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Tissue cloning to agar always contaminate
    #25755391 -

Greetings fellow mushroom grower. I'm new and learning. Recently, I was trying to clone mushroom tissue. But always got contaminate. I'm using SAB. Before cloning, I will Wipe inside SAB with 70% alcohol, Wash hands with sanitizer(kill99%germ) handwash. Clean mushroom with Clorox bleach solution. Wipe hands with 70% alcohol. Flame scalpel red hot. Tear mushroom into half and take the fresh tissue within. Open Petri dishe, drop tissue, and close lid. After 1 or 2 days this is what I got. I've been repeating the same process for tens of times, I always have contaminated.
What do you think is the possible causes?
Is it due to the mushroom tissue, or my SAB, or my hands,  Is it the air not still enough(cause air to stir up contaminants), or all of the above? I'm getting a laminar flow hood built soon, do you think it will solve my problems?
Thanks again for all your help!


Edited by DoMyThing (01/20/19 06:39 AM)

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Re: Tissue cloning to agar always contaminate [Re: DoMyThing]
    #25755430 -

Sorry for your troubles but SAB is not guaranteed to work all the time. Create your flow hood, u can make one for very cheap, if uv done the same proses 10 time then stop and make your flow hood all you problems will be gone.

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Re: Tissue cloning to agar always contaminate [Re: Mr.spores]
    #25755469 -

I've done lots of clones in a SAB. I don't use bleach...for anything. Cleaning the outside of the fruit is unnecessary, warm soapy water is what you should use to spray inside of SAB not iso. Be sure to flame your scalpel red hot. Keep trying, SAB technique takes time to get good at. Lots of practice.

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Re: Tissue cloning to agar always contaminate [Re: Subfinder]
    #25755523 -

I've done lots of clones in a SAB. too...

problem may be with your agar preparation also.


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Re: Tissue cloning to agar always contaminate [Re: WarFlo]
    #25755865 -

i have recently purchased my flow hood.so far i was cloning with my SAB, and mind you its great for tissue culture, agar work etc. but found it a bit difficult to do grain especially if you are using bags not jars.

this is my first clone in my newly bought flow hood..




i am loving it..


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Re: Tissue cloning to agar always contaminate [Re: rams]
    #25756099 -

The contaminants started from the tissue you used. What type of agar are you using? What type of mushrooms are you cloning? Are they fresh? If you are cloning store bought mushrooms, try sourcing better specimens. They have to be fresh. And scalpel sterile. after you flame it, it needs to stay clean so don't touch anything but the clean (inside) part of the tissue.

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Re: Tissue cloning to agar always contaminate [Re: Mr.spores]
    #25756577 -

Mr.spores said:
Sorry for your troubles but SAB is not guaranteed to work all the time. Create your flow hood, u can make one for very cheap, if uv done the same proses 10 time then stop and make your flow hood all you problems will be gone.


Hi, thanks for the advice. Ive ordered my flow hood parts. Waiting for them to arrive. hopefully everything will be good once it's up and running.

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Re: Tissue cloning to agar always contaminate [Re: ScotTony]
    #25756624 -

ScotTony said:
The contaminants started from the tissue you used. What type of agar are you using? What type of mushrooms are you cloning? Are they fresh? If you are cloning store bought mushrooms, try sourcing better specimens. They have to be fresh. And scalpel sterile. after you flame it, it needs to stay clean so don't touch anything but the clean (inside) part of the tissue.


Hi, I totally agree with you about the contaminants started from the tissue. It's some milky slimy thing, not sure what it is called, it smells like sour. FYI, I've grown myself some grey oyster (p.pulmonarius). I'm using own grown mushroom to do it. So they are freshly picked. I washed the whole mushroom with bleach solution because I suspected when I hold and tear them the contaminants stick to my hands and causes contamination. I flame sterilize scalpel red hot every time and didn't let it touches anything.

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Re: Tissue cloning to agar always contaminate [Re: WarFlo]
    #25756647 -

WarFlo said:
I've done lots of clones in a SAB. too...

problem may be with your agar preparation also.


I use home made PDA prep with potato, dextrose, and store bought agar powder. Sterilize >121°C @ >15psi(cause the pressure is not fixed) for >15mins(some time 20mins).

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Re: Tissue cloning to agar always contaminate [Re: DoMyThing]
    #25756698 -

Wait :confused:
Where is this easy and cheap flowhood info?? An actual flowhood is redonkulously expensive..:teareally:

Even a parts list isn't easy to acquire..


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In Peace or Pieces we shall move forward.

Grimm

Edited by McGrimm (01/20/19 05:14 PM)

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Re: Tissue cloning to agar always contaminate [Re: DoMyThing]
    #25757164 -

DoMyThing said:
Hi, I totally agree with you about the contaminants started from the tissue. It's some milky slimy thing, not sure what it is called, it smells like sour. FYI, I've grown myself some grey oyster (p.pulmonarius). I'm using own grown mushroom to do it. So they are freshly picked. I washed the whole mushroom with bleach solution because I suspected when I hold and tear them the contaminants stick to my hands and causes contamination. I flame sterilize scalpel red hot every time and didn't let it touches anything.


Sounds like you are having issues with yeast contamination, and it sounds like your mushroom tissue is too weak (or dead) to overcome it. First of all drop the bleach. It soaks into the mushroom tissue and kills it. Faster then it kills the contaminants. Try not treating your mushroom at all. Second, tell us what species it is. Third, try switching to MEA agar. PDA does not come out with optimal pH in most cases that I have examined. Fourth, if you get contamination every time in a SAB you will get it in a flow hood too, you will just be poorer.


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Re: Tissue cloning to agar always contaminate [Re: McGrimm]
    #25757233 -

I made my well not really a flowhood more of just a hepa filter with air coming thrue but same thing. I spent as little as 200 euro and it works, agar and bags of grain not a problem.

Get a HEPA and carbon filter from amazon build a box out off wood find a high pressure fan of some sort  put it all together and ur done it's not rocket science. If u want more info happy to help

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Re: Tissue cloning to agar always contaminate [Re: DoMyThing]
    #25757766 -

DoMyThing said:
WarFlo said:
I've done lots of clones in a SAB. too...

problem may be with your agar preparation also.


I use home made PDA prep with potato, dextrose, and store bought agar powder. Sterilize >121°C @ >15psi(cause the pressure is not fixed) for >15mins(some time 20mins).


Then mb before you buy your 500$ laminar try uv lamp that one with ozone generation.


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Re: Tissue cloning to agar always contaminate [Re: WarFlo]
    #25761923 -

WarFlo said:
DoMyThing said:
WarFlo said:
I've done lots of clones in a SAB. too...

problem may be with your agar preparation also.


I use home made PDA prep with potato, dextrose, and store bought agar powder. Sterilize >121°C @ >15psi(cause the pressure is not fixed) for >15mins(some time 20mins).


Then mb before you buy your 500$ laminar try uv lamp that one with ozone generation.


May be you misunderstood me, I'm not going to buy a $500 laminar flow hood, I'm going to DIY one actually, with HEPA filter and centrifugal blower enclosed in wooden box. Just like Mr.Spores.

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Re: Tissue cloning to agar always contaminate [Re: DoMyThing]
    #25763919 -

My understanding is that SAB is preffered for clone work as the air can  blow contamination from the fruit to your agar. Tear from the top down and scrap a small sample from near the base with a flamed scaple and get it off your scaple without rubing it all over the plate.

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Re: Tissue cloning to agar always contaminate [Re: Bph]
    #25763989 -

The only way the hood would blow contam from the fruit to the plate would be if the fruit were between the filter face and the plate which is just poor technique. Never put anything between your work and the filter including your hands and scalpel.  I’ve always found it easier to tear from the bottom.  I take samples from the meatiest areas all the way from the base to the cap.  OPs contam is bacteria...happens to the best of us.


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Helpful Threads
stonesun
Mycolorado’s Tamp Fruit Project
Tampanensis Grow with Mycolorado
bw86's Jalisco Grow

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Re: Tissue cloning to agar always contaminate [Re: Mycolorado]
    #25763995 -

:whathesaid:


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Back from my Hiatus :smile:

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Re: Tissue cloning to agar always contaminate [Re: Jooceman]
    #25764768 -

@ OP:

Do you do other agar work in the SAB as well and do you have the same problems?

I use(d) a glovebox but am right now switching to a flowhood. Things I did differently:

- I used to spray in the box with alcohol but that seems like a bad idea now. IMO best to clean the box with bleach and let it dry a bit, try to keep it closed and/or limit air draughts of course.
- What I do/did spray with tho is just boiled water, in order to precipitate particles floating in the air. Then just wait a little for everything to land.
- You can use a few subsequent washes of your cloning tissue in PC'd water.
- But probably you should just practice your routines and improve your techniques since it sounds like you are just accidentally cross-contaminating at some point during excision of the clone tissue... although the satellite contams on the plate do also suggest more general contamination of your agar as well.

Make sure when you take an inner piece of tissue out that your scalpel is well sterilized and that you don't touch anything with the scalpel or tissue and also don't allow any potentially unclean objects to hang above open agar plates nor your cut piece of tissue.
Pay close attention to how you position your fingers and tools etc during this whole process. Mycolorado said it well.

The clone tissue turning deliquiescent (and I guess putrefying from the bacteria?) happened to me before plenty but especially with hard-to-clone species without nice meaty thick pieces.


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