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Offlinekatbusa
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Antibacterial Tea Agar * 4
    #23771470 - 10/25/16 08:57 PM (7 years, 3 months ago)

I just recently completed cleaning a culture of Tarragon Oyster that ferather sent me. One of the methods I used to beat the bacterial infection that accompanied the mycelium was to make tea water agar plates. The reason behind this is because I do not like using antibiotics unless it is absolutley necessary. I guess I get this little tick from working in the wholesale plant business since a young age. There is nothing worse than having to deal with a pest that has a resistance to your pesticide or antibiotic. So I always look for natural, cleaner, safer methods before I go full nuclear.

So with that being said. Tea is not a one shot kills all solution. It is however affective against gram-positive and gram-negative bacteria but there are some bacteria that are resistant.

Here is a good research paper published on the web for our reading:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2763290/

Also check out ferathers Pocket Guide. He is doing some really good work with tea, carbon sources, starch free substrates,and more. He knows his shit and is worth a read!
https://www.shroomery.org/forums/showflat.php/Number/23725598/page/1

Here is my thread where I used tea agar to successfully clean a Tarragon oyster culture.
https://www.shroomery.org/forums/showflat.php/Number/23680797/page/1


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

So lets get onto the meat and potatos. The original post in this thread, is in essence going to be a living document. As I learn, muck up and break things I will post them and my thoughts. I also highly recommend people who are trying out tea to post what they observe and learn as well.

My original recipe is:

25 grams black tea
5 grams malt extract
9 grams agar
500ml water

Prepare it as you would for normal nutrient agar and PC for 25-30min. I used the sandwich method to clean the culture. The agar ends up being a nice dark brown color. Makes seeing mycelium a breeze yet you can still shine light through it.


The original recipe was intentionally heavy on the amount of tea used. I did this to find a known baseline for what worked. From here I plan on lowering the concentration till I find the bottom threshold where the effects of tea are minimal on bacteria. Then from there to find an optimal amount to be used.

Some things I noticed:

*At the 25gram per 500ml concentration the mycelium from a variety of different mushrooms expanded across the agar much slower than the normal half concentration malt extract agar that is used to clean mycelium. I believe this is due to the extra nutrients provided by the tea which I did not take into account.

*The agar seemed to be a bit softer than normal. I believe this could be due to a possible lower PH shift. I have had something similar happen when doing plant tissue culture. I need to verify this in future experiments.

*I have no idea if this is even tea related but I noticed that in two of my Oyster cultures the mycelium switched over to Rhizomorphic growth. I will keep an eye on future cultures to see if I can trend data to support this.

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

I had to delete one of my previous experiments due to being out of town and I wanted to reign in the preparation of the tea agar to be more consistent and controlled across the board. I will be using only pure green tea. No flavors or additives.

The Future preparation method will be

- Add desired amount of tea to 500ml simmering water. Let simmer for 5min
- Strain tea from the water and check water amount. Add more water if needed
    to bring back up to 500ml
- Check PH and adjust if desired or if needed for experiment
- Reheat water to simmering point
- Add 10 grams malt extract, stir to dissolve
- Add 9 grams agar, stir to dissolve
- Autoclave in pressure cooker for 25min
 
              **Done under Laminar Flow Hood**
- Let cool so agar container can be handle without burning ones self
- Gently stir solution to redistribute agar
- Add 20ml of tea agar solution to each  100mm plate
- Let agar plates cool and set to full strength under flow hood
- Use as needed

Each plate I will be using will have an X and Y axis written on the lid. The X will represent horizontal growth and the Y will represent the vertical growth. This will be used to measure the amount of growth across the surface of the agar.

I will also be keeping track of the temperature at the time of measurement and the speed of bacterial and mycelial growth over time. Plus any observations I might have.

The planned Tea concentrations are as follows:

25 grams per 500ml agar solution
20 grams per 500ml agar solution
15 grams per 500ml agar solution
10 grams per 500ml agar solution
5  grams per 500ml agar solution

If I get to the 5 grams concentration and there is still active antibacterial properties I will then step down 1 gram at a time till I see active bacterial growth. Then I will increase the concentration by .5 grams till I see antibacterial characteristics again. Hopefully this will be the threshold of tea and its antibacterial properties.

If at any point in time I notice the antibacterial properties stop before I reach the 5 gram concentration I will treat future experiments in the same way as if I reached the 5 gram concentration to find the threshold of tea's antibacterial properties.

Once the working threshold of tea is established the recipe for the TEK will be updated and future experiments pertaining to mycelium growth speed on tea agar will be conducted. Hopefully a happy medium will be established of good antibacterial properties that will not stunt the growth of mycelium.

I also eventually plan on putting all of the info from these experiments into a format that is easy to read.

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

25 gram per 500ml experiment:

Preparation:

- 25 grams of  green tea to 500ml simmering water. Let simmer for 5min
- Strain tea from the water and check water amount. Add more water if needed
    to bring back up to 500ml
- Did not check PH
- Reheat water to simmering point
- Add 10 grams malt extract, stir to dissolve
- Add 9 grams agar, stir to dissolve
- Autoclave in pressure cooker for 25min
 
              **Done under Laminar Flow Hood**
- Let cool so agar container can be handle without burning ones self
- Gently stir solution to redistribute agar
- Add 20ml of tea agar solution to each  100mm plate
- Let agar plates cool and set to full strength under flow hood

Samples:

-Two samples will be used.
  "Sample 1" -  A known active bacteria colony from a contaminated plate that
      is mycelium free. A smudge from the contamination will be made. The
      contaminated agar wedge will be placed next to the smudge.
 
  "Sample 2" - From known bacteria contaminated straw spawn that will have
      acitve mycelium growing along with the bacteria. 

-Four agar plates were prepared. Two plates of MEA nurtient agar with the 25 gram tea concentration and two plates of the MEA nutrient agar without tea.

11/27/2016:
     
    Sample 1 - Samples were established on tea and tea free MEA agar.
           
          Tea:
              - Measurments: X- 8.82mm  Y- 12.03mm
              - Temp: 70 Deg F

          No Tea:
              - Measurements: X- 8.10mm Y- 13.02mm
              - Temp: 70 Deg F

   
      Sample 2 -
       
          Tea:
              - Measurements: X- 22.72mm  Y- 17.70mm
              - Temp: 70 Deg F

          No Tea:
              - Measurements: X- 18.59mm Y- 19.54mm
              - Temp: 70 Deg F

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

11/30/2016:
     
    Sample 1
           
          Tea:
              - No reorganization or no new growth noticed with the bacteria
              - Measurments: X- 8.82mm  Y- 12.03mm
              - Temp: 71 Deg F
             

          No Tea:
              - Noticed the bacterial colony has reorganized and has also
                spread.
              - Measurements: X- 13.15mm Y- 19.02mm
              - Temp: 71 Deg F
             

   
      Sample 2 -
       
          Tea:
              - No new bacterial growth noticed. The straw has begun to
                slightly fuzz up from the mushroom mycelium
              - Measurements: X- 22.72mm  Y- 17.70mm
              - Temp: 71 Deg F
             
         
        No Tea:
              - The straw has begun to fuzz up from the mushroom mycelium. A
                very small string colony of bacteria has been noticed. Also
                a very small circular colony of bacteria has been noticed.
                However very hard to take a photo of.
              - Measurements:
                        String Colony: X- .47mm Y- 1.42mm
                        Circular Colony: X- 1.05mm Y- 1.03mm
              - Temp: 71 Deg F



     
         
     

   
 









Edited by katbusa (11/30/16 02:58 PM)


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OfflineQuadman
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Re: Antibacterial Tea Agar [Re: katbusa]
    #23771507 - 10/25/16 09:09 PM (7 years, 3 months ago)

Quote:



*At the 25gram per 500ml concentration the mycelium from a variety of different mushrooms expanded across the agar much slower than the normal half concentration malt extract agar. I believe this is due to the extra nutrients provided by the tea which I did not take into account.




I'm confused, did you mean to say faster?


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Offlinekatbusa
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Re: Antibacterial Tea Agar [Re: Quadman]
    #23771548 - 10/25/16 09:24 PM (7 years, 3 months ago)

No sir. The mycelium was slow to spread on the original formulation. When cleaning mycelium I use half or a little less than half of the called for nutrients. The reason for this is that the mycelium expands faster and has a chance to out run molds so you can get a small clean sample from the leading edge.

Now if you have a clean culture then I guess the speed at how it expands wouldn`t matter as much.


Edited by katbusa (10/25/16 09:25 PM)


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OfflineQuadman
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Re: Antibacterial Tea Agar [Re: katbusa]
    #23771634 - 10/25/16 10:05 PM (7 years, 3 months ago)

Ok , wanted to make sure I understood. Thanks


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


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OfflineFerather
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Re: Antibacterial Tea Agar [Re: katbusa]
    #23772284 - 10/26/16 04:06 AM (7 years, 3 months ago)

Very nice writeup, looking forward to more results and tests. Starting a few tests myself.
The extra nutrition part is true, 25g tea is like 60g starch free grain flour.


Quote:

katbusa said:
My one plate of Summer Oyster actually over grew the bacteria while in the fridge. Strong strain!



Summer oyster is more aggressive than Pink oyster, plus it generates a higher yield.


Edited by Ferather (10/26/16 06:42 AM)


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Offlineadadada
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Re: Antibacterial Tea Agar [Re: Ferather]
    #23772375 - 10/26/16 05:39 AM (7 years, 3 months ago)

What is this sandwich method that you speak of?  I searched through the forum and didn't find anything about it.

Quote:

I used the sandwich method to clean the culture.




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Offlinefilyep
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Re: Antibacterial Tea Agar [Re: adadada]
    #23772382 - 10/26/16 05:46 AM (7 years, 3 months ago)

I believe it's where you sandwich the transferred agar wedge between two antibacterial agar wedges. One being the receiving dish of agar and the other a wedge that you cut from another dish of antibacterial agar to cover the contaminated wedge. Hope that makes sense.

Or you do your transfer and make yourself a sandwich.  :smile:


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Offlinefilyep
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Re: Antibacterial Tea Agar [Re: filyep]
    #23772384 - 10/26/16 05:48 AM (7 years, 3 months ago)



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Offlineadadada
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Re: Antibacterial Tea Agar [Re: filyep]
    #23772394 - 10/26/16 05:57 AM (7 years, 3 months ago)

Thank you!  Very interesting approach and a handy tool for cleaning cultures!

Now if only there was a similar method for separating out trich :smile:


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OfflineFerather
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Re: Antibacterial Tea Agar [Re: adadada]
    #23772429 - 10/26/16 06:44 AM (7 years, 3 months ago)

Quote:

adadada said:
Thank you!  Very interesting approach and a handy tool for cleaning cultures!

Now if only there was a similar method for separating out trich :smile:



Cant see why we cant try that after, just a case of energy source.
Something trich hates and your sample likes.


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

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

https://www.shroomery.org/forums/showflat.php/Number/27857366#27857366

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OfflineFerather
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Re: Antibacterial Tea Agar [Re: Ferather]
    #23772860 - 10/26/16 10:12 AM (7 years, 3 months ago)

"Cinnamon verum bark has the highest antimicrobial activity, particularly against antibiotic resistant strains." -- Source.

New recipe: 20g of raw black tea, 1 teaspoon of ground cinnamon.



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

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

https://www.shroomery.org/forums/showflat.php/Number/27857366#27857366

DTS DCH Driver for Realtek [DTS:X] - Unlocked and reprogrammed.


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Invisibleflyontoast
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Re: Antibacterial Tea Agar [Re: filyep] * 1
    #23773521 - 10/26/16 02:39 PM (7 years, 3 months ago)

Quote:

katbusa said:
25 grams black tea
5 grams malt extract
9 grams agar
500ml water




Does it have to be black tea?

Quote:

filyep said:
Or you do your transfer and make yourself a sandwich.  :smile:



Can I use a SAB or do you need a flow hood for this? :wink:


--------------------
:mushroom: My trade list :mushroom:
Looking for strong terrestrial fruiters for an outdoor beds experiment:
Agaricus Bitorquis, Agaricus Augustus, Agaricus blazei/subrufescens, Stropharia Rugoso-annulata, Clitocybe Nuda (blewits), and any species or other genus that you think work outdoors.
Also, any commercially viable Pleurotus, cold or hot strains.
Thanks for the Q&A, trades, and all the posters & teachers that have come before us  :skol:


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Offlinekatbusa
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Re: Antibacterial Tea Agar [Re: flyontoast]
    #23773564 - 10/26/16 02:58 PM (7 years, 3 months ago)

No it does not have to be black tea. It can be either black or green. Green tea however seems to have better antibacterial properties. I chose black tea because my store was out of raw unflavored green tea.

Just an FYI even if you use green tea. It will end up darker much like normal tea after the PC process.

You do not need a Laminar flow hood for this. A SAB will work just fine. Just don't forget to add the pickle!! LOL


Edited by katbusa (10/26/16 03:02 PM)


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Offlinekatbusa
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Re: Antibacterial Tea Agar [Re: Ferather]
    #23773567 - 10/26/16 03:00 PM (7 years, 3 months ago)

Quote:

Ferather said:
Quote:

adadada said:
Thank you!  Very interesting approach and a handy tool for cleaning cultures!

Now if only there was a similar method for separating out trich :smile:



Cant see why we cant try that after, just a case of energy source.
Something trich hates and your sample likes.




Mold has always been a royal pain in the ass. I still have to rely on taking alot of samples till I end up with one or two that give cleanish mycelium to work with. I wish there was something we could do about slowing mold down.


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Offlinekatbusa
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Re: Antibacterial Tea Agar [Re: Ferather]
    #23773589 - 10/26/16 03:06 PM (7 years, 3 months ago)

Quote:

Ferather said:
"Cinnamon verum bark has the highest antimicrobial activity, particularly against antibiotic resistant strains." -- Source.

New recipe: 20g of raw black tea, 1 teaspoon of ground cinnamon.







Cinnamon 0_0. Holy smokes let us know how this works. I use cinnamon often after planting seeds in trays to prevent damping off disease which is a type of fungus. You might be on to something.. I wonder how trich and good mycelium will react to this.


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OfflineQuadman
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Re: Antibacterial Tea Agar [Re: katbusa]
    #23773612 - 10/26/16 03:12 PM (7 years, 3 months ago)






Mold has always been a royal pain in the ass. I still have to rely on taking alot of samples till I end up with one or two that give cleanish mycelium to work with. I wish there was something we could do about slowing mold down.



:whathesaid: Feather you need to reverse engineer something for us:grin:


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OfflineQuadman
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Re: Antibacterial Tea Agar [Re: Quadman]
    #23773628 - 10/26/16 03:15 PM (7 years, 3 months ago)

I never heard of that Katbusa , always used Benlate . Wonder how mycelium would react to that ?


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Offlinekatbusa
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Re: Antibacterial Tea Agar [Re: Quadman]
    #23773660 - 10/26/16 03:23 PM (7 years, 3 months ago)

Quote:

Quadman said:





Mold has always been a royal pain in the ass. I still have to rely on taking alot of samples till I end up with one or two that give cleanish mycelium to work with. I wish there was something we could do about slowing mold down.



:whathesaid: Feather you need to reverse engineer something for us:grin:




If anyone can do it, Ferather would be the one. His cinnamon idea is brilliant and makes sense. I hope it works


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Offlinekatbusa
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Re: Antibacterial Tea Agar [Re: katbusa]
    #23773670 - 10/26/16 03:28 PM (7 years, 3 months ago)

I know benlate / benomyl is systemic. Isn't is also broadspectrum for fungi?

I would imagine it would toast good fungi.


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OfflineFerather
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Re: Antibacterial Tea Agar [Re: katbusa]
    #23773744 - 10/26/16 03:50 PM (7 years, 3 months ago)

As far as I see it cinnamon could be hit and miss like pine wood. Not all oyster's will eat pine, due to pine oils.
Cinnamon oil stops mold and bacteria for up to 60 days, so wee need to balance it, not too rich.

I don't mind slow as long as it works as intended, to clean samples.


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

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

https://www.shroomery.org/forums/showflat.php/Number/27857366#27857366

DTS DCH Driver for Realtek [DTS:X] - Unlocked and reprogrammed.


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