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Invisiblepacmanbreed
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24610756 - 09/07/17 04:26 AM (6 years, 5 months ago)

Cubes exhibits tementose on growth on t-gel.
This are 6 days from innoc. About half inch from the center.

bacterial donor brf plate


1st tgel cube wedge


2nd tgel cube wedge.

I think some spores has a hard/delayed time germinating on t-gel specially on high PH. Since Bacteria lowering the ph gives a window for eg. "green machine spores" to germinate.
Also when combating piggy backing bacteria from it I think that its better to control condensation and water pooling to maximize its effect and use toothpicks to leave the bacterial spore behind.


wood loving species loves the tea so much.
Garnoderma after 6 days. About 1.5+ inch from innoc.

1st wedge


2nd wedge

Hope Its not too late to plant toothpicks on them.
I will be away from home for a month due to work.
-Is it advisable to leave them colonizing and just transfer the toothpicks to new plates after a long 1.5 month?

Edited by pacmanbreed (09/07/17 04:31 AM)

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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: pacmanbreed]
    #24611081 - 09/07/17 08:57 AM (6 years, 5 months ago)

The cubensis version, is this without sugar? If without, its done quite well for a sugar free media.
Obviously not the best growth ever, but it seems to have helped clean the sample.

The woodloving is doing well due to laccase as discussed.

:takingnotes:


Usually I would add the pegs, allow them to colonize and then refrigerate.
It should be ok to add the pegs and leave them if you have to.


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Invisiblepacmanbreed
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24612860 - 09/08/17 01:10 AM (6 years, 5 months ago)

Quote:

Ferather said:
The cubensis version, is this without sugar? If without, its done quite well for a sugar free media.
Obviously not the best growth ever, but it seems to have helped clean the sample.

The woodloving is doing well due to laccase as discussed.

:takingnotes:

Usually I would add the pegs, allow them to colonize and then refrigerate.
It should be ok to add the pegs and leave them if you have to.




Thanks for the reply brother.
Yes its sugar less.
(B.) 50g Water, 50g Extract, 2.5g Agar, 0.2g Gelatin, 0.50g Dogfood.
Those are opened sacrificial cube plates for picture and control.
"Some are about 0.75-1" growth. From Recipe (A.) 0.75g DF agar recipe i posted".

Just finished cooking my pegs ready for instalation before leaving my work for a month.
Ill transfer the pegs to higher nutrition t-gel nextround.
50g Water, 50g Extract, 2.5g Agar, 0.2g Gelatin, 1g sugar 1g Dogfood.

Edited by pacmanbreed (09/08/17 01:14 AM)

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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: pacmanbreed] * 1
    #24613154 - 09/08/17 07:29 AM (6 years, 5 months ago)

Wow ok, and it's still growing after transfer 3, that is very interesting. There is also no starch in the tea extract, T-Gel.
Since the cubensis shouldn't be producing laccase, it's running off the proteins in gelatin and dog food.

Proteins are composed of C-H-O-N, for the mycelium that is like sugar with nitrogen attached.
Based on the fact that most sugar's would otherwise count as a carbon source.

Humans also generate energy from proteins and C-H-O based vitamins.
100% protein or vitamins will produce a bad C:N ratio.

Ideally used as an additive to 100% C-H-O.


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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: Ferather] * 1
    #24613215 - 09/08/17 08:21 AM (6 years, 5 months ago)

Here is the composition of malt extract, and how to make an antibacterial agar recipe.
Copy the macro-micro nutrients, add a carbon source your mycelium likes.

Ideally the carbon source should be disliked by competitors.
This will be much harder for cubensis, no laccase.

Here is tea leaves, and tea extract.


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Invisiblepacmanbreed
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24614658 - 09/08/17 08:28 PM (6 years, 5 months ago)

Quote:

Ferather said:
Here is the composition of malt extract, and how to make an antibacterial agar recipe.
Copy the macro-micro nutrients, add a carbon source your mycelium likes.
Ideally the carbon source should be disliked by competitors.
This will be much harder for cubensis, no laccase.
Here is tea leaves, and tea extract.



Pardon for my noobness.
Thank you for the links brother.
Im Thinkering of cellulose/other additive as C source, since coprophilic also digest this in nature.
:poopontoast:

Quote:

Ferather said:
Wow ok, and it's still growing after transfer 3



The Cubes are still on 1st transfer.

Had absorb that logical noob friendly explanation,
I think I made an out balance C:N ratio by using high protien DF & not adding sugar with tea as the only C source.
I think excess protien as N source is also attrative for competitors which i observed when ever i used DFA vs PDA based on mold germintion time leading to sooner sporulation..
Will source yeast extract next round for n and micro.
Ill be careful on N ratio nextime.


Based on your hard reseach, tea and gelatin is around 30:1 ratio.
Quote:


-what is outcome C:N ratio of my mix?
50g Water, 50g Extract, 2.5g Agar, 0.2g Gelatin, 0.50g Dogfood

-if youll use this on dung lovers, would you target it at 20:1 ratio?



.


Hope you dont mind my questions brother.
Ill try to use them as learning ground with trials.
Still absorbing computation of macro micro additives.
Eg. Homogenized HUMUS = 10:1.
Im still reading papers/links in regards to this.
Love how you used 50:1 ratio specially the pellets for wood lovers.

Edited by pacmanbreed (09/09/17 09:58 PM)

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Invisiblepacmanbreed
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24614956 - 09/08/17 10:42 PM (6 years, 5 months ago)

By the way an update before leaving home.
The leading edge of 1st t-gel cube sacrificial plate has grown to 1" and now visible after 2 days.
Also no signs of contam germination yet from opening it in a high spore load area. Will keep this control on how long till the landed green machine germinates on 7-7.5ph tea. They usually germinate in 3-5 days on DFA


1st tgel cube wedge comparison

Edited by pacmanbreed (09/09/17 10:02 PM)

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OfflineBigbadwooof
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Re: Cleaning dirty cultures with tea agar [Re: pacmanbreed]
    #24616398 - 09/09/17 03:48 PM (6 years, 5 months ago)

My cultures look incredibly faint, and wispy on tea agar. They look like mold mycelium. Very tomentose. Maybe I put more tea in than you fellas.


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Invisiblepacmanbreed
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Re: Cleaning dirty cultures with tea agar [Re: Bigbadwooof]
    #24616589 - 09/09/17 05:24 PM (6 years, 5 months ago)

Quote:

Bigbadwooof said:
My cultures look incredibly faint, and wispy on tea agar. They look like mold mycelium. Very tomentose. Maybe I put more tea in than you fellas.



Did you add a Nitrogen source to this?
Mine look tememtose aswell. I guess its the tannins/tanic suppression nature of tea.

Ferather is right, I feel that we Just need to balance c:n ratios while using the teas macro/micro.
i feel that T-MEYA is standard starting/learning ground for this. But still can use any sources from our brother's pocket guide.

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OfflineBigbadwooof
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Re: Cleaning dirty cultures with tea agar [Re: pacmanbreed]
    #24617478 - 09/10/17 03:40 AM (6 years, 5 months ago)

Malt Extract Yeast Agar?

I have actually considered adding a bit of coffee to it... I suppose then lime would become more of a necessity. I have some Bat Guano based marijuana fertilizer from fox farm... Maybe that would be a good nitrogen source.

As you can see, my approach to agar is much less scientific that some of you lol. In fact, the only time I measure my nutrient source, is when I'm using LME, because that shit is fucking expensive. Otherwise, I just use some watered down grain water, or something. I think I added grain juice to my tea agar for the first round I made. I don't remember.

Whatever I'm doing, it's working just fine, and I'm happy. I'm inoculating a bunch of jars now, with a beautiful looking clean culture. I think the bacteria had become resistant to gentamycin... I think I used gent on the master slant, years ago. Probably not the wisest decision lol!


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Invisiblepacmanbreed
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Re: Cleaning dirty cultures with tea agar [Re: Bigbadwooof]
    #24617670 - 09/10/17 07:46 AM (6 years, 5 months ago)

Quote:

Bigbadwooof said:
Malt Extract Yeast Agar?




yes just for observation purpose.
But we can exclude maltose and use other plenty of C sources around the kitchen.
Tea already has plenty of C for energy without starches and sugar but lacks in N.
Code:
Ferather said:
3g of sugar for every 100g tea leaves.
Potential energy and N sources (incomplete) for tea.
Polyphenols, varied: [ C6 | H5 | O1 ] x.
Cellulose, varied: [ C6 | H10 | O5 ] x.

Theophylline: C7 | H8 | N4 | O2.
Theobromine: C7 | H8 | N4 | O2.
Caffeine: C8 | H10 | N4 | O2.
Theaflavin: C29 | H24 | O12.
Tannins: C76 | H52 | O46.
Catechin: C15 | H14 | O6.
Carotene: C40 | H56.





Quote:

Bigbadwooof said:
I have actually considered adding a bit of coffee to it...




Minimal for N balance,
I feel that i will omit coffee since i always drink it  :drinkcoffee: , tea already has cafeine/sugar/macro/micro & its already acidic in nature around ph-4 with bacterial suppression properties due to tannins eg. "Camellia sinensis contain polyphenolic components with activity against a wide spectrum of microbes".
Ill use other N sources.
It seems thats cubes also love protien as N source.
Thats why gelatin&yeastextract is added to the mix.
Maybe its also the reason why it shows robust rizho in DFA due to protease production.



Quote:

Bigbadwooof said:
I suppose then lime would become more of a necessity.




Code:
RogerRabbit said:
mushroom metabolites will swing the pH acidic during colonization.

If you're using well water with a high pH over 8 or so,
a bit of vinegar or citric acid will lower it to neutral
- which is a good starting point.

I often say mycelium will colonize a bulk substrate fastest at around 5 to 6,
- to get people to avoid using lime which is recommended in so many teks.
for no reason because it often raises the pH too much, slowing colonization.

Most bulk substrate ingredients are already in the /- pH 6 range.
- so no adjustment is necessary.
RR



I feel this is the reason why our cubes exhibit very slow tementose growth in this agar mix..
They need to acidify the substrate 1st to around Ph of 5 - 6.5 to release carbon-acid bonds.
The Advantage of getting to ph of 7 is to increase suppressional properties and give a delayed/hard time for landed spores to release the sugars within after they germinate to prevent sporultion/muliply
Code:
Ferather said:
It works in the way of alternative carbon sources (non-sugar),
100g of black tea has 3g natural sugar no starch.
As it stands without the trace carbohydrates,
it's very nutritional and soluble, even cheap black tea.

The natural carbon containing acids breakdown at about ph 6.5, so pH 7-7.5 is ideal.
You can still get some bacteria but total activity should be reduced overall.
Here is some composition links (external),



Also Lime must be used in minimum amount just to balance out the acidic ph of tea.
Specially since some have high magnesium content based on sources. Unlike pure calcium oxide/CaO.
Better is to used egg shells in agar work to be safe, which is also a 1C source base on lewis structure.
Calcium carbonate: [Ca | C | O3]
Or 2c for Calcium bicarbonate: [ Ca | C | H | O3 ] 2.

Quote:

Bigbadwooof said:
I have some Bat Guano based marijuana fertilizer from fox farm... Maybe that would be a good nitrogen source.




Yes i think its good macro micro source aswell.
Better to use organic n source, i red from a paper that inorganic N reduces cellulase activty.
Code:
Ferather said:
Example nitrogen materials:
Proteins, Vitamins, Caffeine, Ureic, Ammonical.





Code:
Bigbadwooof said:
As you can see, my approach to agar
is much less scientific that some of you lol.
In fact, the only time I measure my nutrient source,
Is when I'm using LME,
because that shit is fucking expensive.


Same here brother.
Curiosity - community testing - observation- sharing - communication leads to greater knowledge & teks.

Glad elder ferather shared some of his leg work on this for community testings.
Im still gasping/absorbing to properly use our little stock in the kitchen.

I even cant source Lme from here. Importation is badly expensive and not an option for a kitchen grower.



Code:
Bigbadwooof said:
I think the bacteria had become resistant to gentamycin.
I think I used gent on the master slant, years ago.
Probably not the wisest decision lol!


Thank you for sharing ur observation.
Glad that saved me from start and some $$$$.


Hoping that elder brothers with greater understanding specially ferather shares a table of Potential energy and N sources of barley malt extract For easier comparsions to this. :smile:
Code:
Ferather said:
Potential energy and N sources (incomplete) for tea.

Polyphenols, varied: [ C6 | H5 | O1 ] x.
Cellulose, varied: [ C6 | H10 | O5 ] x.

Theophylline: C7 | H8 | N4 | O2.
Theobromine: C7 | H8 | N4 | O2.
Caffeine: C8 | H10 | N4 | O2.
Theaflavin: C29 | H24 | O12.
Tannins: C76 | H52 | O46.
Catechin: C15 | H14 | O6.
Carotene: C40 | H56.




Sorry for the long post. Ill use this for a reference while learning.

Edited by pacmanbreed (09/14/17 04:37 AM)

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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: pacmanbreed] * 1
    #24617995 - 09/10/17 11:17 AM (6 years, 5 months ago)

So much input and good understanding. To clarify cubensis will mostly ignore the tea for carbon, but not the macro-micro nutrients.
Since the cubensis cannot digest the inhibitory materials, it will restrict the growth much like gentamicin or other.

For a woodlover, if not too much (too rich), it will digest the inhibitory materials, and grow pretty normal.
Those who have had good success using tea extract, used it like gentamicin, with normal agar.

Unless you are growing a woodlover, I'd take the route of treating it like gentamicin.


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Growing mushrooms, general guide and information (Ferather's Journal), https://ibb.co/rG3rML2

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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: Ferather] * 1
    #24618026 - 09/10/17 11:29 AM (6 years, 5 months ago)

I'm not 100% sure about the C:N that is best for cubensis, as far as I know 20:1 promotes fruiting, higher promotes colonization.
Since your using agar you probably want around 40:1 - 60:1 for a good speed. 100% grain is about 15:1 - 20:1.

2% nitrogen content total, 70% carbohydrates (44.45% carbon) = 31.115% carbon.

31:2 (31 /2 and 2 /2) = 15.55:1


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Growing mushrooms, general guide and information (Ferather's Journal), https://ibb.co/rG3rML2

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Invisiblepacmanbreed
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24618596 - 09/10/17 03:31 PM (6 years, 5 months ago)

Thanks for the guidance brother.
This will save me some precious time and efforts.
Since im goin to probio route & been composting for a year now.

Been drinking my coffee lately. :takingnotes:
Will limit it upon reading hydrate of carbons(carbohydate) glycogen etc.

Quote:

Ferather said:
2% nitrogen content total, 70% carbohydrates (44.45% carbon) = 31.115% carbon
31:2 (31 /2 and 2 /2) = 15.55:1



-(44.45% carbon) = average weight of C in most grain?

If so based on nutritional labels like this per 100g

-1.89% nitrogen content total, 72.8% carbohydrates (44.45% C weight) = 32.359% carbon
32.359:1.89(32.359/1.89 & 1.89/1.89) = 17.12:1

This is nice.
Im thinking of using the some of the sugar for probio ferment for my family to balance it to 30:1 before innoc.

Sorry for the bother and slightly being off topic.

Edited by pacmanbreed (09/11/17 08:18 AM)

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Invisiblepacmanbreed
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Re: Cleaning dirty cultures with tea agar [Re: pacmanbreed]
    #24622503 - 09/11/17 11:37 PM (6 years, 5 months ago)

Update:
11 days since innoc of 2 sacrificial plate outside SAb.
The one finally have contam germinated i can see sectoring at 11oclock.
The other one is almost fully colonized without visible contam.

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Offlinetombosley8
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Re: Cleaning dirty cultures with tea agar [Re: pacmanbreed]
    #24622510 - 09/11/17 11:40 PM (6 years, 5 months ago)

why so much contam?


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Invisiblepacmanbreed
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Re: Cleaning dirty cultures with tea agar [Re: tombosley8]
    #24623976 - 09/12/17 04:43 PM (6 years, 5 months ago)

Quote:

tombosley8 said:
why so much contam?




They are sacrificial plates which are periodically opened in high spore load room to test the tea tannins effect on spore germination.

11 days sporulation is not quite bad.
I usually get contam sporulation around 3-4 days in dFA

Edited by pacmanbreed (09/12/17 04:44 PM)

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OfflineBigbadwooof
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Re: Cleaning dirty cultures with tea agar [Re: pacmanbreed]
    #24625341 - 09/13/17 05:57 AM (6 years, 5 months ago)

Thanks for the informative post. You ought to post around here more often! :smile:


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Re: Cleaning dirty cultures with tea agar [Re: Bigbadwooof] * 1
    #24629978 - 09/14/17 07:22 PM (6 years, 5 months ago)

Yes it can happen, the green machine is a wood lover, and you are using its natural germination carbon :wink: .
That is a good test, 11 days of exposed situations, not bad. My mold test interestingly stalled.

The oyster mycelium overgrew it eventually, but that is wood lover vs wood lover.
Also the growth on the essentially protein agar seems usable, interesting.

Also your conversion of millet looks correct to me.

Composition of Gelatin (change to 100g).


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Edited by Ferather (09/14/17 07:42 PM)

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Re: Cleaning dirty cultures with tea agar [Re: Ferather] * 1
    #24630206 - 09/14/17 08:57 PM (6 years, 5 months ago)

Brown rice: 76% Carbs (inc fibre), 33.782% Carbon. 1.513 Nitrogen (usable).

33.782 / 1.513 = 22.32:1, Quite optimal for fruiting, faster in ratio, do 50/50 dry to dry cellulose-other to achieve: 44.64:1 (faster).

Starch and cellulose (plant fibre) are composed of the same components (sugars).


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