible Seeds Bulk Cannabis Seeds   PhytoExtractum Kratom Powder for Sale   Myyco.com Isolated Cubensis Liquid Culture For Sale   Mushroom-Hut Substrate Bags   Unfolding Nature Unfolding Nature: Being in the Implicate Order

Jump to first unread post Pages: 1 | 2 | Next >  [ show all ]
Some of these posts are very old and might contain outdated information. You may wish to search for newer posts instead.
Offlinefir3fly86
Stranger

Registered: 06/20/17
Posts: 82
Last seen: 6 years, 4 months
Agar optimum transfer time?
    #24604669 - 09/05/17 12:36 AM (6 years, 6 months ago)

Hi all,
Newbie here =)

Attempting first monotub tek growth.
Have another thread under a different subject but would really like this particular stumbling block cleared up if thats ok.

Currently learning agar, and learning first hand just how many agar plates I'll be needed before I can proceed to grain, feeling a little demoralized but will keep on going for sure till I get it right.

My issue is, now having 3 generations of plates (most unsuccessful and growing some bacs), what is the best way to ensure you transfer healthy tissue before bacs catch up?

My plates aren't easy to see through and I have them all in ziplock bags too so its hard to tell.
The only thing thats evident on the transferred agars is initial upward growth of hairs from the center.

So my question is...roughly speaking, at what point would be it ideal to make the next transfer?

Thanks

Extras: Filter Print Post Top
InvisibleMateja
 Unread Journal User Gallery


Registered: 07/14/16
Posts: 7,948
Loc: Here
Re: Agar optimum transfer time? [Re: fir3fly86]
    #24604811 - 09/05/17 03:05 AM (6 years, 6 months ago)

First, recognize that mushroom mycelium
is growing on your plate, then when it has grown out to the size
of a small/medium size coin go ahead and repeat the process.

The way i make Agar and it comes out pretty clear is i
just grind up some dry dog food (any kind) and mix with water at
2% strength. I boil everything up for 60-120 sec so that most
of the dog food dissolves, then strain everything and mix with agar.

You will have no problems seeing the tiniest dot from the "other" side of the plate with that mix. You could even go 1% dog food I'm sure.

This recipe has worked for me every time, I'm a beginner. But to be sure wait for the more experienced guys to wake up from the other side of the world :smile:


--------------------
Cakes inside Water Tub

Edited by Mateja (09/06/17 04:07 PM)

Extras: Filter Print Post Top
InvisibleMateja
 Unread Journal User Gallery


Registered: 07/14/16
Posts: 7,948
Loc: Here
Re: Agar optimum transfer time? [Re: Mateja]
    #24604814 - 09/05/17 03:09 AM (6 years, 6 months ago)

And last but not least, you question has probably been answered
too many times in this forum. Click on the: "Search Post" icon and
you will have more success than if you ask in a thread :smile:


--------------------
Cakes inside Water Tub

Extras: Filter Print Post Top
InvisibleLotKid
Never.Trust.A.Prankster
Male User Gallery

Registered: 01/07/17
Posts: 8,169
Loc: Shakedown St.
Trusted Cultivator
Re: Agar optimum transfer time? [Re: Mateja]
    #24604819 - 09/05/17 03:18 AM (6 years, 6 months ago)



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

:mushroom2: LotKid's PE Tips        :shpongle:    LotKid's Chocolates :mushroom2:
:dancingbear: :dancingbear:

Extras: Filter Print Post Top
Offlinefir3fly86
Stranger

Registered: 06/20/17
Posts: 82
Last seen: 6 years, 4 months
Re: Agar optimum transfer time? [Re: Mateja]
    #24604847 - 09/05/17 03:54 AM (6 years, 6 months ago)

Quote:

Mateah said:
First, recognize that mushroom mycelium
is growing on your plate, then when it has grown out to the size
of a small/medium size coin go ahead and repeat the process.

The way i make Agar and it comes out pretty clear is i
just grind up some dry dog food (any kind) and mix with water at
4% strength. I boil everything up for 60-120 sec so that most
of the dog food dissolves, then strain everything and mix with agar.

You will have no problems seeing the tiniest dot from the "other" side of the plate with that mix. You could even go 2% dog food I'm sure.

This recipe has worked for me every time, I'm a beginner. But to be sure wait for the more experienced guys to wake up from the other side of the world :smile:




Hey there :smile: Thanks for the reply. Ive been using so far potato flakes with agar, this is an interesting alternative.

Do you use any food colouring at all? Or leave as is?

Thanks!

Extras: Filter Print Post Top
Offlinefir3fly86
Stranger

Registered: 06/20/17
Posts: 82
Last seen: 6 years, 4 months
Re: Agar optimum transfer time? [Re: LotKid]
    #24604848 - 09/05/17 03:56 AM (6 years, 6 months ago)

Quote:

LotKid said:
Op, read this.

https://www.shroomery.org/forums/showflat.php/Number/18430998




Thanks will check this out! :smile:

Extras: Filter Print Post Top
InvisibleMateja
 Unread Journal User Gallery


Registered: 07/14/16
Posts: 7,948
Loc: Here
Re: Agar optimum transfer time? [Re: fir3fly86] * 1
    #24606463 - 09/05/17 04:46 PM (6 years, 6 months ago)

Quote:

fir3fly86 said: Do you use any food coloring?




No, i personally think contams are easier to spot on a clear plate!

I will make DFA (Dog Food Agar) tomorrow morning and will do several
at different strengths from 1-4% and i will post pics here so
you can see the end result and how clear the plates are :smile:


--------------------
Cakes inside Water Tub

Edited by Mateja (09/05/17 04:54 PM)

Extras: Filter Print Post Top
Offlinefir3fly86
Stranger

Registered: 06/20/17
Posts: 82
Last seen: 6 years, 4 months
Re: Agar optimum transfer time? [Re: Mateja]
    #24607631 - 09/06/17 01:41 AM (6 years, 6 months ago)

Quote:

Mateah said:
Quote:

fir3fly86 said: Do you use any food coloring?




No, i personally think contams are easier to spot on a clear plate!

I will make DFA (Dog Food Agar) tomorrow morning and will do several
at different strengths from 1-4% and i will post pics here so
you can see the end result and how clear the plates are :smile:




That would be awesome thank you! Look forward to seeing them.

Extras: Filter Print Post Top
InvisibleMateja
 Unread Journal User Gallery


Registered: 07/14/16
Posts: 7,948
Loc: Here
Re: Agar optimum transfer time? [Re: fir3fly86]
    #24609567 - 09/06/17 06:29 PM (6 years, 6 months ago)

Just finished pouring 40 plates DFA.

20 plates with 1% dog food and 20 plates with 2% dog food.
To be honest they seem equally clear both strengths.

Here is one of the plates with 2% DFA, with a Norwegian 100 crown bill behind it.

Also this plate has condensation covering the entire plate,
when the condensation clears up tomorrow it will look as clear as day :smile:


Also take bod's word into consideration
Quote:

bodhisatta said:


if you make agar with meat/protein and some contamination grows on it, it might be a smart idea to wrap the plate extra well and toss it out. you can easily grow up astronomical numbers of human pathogenic bacteria. don't play microbiologist and make slides with shit that grew on a meat agar plate is my suggestion




I wonder if the solution to this would be meat free dog food :tongue:
Next time i will get a sample of the vegetarian dog food they have at the pet store and try it out.


--------------------
Cakes inside Water Tub

Extras: Filter Print Post Top
Offlinefir3fly86
Stranger

Registered: 06/20/17
Posts: 82
Last seen: 6 years, 4 months
Re: Agar optimum transfer time? [Re: Mateja]
    #24688145 - 10/06/17 11:15 AM (6 years, 5 months ago)

awesome thank you for posting these ! look great, going to have to give this a try.

Extras: Filter Print Post Top
Offlinefir3fly86
Stranger

Registered: 06/20/17
Posts: 82
Last seen: 6 years, 4 months
Re: Agar optimum transfer time? [Re: fir3fly86]
    #24688159 - 10/06/17 11:19 AM (6 years, 5 months ago)

Ok...so ... tried to transfer again and see what I could salvage...I'm still not too sure on when the best time to do the next transfer is.

For example this one pictured below is about 1 week old now, I have 2 others that look very similar to this. Question is...they all seem to be first growing upwards? Is this normal?

I imagine in a few days it will start to fan out, but I don't want to leave it too long for contams to catch up?



Sorry for the poor qual, the sides of the agars are now becoming opaque..need to change them.

What do you guys think? Should I transfer or is it too soon?

Thanks!

Extras: Filter Print Post Top
InvisibleMateja
 Unread Journal User Gallery


Registered: 07/14/16
Posts: 7,948
Loc: Here
Re: Agar optimum transfer time? [Re: fir3fly86]
    #24690271 - 10/07/17 07:23 AM (6 years, 5 months ago)

Hmm, the mys should not be growing upward it should be 3-5 cm in diameter if you transferred it a week ago. What agar recipe did you use?


--------------------
Cakes inside Water Tub

Extras: Filter Print Post Top
Offlinefir3fly86
Stranger

Registered: 06/20/17
Posts: 82
Last seen: 6 years, 4 months
Re: Agar optimum transfer time? [Re: Mateja]
    #24693177 - 10/08/17 05:23 AM (6 years, 5 months ago)

Quote:

Mateah said:
Hmm, the mys should not be growing upward it should be 3-5 cm in diameter if you transferred it a week ago. What agar recipe did you use?




thanks for reply Mateah. This is still my original agar recipe potato flakes, agar agar, drop of honey and food colouring. Roughly following:

2.5g agar (1.38%)
5ml potato flakes (2.77%)
180ml water
drop of honey


Do you recommend I try another? All of them seem to be initially growing up :/ Also wondeirng...it always seems to form 2 layers even though I heat them up and mix well, on pouring it always forms the 2 layers, is this a factor maybe?

Thanks, any advice appreciated

Edited by fir3fly86 (10/08/17 05:24 AM)

Extras: Filter Print Post Top
InvisibleMateja
 Unread Journal User Gallery


Registered: 07/14/16
Posts: 7,948
Loc: Here
Re: Agar optimum transfer time? [Re: fir3fly86]
    #24693356 - 10/08/17 08:08 AM (6 years, 5 months ago)

2% agar is the strength I use along with 2% nutrients. 300ml water 6g dog food 6g agar, works both for germination/vegetative growth and lately I’ve had A LOT of pins to clone from those plates.

You can substitute dog food for cat food, rabbit food, hamster food etc.

Your agar recipe works tho, you have growth and you should make another transfer already from that latest transfer that is a week old! Sometimes it takes a few transfers before more organized growth appeares. Just keep transferring every 3-6 days if you’re looking to obtain clean plates quick! GL :smile:


--------------------
Cakes inside Water Tub

Extras: Filter Print Post Top
Offlinefir3fly86
Stranger

Registered: 06/20/17
Posts: 82
Last seen: 6 years, 4 months
Re: Agar optimum transfer time? [Re: Mateja]
    #24693414 - 10/08/17 09:02 AM (6 years, 5 months ago)

Quote:

Mateah said:
2% agar is the strength I use along with 2% nutrients. 300ml water 6g dog food 6g agar, works both for germination/vegetative growth and lately I’ve had A LOT of pins to clone from those plates.

You can substitute dog food for cat food, rabbit food, hamster food etc.

Your agar recipe works tho, you have growth and you should make another transfer already from that latest transfer that is a week old! Sometimes it takes a few transfers before more organized growth appeares. Just keep transferring every 3-6 days if you’re looking to obtain clean plates quick! GL :smile:




Great thanks! :smile: Will do, so even though there is no clear edges I just grab a piece that looks relatively 'ok' and transfer I guess? Until things grow out a little better?

Extras: Filter Print Post Top
InvisibleMateja
 Unread Journal User Gallery


Registered: 07/14/16
Posts: 7,948
Loc: Here
Re: Agar optimum transfer time? [Re: fir3fly86]
    #24693649 - 10/08/17 10:40 AM (6 years, 5 months ago)

I usually transfer to two or three new plates from the same donor plate. That way I can use one plate to let the culture grow out pretty big and watch it grow more organized, I can use one plate to let colonize the entire plate and then pin so I can take my clones, and one of the plates I keep around enough to have some growth to transfer onto new plates if I feel the need to.

The point is, plan ahead what you need to culture for. If I want to make LI for example I will transfer to one or more plates to later use as inoculate and I will also transfer a backup to a new plate. You get the picture :smile:


--------------------
Cakes inside Water Tub

Extras: Filter Print Post Top
Offlinefir3fly86
Stranger

Registered: 06/20/17
Posts: 82
Last seen: 6 years, 4 months
Re: Agar optimum transfer time? [Re: Mateja]
    #24693725 - 10/08/17 11:13 AM (6 years, 5 months ago)

Quote:

Mateah said:
I usually transfer to two or three new plates from the same donor plate. That way I can use one plate to let the culture grow out pretty big and watch it grow more organized, I can use one plate to let colonize the entire plate and then pin so I can take my clones, and one of the plates I keep around enough to have some growth to transfer onto new plates if I feel the need to.

The point is, plan ahead what you need to culture for. If I want to make LI for example I will transfer to one or more plates to later use as inoculate and I will also transfer a backup to a new plate. You get the picture :smile:




Ok great thanks! Duly noted =)

Just to keep you in on the loop...and...for a little learning myself, while I am waiting for my new spore agars to germinate, I did some transfers on the 3 agars I have left.

A was the healthiest looking one that I had some hopes on, this is the one thats been growing upwards though, what do you think? There does now seem to be some horizontal growth, so I took the transfers from those areas...the vertical growing bit looks like its covered almost in a cobwebby looking structure...






B...well...looks very wrong to me..didnt do any transfers



C...also doesn't look great? Didnt transfer



Any opinions appreciated =)

Edited by fir3fly86 (10/08/17 11:15 AM)

Extras: Filter Print Post Top
InvisibleMateja
 Unread Journal User Gallery


Registered: 07/14/16
Posts: 7,948
Loc: Here
Re: Agar optimum transfer time? [Re: fir3fly86]
    #24694082 - 10/08/17 01:08 PM (6 years, 5 months ago)

Have you seen this post? Stropharis shows how and what to transfer :smile:

Quote:

Stropharis said:
Stro's Cleaning and Isolating On Agar




I recommend putting all your spores and clones on agar first, by doing so, you can ensure that you are working with a clean culture before you introduce it to grains, and it will also take out the guess work of spore viability.

So once you have a culture growing on agar, you will now want to make sure that it is clean and free of contaminates. 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, if you do not have one, it is a very easy build using Stro's Still Air Box. Ideally though, given that you have sufficient time and/or money, I recommend building a laminar flow hood. However, their will still be circumstances that make a still air box the more suitable choice. If you are going to build one, Stro’sFlowhood Build may be of use to you, building it was a very rewarding process. If you do decide to use it as a reference, please share your progress with me.

Some other items needed include paper towels, isopropyl alcohol, lighter, alcohol lamp or similar, gloves, scalpel, new dishes, and parafilm or cling wrap, and your cultures of course.

To demonstrate how I clean up my cultures when they need it, 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 what clone was taken carelessly, the photos speak for themselves.

When taking A, I swung my un sterilized scalpel like a battle axe dismembering the pin and watched it fall onto the substrate below, then I stabbed it while doing my best Braveheart scream and preceded to gently place it on the plate. I figured this was a fairly un-climactic ending so I shook the dish and watched the nasty little pin roll around contaminating the entire medium surface.

You would ideally want to do a transfer from a dish as soon as you see growth to avoid spreading any contaminants during the transfers. The less there is growing on your dish before the transfer the better.

So now we need to clean these up right? In order to do that, we will be transferring contaminant free, healthy mycelium growth to a fresh plate. 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, you will be working with 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 taken 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 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.



All right, that covers that for the most part. Lets talk sectoring; I will go ahead and use the images that I 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 or concerned about "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 perimeter 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 we can see six.



I guess my point is, that 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 appearances speak for themselves. The colonization times were faster and characteristics are distinctive. If I don't keep medium and conditions consistent, 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 parameter 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 the 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 distinguish itself so much after saying that.





- Stro






--------------------
Cakes inside Water Tub

Edited by Mateja (10/08/17 01:09 PM)

Extras: Filter Print Post Top
Offlinefir3fly86
Stranger

Registered: 06/20/17
Posts: 82
Last seen: 6 years, 4 months
Re: Agar optimum transfer time? [Re: Mateja]
    #24694282 - 10/08/17 02:27 PM (6 years, 5 months ago)

Yes have seen the post its great! :smile: I just dont seem to get anything near resembling those strong rhizomorphic strands, but...will follow your advice and be patient and make sure I keep up the transfers on time, fingers crossed :laugh:

Extras: Filter Print Post Top
InvisibleMateja
 Unread Journal User Gallery


Registered: 07/14/16
Posts: 7,948
Loc: Here
Re: Agar optimum transfer time? [Re: fir3fly86]
    #24694445 - 10/08/17 03:36 PM (6 years, 5 months ago)

Just let a plate grow out and you will see all kinds of mycelium :smile:


--------------------
Cakes inside Water Tub

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

Shop: Kraken Kratom Red Vein Kratom   MagicBag.co All-In-One Bags That Don't Suck   North Spore Bulk Substrate   Bridgetown Botanicals CBD Concentrates   Left Coast Kratom Buy Kratom Extract   Original Sensible Seeds Bulk Cannabis Seeds   PhytoExtractum Kratom Powder for Sale   Myyco.com Isolated Cubensis Liquid Culture For Sale   Mushroom-Hut Substrate Bags   Unfolding Nature Unfolding Nature: Being in the Implicate Order


Similar ThreadsPosterViewsRepliesLast post
* Agar Anonymous 1,547 8 04/15/04 10:15 AM
by Bi0TeK
* Re: mold in agar plate Anonymous 12,415 3 12/04/99 12:35 AM
by Anonymous
* Agar to Rice flour substrates foxxybrown 2,147 3 09/01/03 05:00 AM
by eggy
* Fruit on Agar?? Yarry 5,120 17 01/22/04 09:44 PM
by Anonymous
* golden teacher agar asking fugu 3,083 6 01/31/03 12:59 PM
by debianlinux
* agar/peroxide conc. iudexk 1,334 1 08/27/01 01:17 PM
by jonnyshaggs420
* Potato Agar Culture Tubes! *NEW* r2_ 1,743 13 01/01/06 03:21 PM
by r2_
* Why grow on agar? stcore 2,021 1 07/29/02 05:03 PM
by bluhoney

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
3,905 topic views. 27 members, 123 guests and 96 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 0.028 seconds spending 0.008 seconds on 16 queries.