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OfflineBlueDisa
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Bacteria inhibiting spore germination
    #24329582 - 05/18/17 12:20 PM (4 months, 4 days ago)

Good Day Shroomers

I have some prints of different species that I am trying to germinate.

Some of the prints are contaminated with bacteria.

When I put spores on agar to germinate, the bacteria germinates first and consequently my spores do not germinate (as the bacteria grows all over the spores) so I do not even have a chance to isolate some healthy mycelium. How do I get these infected spore prints to germinate and produce some mycelium? I have read about antibiotic agar, but I live rural and won't be able to get any of the mycologically useful antibiotics any time soon.

I have got some Wine Caps that managed to germinate right through the bacterial contamination, I did an agar sandwich with them and got some clean mycelium.

Are there any teks I could follow to help with this problem.


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InvisiblebodhisattaM
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Re: Bacteria inhibiting spore germination [Re: BlueDisa]
    #24329596 - 05/18/17 12:24 PM (4 months, 4 days ago)

How are you putting the spores on the agar?

I suggest if from a spore print to take a loop to remove some spores from the print then take a small amount of sterilized water in a shot glass and dip the loop in. This dilutes the concentration or spores down and also bacteria. Then dip a new sterile loop into the water/spore solution then make streak plates.

Streaking out spores rather than blobbing a drop in the middle of a plate will greatly increase your chance of success


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Invisible36fuckin5
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Re: Bacteria inhibiting spore germination [Re: bodhisatta]
    #24329712 - 05/18/17 01:04 PM (4 months, 3 days ago)

Quote:

bodhisatta said:
How are you putting the spores on the agar?

I suggest if from a spore print to take a loop to remove some spores from the print then take a small amount of sterilized water in a shot glass and dip the loop in. This dilutes the concentration or spores down and also bacteria. Then dip a new sterile loop into the water/spore solution then make streak plates.

Streaking out spores rather than blobbing a drop in the middle of a plate will greatly increase your chance of success




Exactly what I was gonna suggest. With clean prints I don't bother with this, but if it's an outdoor print or something just not grown in the cleanest place then streaking is the way to go.


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OfflineBlueDisa
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Re: Bacteria inhibiting spore germination [Re: 36fuckin5]
    #24331716 - 05/19/17 02:37 AM (4 months, 3 days ago)

I have just been scraping spores off prints with a scalpel blade and placed it all in one spot on the dish , so that is probably my mistake.

Thank you for the advice, I will streak some plates and update with results. :smile:


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OfflineAlan RockefellerM
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Re: Bacteria inhibiting spore germination [Re: BlueDisa]
    #24332040 - 05/19/17 06:22 AM (4 months, 3 days ago)

You can mail order antibiotics for your agar, here are the ones I use:

http://www.the-odin.com/ampicillin-sodium/

http://www.the-odin.com/kanamycin-monosulfate/

I did a test yesterday where I tried adding them before and after pressure cooking, and then put 3 species of bacteria on the dishes.  Will know in a couple days if you can autoclave them or if you need to add them afterwards.  I suspect that you can autoclave them.


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Offlinegillaputty
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Re: Bacteria inhibiting spore germination [Re: Alan Rockefeller]
    #24333168 - 05/19/17 04:15 PM (4 months, 2 days ago)


Use dasani to reintake the minerAL intake of the gullet of the Mushroom veils.


Edited by gillaputty (05/19/17 05:50 PM)


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InvisiblebodhisattaM
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Re: Bacteria inhibiting spore germination [Re: gillaputty] * 6
    #24333208 - 05/19/17 04:32 PM (4 months, 2 days ago)

Quote:

gillaputty said:
Try excluding potatoes flakes in your agar.

Use dasani to reintake the minerAL intake of the gullet of the Mushroom veils.



:smbfacepalm:


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Re: Bacteria inhibiting spore germination [Re: bodhisatta]
    #24333757 - 05/19/17 08:20 PM (4 months, 2 days ago)

thanks Alan! let us know how that turns out, I've never worked with those antibiotics

I have only used gentamicin and chloramphenicol, and only VERY rarely, since well-timed transfers and streaking techniques usually make it pretty simple to separate from bacteria

I've only come across two prints that were so dirty they required antibiotics  (due to the bacteria suppressing it). also, sometimes I use them as a precaution on the final plate before making master slants


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Offlinegillaputty
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Re: Bacteria inhibiting spore germination [Re: c10h12n2o]
    #24333773 - 05/19/17 08:26 PM (4 months, 2 days ago)

How about it mixed.

Try 5 ml solution.
It exists to he six ml currently in my make-up.

Change the colingal passage way up and reuptake nakephederyn.


Edited by gillaputty (05/19/17 08:27 PM)


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Offlinegillaputty
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Re: Bacteria inhibiting spore germination [Re: gillaputty]
    #24334142 - 05/19/17 10:36 PM (4 months, 2 days ago)

:3stooges:


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Invisible36fuckin5
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Re: Bacteria inhibiting spore germination [Re: gillaputty]
    #24339675 - 05/22/17 05:56 AM (4 months, 6 hours ago)

Quote:

gillaputty said:

Use dasani to reintake the minerAL intake of the gullet of the Mushroom veils.




Are these even words? WTF are you talking about?


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OfflineLennybernadino
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Re: Bacteria inhibiting spore germination [Re: Alan Rockefeller]
    #24341151 - 05/22/17 07:51 PM (3 months, 30 days ago)

Any luck with autoclaving your two trial antibiotic media?


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OfflineAlan RockefellerM
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Re: Bacteria inhibiting spore germination [Re: Lennybernadino]
    #24386992 - 06/08/17 03:30 AM (3 months, 14 days ago)

Quote:

Lennybernadino said:
Any luck with autoclaving your two trial antibiotic media?





The antibiotics still worked after being autoclaved.

I was using Ampicillin and Kanamycin.    I am going to start adding chlorampheticol too - bacteria that are resistant to the ampicillin type antibiotics are pretty common in nature, far fewer are resistant to chlorampheticol, and you don't need to add very much.


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Invisiblec10h12n2oS
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Re: Bacteria inhibiting spore germination [Re: Alan Rockefeller]
    #24387020 - 06/08/17 04:02 AM (3 months, 14 days ago)

great info as always, thanks alan!

i use gent + chloramph in combination when i use antibiotics (which isnt very often), really handy for knocking out hitchhiking bacteria or on the last plate before making slants


--------------------
"Elections are futures markets in stolen property."
-- H. L. Mencken

“And those who were seen dancing were thought to be insane by those who could not hear the music.”

"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"

― Friedrich Nietzsche


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OfflineChwyn
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Re: Bacteria inhibiting spore germination [Re: c10h12n2o]
    #24387750 - 06/08/17 12:12 PM (3 months, 14 days ago)

A user named ferather showed me this the other day. this is an old thread but id like to add.

Antibacterial agar: https://www.shroomery.org/forums/showflat.php/Number/23771470

although semi-experimental it does work.
It also works with gelatin + agar + lime + ME mixes.


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