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Offlinepacmanbreed
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Re: Cleaning dirty cultures with tea agar [Re: Ferather] * 1
    #24638782 - 09/17/17 05:21 PM (6 years, 4 months ago)

Quote:

Ferather said:
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).




The cubes also overgrew the green. I think its because of the ph of tea. (I feel that its Aspergillus from DF/corn vs trichoderma based on looks since i also keep contaminated plates for reference)
Interestingly my bacterial problem minimized and i can see sectoring clearly while the sectors grew flat on agar..
From this:

To this:


Good info, The gelatin has plenty of aminos and N for them. I think fast-carbon/sugar is what i lack from this recipe to give them a boost for transfers.
(B.) 50g Water, 50g Extract, 2.5g Agar, 0.2g Gelatin,
0.50g Dogfood.
Ill omit dogfood/starch and replace it with sugar next round.
I guess im getting the contam on the df high population load.
My bad last cooked plate didnt solidify, i forgot to balance the ph by cao3..

I think simpe tea+cao3+yeastnutrient+sugar is a good combo for macro+micro..


Edited by pacmanbreed (09/17/17 05:35 PM)


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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: pacmanbreed]
    #24641101 - 09/18/17 02:59 PM (6 years, 4 months ago)

Thank you, I am currently trying another run from peg, using 2g of gelatin, since it also counts as a carbon source.
The only issue is the mixture needs more agar than normal, the media set very soft, its more like jelly.

The good news is, it does not liquefy when being digested, and appears to become harder.

I am tempted to rip open the tea bag and add directly to the agar powder.


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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24641176 - 09/18/17 03:27 PM (6 years, 4 months ago)

On a separate note, the cellulose agar alternative can produce some very nice genetics, essentially isolating growth on end media.
For a quick recipe, where used like agar for body, 25g dry paper pellets and 2-3g malt extract (or other agar additives).

You can cut down on the water content, as agar is around 95% water, ideal for attracting unwelcome germinating.
For example, 50% water, that's about 25g water added, 66% = 50g added, 75% = 75g added, and so on.

Use the weight system, as ratios of both water and nutrients come in grams, not volume.


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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24643076 - 09/19/17 05:48 AM (6 years, 4 months ago)

Forgot to mention, I am using CaCO3 (Calcium carbonate) with my agar, end result is pH 6.5, roughly.
I only got one spot of mold after 8 days, which stalled, no bacteria or yeast whatsoever.

I ran a test with CaCO3 as the only carbon source, with added Ma-Mi nutrients.
Its about 25% the strength of sugar per gram as a carbon source.

   


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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24643107 - 09/19/17 06:15 AM (6 years, 4 months ago)

Moving back to plain WL-Tek (enriched, no added carbon), you can also use tissue samples.
The mycelium will use stored energy as it's starter carbon, it takes about 3-5 days.

I have also done, with added tea, and wild tissue (Image 2).

   



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Edited by Ferather (09/19/17 06:40 AM)


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Offlinepacmanbreed
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24643282 - 09/19/17 08:12 AM (6 years, 4 months ago)

Thanks brother.
Quote:

Ferather said:
On a separate note, the cellulose agar alternative can produce some very nice genetics, essentially isolating growth on end media.
For a quick recipe, where used like agar for body, 25g dry paper pellets and 2-3g malt extract (or other agar additives).




Nice just like cattitives.

Quote:

Ferather said:
You can cut down on the water content, as agar is around 95% water




Confirm it. I guess this is for pure paper pellets water holding capacity = 95%?
Eg. 25g pellet + 25g water = 50% water?

Sorry for bumping again brother.
Would love to try this as soon as  got home.
Would try pure cellulose or pupal casing.


Edited by pacmanbreed (09/19/17 08:21 AM)


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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: pacmanbreed] * 1
    #24643980 - 09/19/17 02:14 PM (6 years, 4 months ago)

No I mean a normal agar recipe will be around 95% water content as an end media.
With the pellets you are able to select your content from a given range.

For example: 2g ME + 2g Agar is 4g dry + 100g water (96%).
Pellets you can do 50% water, 66%, 75% so on.

----

Content range:

x 1 Dry Substrate | 1 Part(s) | 50% Water.
x 2 Dry Substrate | 2 Part(s) | 66% Water.
x 3 Dry Substrate | 3 Part(s) | 75% Water.
x 4 Dry Substrate | 4 Part(s) | 80% Water.


Usually I would remove the water present in pellets, but this can be ignored @ 5g per 100g.

So let me convert that into a working range:

A) 25g dry, x1 = 25g water, 50%. 1/2 dry, 1/2 water.
B) 25g dry, x2 = 50g water, 66%. 1/3 dry, 2/3 water.
C) 25g dry, x3 = 75g water, 75%. 1/4 dry, 3/4 water.

A) 25g + 25g, 50g total weight.
B) 25g + 50g, 75g total weight.
C) 25g + 75g, 100g total weight.


Tip: If you weigh your containers-setup, you can measure after cook content.


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Edited by Ferather (09/19/17 02:48 PM)


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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24651141 - 09/22/17 08:01 AM (6 years, 4 months ago)

The 2g 240B gelatin T-Gel is working on Tarragon oyster, I'm using an 8 month old peg transfer, it regenerated in 3 days.

So far no contamination, here's the interesting part, there's no sugar so no I'm not pressure cooking.
I am also essentially assembling the entire media open air, however covered for cooling.

So far all attempts have produced usable mycelium, and in one case stalled mold.
As mentioned the Tarragon mycelium quickly devoured the stalled mold.

While I don't suggest what I did, it does show the usability.


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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24651153 - 09/22/17 08:08 AM (6 years, 4 months ago)

Additional recipe:

> Extract: 3g (1 bag) Black tea + 125g CaCO3 water, check the end pH.

> Recipe (C): 100g Extract, 3-4g Agar, 2g 240B Gelatin.

----

Additional notes:

Black tea, is normal green tea that has been oxidized, when tea oxidizes it turns very black.
When mycelium oxidizes tea, it also turn's it black, and you will see a dark ring.

Black tea is oxidized and easier to decay, as if done by laccase.
The good news is decay can occur without laccase.

Decay will also occur above pH 6.5.

----

Inhibitory materials present in plants are acidic, hence the fact most plants are acidic.
If these materials where not acidic or tolerant, they would decay (useless).

Acidic materials will happily breakdown via pH into carbon-other.

----

In short there are other natural methods of decay.

---

Side note: Cubensis is primary to cellulose, and requires many Ma-Mi nutrients.
In nature cubensis likes the nitrogen present in dung (quick-easy).

No laccase means it will have a tough time on some materials.
Especially if that material is void of fast carbon.

----

Breaking down a viable carbon source via pH using another viable carbon source = clever!


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Edited by Ferather (09/23/17 09:26 AM)


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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24661404 - 09/26/17 12:34 PM (6 years, 4 months ago)

It seems 2g 240B gelatin is too much nitrogen, or there is not enough carbon in the tea extract.
Essentially the nitrogen ratio is far too high, growth is very good, but extremely slow.

I would therefore give the limit of around 0.8g 240B gelatin per 100g water.
If more is used, additional usable carbon will need to be added.

I'm going to try again, but using all of the tea, from dry.


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OfflineBigbadwooof
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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24661523 - 09/26/17 01:03 PM (6 years, 4 months ago)

Quote:

Ferather said:
It seems 2g 240B gelatin is too much nitrogen, or there is not enough carbon in the tea extract.
Essentially the nitrogen ratio is far too high, growth is very good, but extremely slow.

I would therefore give the limit of around 0.8g 240B gelatin per 100g water.
If more is used, additional usable carbon will need to be added.

I'm going to try again, but using all of the tea, from dry.




I have to ask, why are you guys so interested in using gelatin instead of agar?


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OfflineFerather
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Re: Cleaning dirty cultures with tea agar [Re: Bigbadwooof]
    #24661936 - 09/26/17 03:47 PM (6 years, 4 months ago)

It's not instead of agar, its being used as an additive along with the agar powder.
This is because mycelium will break it down for carbon and nitrogen.

Using just gelatin as the body, will turn to a liquid.


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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24666382 - 09/28/17 08:44 AM (6 years, 4 months ago)

Here is something I might try with T-Gel instead of calcium carbonate water, I can use generic indigestion tablets.
Cheap and ideal to use for agar, each tablet contains 500mg calcium carbonate, 524mg glucose.

The calcium carbonate will neutralize the pH (acidity) and add more carbon.

       


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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24678427 - 10/02/17 03:33 PM (6 years, 3 months ago)

Here is the 2g gelatin test, much slower due to high nitrogen ratio, as I mentioned.
Due to the slowness, the black laccase oxidization ring is much larger.

It has a sweet smell, I hope due to glycogen production?

     

     


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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24680833 - 10/03/17 12:10 PM (6 years, 3 months ago)

Quote:

Ferather said:
Here is the 2g gelatin test, much slower due to high nitrogen ratio, as I mentioned.
Due to the slowness, the black laccase oxidization ring is much larger.

It has a sweet smell, I hope due to glycogen production?

     

     




Oxidation ring? I haven't been following this thing from the beginning.


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Re: Cleaning dirty cultures with tea agar [Re: Bigbadwooof]
    #24681045 - 10/03/17 01:20 PM (6 years, 3 months ago)

Laccases (EC 1.10.3.2) are copper-containing oxidase enzymes found in many plants, fungi, and microorganisms.
Laccases act on phenols and similar molecules, performing one-electron oxidations, which remain poorly defined.

... Other laccases produced by the fungus Pleurotus ostreatus, play a role in the degradation of lignin ...

... Laccases require oxygen as a second substrate for their enzymatic action ...

Source.


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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24681055 - 10/03/17 01:22 PM (6 years, 3 months ago)

Black tea is a type of tea that is more oxidized than oolong, green and white teas.
Black tea is generally stronger in flavor than the less oxidized teas.

Source.


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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24704998 - 10/12/17 02:59 PM (6 years, 3 months ago)

Well the sweet smell as disappeared along with the oxidization ring, no signs of any contamination.
So in conclusion, the laccase and mycelium was converting materials into simple sugars.

I will add some wood pegs, and transfer some growth, takes 2-5 days.

Previous image, with laccase oxidization, and currently.

   


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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24707034 - 10/13/17 12:15 PM (6 years, 3 months ago)

Next test recipe, cellulose agar alternative:

----

pH 7.5-8, No sugar and no starch.

0.1g > YN, soluble nutrients.
1g > MG, soluble nutrients.
75g > Boiling hot water.
25g > Paper pellets.
2g > 240B gelatin.
2-4g > CaCO3.

About 16:1 carbon to nitrogen.

----

Inoculated with a Tarragon peg: 16/10/2017 | 20:42 | UK format.

----


Basic nutrients in paper pellets:

Potassium, Phosphorus, Magnesium, Chromium, Copper, Iron, Manganese, Silicon.

----

Soluble nutrients provided by Miracle-Gro and Yeast nutrient:

   

   

----

Gelatin nutritional composition

----

Carbon sources:

Cellulose, Hemicellulose.
Calcium carbonate.
Proteins.

----

17/10/2017 | 14:30 | UK format.

Regeneration after about 12 hours, some growth.

   


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Edited by Ferather (10/17/17 07:31 AM)


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Re: Cleaning dirty cultures with tea agar [Re: Ferather]
    #24717835 - 10/17/17 07:20 PM (6 years, 3 months ago)

I wonder if gentamycin+tea agar would slow growth too much.


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