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Offlinegone-pear-shaped
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Diluting spores in sterile water before plating?
    #27136514 - 01/09/21 12:16 AM (1 month, 26 days ago)

The first time I inoculated agar from a spore print using an inoculation loop, the spores were clumped together and many regions of the plate had no spores at all. I recall reading that a tiny amount of spores could be picked up from the print and mixed into a ten mL of sterile warm water (with the tiniest bit of surfactant?) and for each plate inoculated, the loop would be dipped into that water before streaking the plate. Or I guess a sterile syringe or pipette could be used.

If I do this, I would sterilize a 100 mL media jar containing water plus a polypropylene disposable pipette. The surfactant would be Triton X-100 or Tween 80.

Does this technique work well? Is there a name for this, or something I should search for? Should I not get the inoculation loop wet with agar, to prevent the spores clumping and not mixing with the water? Is a swab used instead of a loop to transfer from print to water?


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OfflineDoctor MarioS
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Re: Diluting spores in sterile water before plating? [Re: gone-pear-shaped]
    #27136537 - 01/09/21 12:34 AM (1 month, 26 days ago)

You can swab the print with a loop and streak the agar with it. No need to put the spores in water. When you see spores what you're actually seeing is probably billions of spores in area. Just because you can't see them doesn't mean they aren't there.


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Offlinegone-pear-shaped
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Re: Diluting spores in sterile water before plating? [Re: Doctor Mario]
    #27136736 - 01/09/21 04:13 AM (1 month, 26 days ago)

Quote:

Doctor Mario said:
You can swab the print with a loop and streak the agar with it. No need to put the spores in water. When you see spores what you're actually seeing is probably billions of spores in area. Just because you can't see them doesn't mean they aren't there.



The issue is that I got germination from very few points per plate, and that made it difficult to know whether hyphae had mated. Do you think I had a bad print or made bad agar?


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Re: Diluting spores in sterile water before plating? [Re: gone-pear-shaped] * 1
    #27136836 - 01/09/21 07:16 AM (1 month, 26 days ago)

For me adding unneeded liquid to the surface of the agar is a pia. Don't worry about spores clumping too much, just try and be more delicate when picking up spores with the loop.

Transfer any good looking growth from the germ plate and start your selecting or whatever.

in my nooby opinion I think spending too much time on agar is a fools game. You want to get fruit asap so you can clone. You can get the nicest most isolated/wahtever myc growth ever on agar, but have no idea what sort of fruit it produces.

Germinate, transfer, fruit when clean.


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OfflineDoctor MarioS
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Re: Diluting spores in sterile water before plating? [Re: gone-pear-shaped]
    #27137168 - 01/09/21 11:19 AM (1 month, 26 days ago)

As long as the print wasn't exposed to extreme temperatures for to long it should be fine. What was your agar recipe and how long did you wait for germination? It can take up to 3 weeks though I usually see something around 9 or 10 days if not sooner.

This is my favorite agar tek. You don't have to have blender or boil the water on the stove. I put the ingredients in the media bottle, add the water, and microwave it until its about to boil over. Shake the bottle real good and of theres any clumps floating around microwave it again in small bursts and then shake it again. Other than that, I do everything else the same.

For germinating spores try making the agar a bit lighter. Use 9g Agar per 500mL water instead of 10.


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Offlinegone-pear-shaped
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Re: Diluting spores in sterile water before plating? [Re: Doctor Mario]
    #27139058 - 01/10/21 07:31 AM (1 month, 25 days ago)

Quote:

Doctor Mario said:
As long as the print wasn't exposed to extreme temperatures for to long it should be fine. What was your agar recipe and how long did you wait for germination? It can take up to 3 weeks though I usually see something around 9 or 10 days if not sooner.



It was some time ago and I don't remember the agar recipe, and due to personal issues I did not continue to the next steps. But for example, this plate was the worst of it (and apologies for the condensation):

It looked as though as few as one spore germinated. So that plate couldn't be used because I couldn't tell whether it would be capable of producing a fruiting body. Does that happen sometimes, or is it indicative of something really wrong?


Edited by gone-pear-shaped (01/10/21 07:32 AM)


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