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Offlinesomanytims
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Is this mycelium on my agar?
    #24918359 - 01/16/18 03:53 PM (3 years, 3 months ago)

First attempt at agar plates..

From a little more research, I now realize that I completely misunderstood how I should streak the plates - so they're not great (will be rectified when my next load of plates arrive!).

However, I do have some form of growth and I'm interested as to whether or not it is in fact mycelium growth or something else.

Either way - it's not looking very strong (which could be down to my agar recipe or the temperature in my basement!), but if it is mycelium - then I'll start trying agar to agar transfers on the next run rather than dipping back into the spawn syringe.





Also.. those thick yellow lines.. is that actually some form of infection?  It seems to closely follow the lines that I used when performing my initial streaks and had thought it was just the agar being agitated in some way.. but the way it's bubbled up now - I'm not sure?


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Invisiblehamloaf
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Re: Is this mycelium on my agar? [Re: somanytims]
    #24918370 - 01/16/18 03:56 PM (3 years, 3 months ago)

The white circles look like mushroom myce.
The greasy yellow lines are bacteria.


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Invisiblementat
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Re: Is this mycelium on my agar? [Re: somanytims]
    #24918377 - 01/16/18 04:00 PM (3 years, 3 months ago)

the white discs looks like a decent wake up, and not really surrounded by the bacteria.

With proper sterile procedures you could transfer to new plates.


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Offlinesomanytims
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Re: Is this mycelium on my agar? [Re: mentat]
    #24918427 - 01/16/18 04:18 PM (3 years, 3 months ago)

That's a crazy amount of bacteria.. it basically follows the path of my inoculation loop.

I soaked in 70% alcohol, flamed in alcohol burner.. then into the SAB where it was used straight away.

My inoculation loop technique is rubbish at the moment - managed to dig a few holes in the agar as I went along - but still.. to get bacteria the whole way across it.. something must be going wrong?

Oh - the other vector.. i'm using a small shot glass to dip the loop into.

The shot glass gets wiped with 70% alcohol before being placed in the SAB.

Syringe gets heated to glowing over alcohol burner, quick squirt to cool it down.. then a couple of ml squirted into the shot glass inside the SAB.

To pick up the liquid I'm just dipping the loop into the shot glass and swirling it around a bit.

I suppose theres one final vector.. The syringes are from ebay so could be just full of contamination?


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Re: Is this mycelium on my agar? [Re: somanytims]
    #24918447 - 01/16/18 04:27 PM (3 years, 3 months ago)

if the spores are not older than a couple of years, they wake even if i sprinkle them right off the foil onto agar.

if i use a vial or syringe, i drop 3 tiny droplets onto agar right out of the syringe, but not swipe.

this works for me, minimizing my chances of contamination.

but you are good, most of the edges of those mycelial growth seems clean. just transfer them carefully and it will reward you for it


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Offlinesomanytims
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Re: Is this mycelium on my agar? [Re: mentat]
    #24918515 - 01/16/18 05:02 PM (3 years, 3 months ago)

Cool! :smile:

Will attempt a transfer tomorrow and see how they get on.

How do you prevent the drops from moving all over the plate?

I tried one plate with a drop and it ended up running all over the place.


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OfflineIcezizim
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Re: Is this mycelium on my agar? [Re: somanytims]
    #24918556 - 01/16/18 05:16 PM (3 years, 3 months ago)

Quote:

somanytims said:
Cool! :smile:

Will attempt a transfer tomorrow and see how they get on.

How do you prevent the drops from moving all over the plate?

I tried one plate with a drop and it ended up running all over the place.




Inoculation loop is probably the best way, so you can transfer from contams pretty easy. Although you could probably use the tip of the syringe to make a little dimple in the agar and try to set a single drop of solution in that.


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Offlinemushpunx
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Re: Is this mycelium on my agar? [Re: Icezizim]
    #24918575 - 01/16/18 05:22 PM (3 years, 3 months ago)

"The shot glass gets wiped with 70% alcohol before being placed in the SAB.

Syringe gets heated to glowing over alcohol burner, quick squirt to cool it down.. then a couple of ml squirted into the shot glass inside the SAB."

This is your problem right here. Alcohol does not sterilize, it only sanitizes. You would have to wrap that shot glass in foil and run it thru a PC cycle to sterilize it. The glass was not sterile.

But the glass is totally unnecessary anyways.

To use spore solution on agar, sterilize the needle and place a drop of solution on the plate.
Then sterilize your inoculation loop and swipe the spores.


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Offlinesomanytims
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Re: Is this mycelium on my agar? [Re: mushpunx]
    #24918585 - 01/16/18 05:26 PM (3 years, 3 months ago)

Quote:

mushpunx said:
This is your problem right here. Alcohol does not sterilize, it only sanitizes. You would have to wrap that shot glass in foil and run it thru a PC cycle to sterilize it. The glass was not sterile.




Thanks! Makes perfect sense.

Will incorporate it into my next batch.


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Invisiblementat
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Re: Is this mycelium on my agar? [Re: somanytims]
    #24919778 - 01/17/18 03:28 AM (3 years, 3 months ago)

Quote:

somanytims said:

How do you prevent the drops from moving all over the plate?

I tried one plate with a drop and it ended up running all over the place.




i just don't tilt or disturb the plate. lift carefully and put it in the place where it could do the job. i do not preserve spore suspention plates upside down for that reason.

and yes.. alcohol is the problem indeed.
i made once a vial for myself, from an old spore suspension vial. i washed it, put peroxide in it over night, then poured and dried in front of the flowhood and sterilized with flame inside. worked with sterile water and it worked, when i tested it did not get infected on agar.


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Offlinemushpunx
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Re: Is this mycelium on my agar? [Re: somanytims]
    #24919895 - 01/17/18 06:09 AM (3 years, 3 months ago)

Quote:

somanytims said:
Cool! :smile:

Will attempt a transfer tomorrow and see how they get on.

How do you prevent the drops from moving all over the plate?

I tried one plate with a drop and it ended up running all over the place.





By placing a drop of spore solution on the plate, and then streaking it in a zig zag line across the plate with an inoculation loop.


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