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Invisiblebrindle foxx
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Issues with spores and swabs
    #26809551 - 07/07/20 05:43 PM (3 years, 7 months ago)

I keep trying to put different spores onto agar via innoc loop and shaving off the print onto the agar. Pressing in and streaking. Seems a contam will form elsewhere and nothing happens bear the spores.

Same issue with APE swab where I rub and rub on the agar surface and nothing.
Even tried clipping the swab and sticking it into a hole in the center of plate still nothing.

Do I need to add water or activate these spores a different way? Possibly go to spore syringe and drop that on agar and clean it up?


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Offlinepolaritymind
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Re: Issues with spores and swabs [Re: brindle foxx]
    #26809680 - 07/07/20 06:52 PM (3 years, 7 months ago)

Is this all one spore print youre talking about? Sounds a bit like old and dead spores to me, or maybe your tools are too hot from flame sterilizing and kill the spores. In that case cool them in the target plate first.
Or a method I recently learned about it using a swab in a shotglass with water covered in cling wrap, microwaved, cooled in the SAB and then use the wet swab, that also transports some more spores than physical rubbing. But tbh I usually found that spores are small enough that even if it looks like nothing is on the tool there still usually is growth after.

What I also used to do is scrape the spores onto the plate, letting the black flakes drop directly onto the oponed plate. This is very excessive and not neccesary though, just another idea.


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OfflineDoyledozo
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Re: Issues with spores and swabs [Re: polaritymind]
    #26809725 - 07/07/20 07:12 PM (3 years, 7 months ago)

I've never gotten spores from a swab to terminate from a dry streak. Also don't have much luck taking dry spores off a print with a loop. I got myself a box of sterile swabs. With those I can streak with spores from a syringe or dampen it with condensation from my receiving plate and pick up a small bit from a print. For spore swabs I use flamed tweezers to pluck a tuft of cotton and push it into the agar. You may want to use a softer agar mix for germ plates.


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Invisiblesandman420
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Re: Issues with spores and swabs [Re: Doyledozo]
    #26809754 - 07/07/20 07:23 PM (3 years, 7 months ago)

Yea softer agar for stubborn spores. Contamming in other places sounds like you're contaminating your plates during inoc with sloppy SAB procedures. The presence of the contams may somehow not allow the spores to germinate maybe.

When I use a loop I do not streak it much, only like a 1/4" little dab because spores tend to glob up on it and not really streak right anyway. No pressing in to pierce the agar or anything not sure if I misunderstood that.


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Edited by sandman420 (07/07/20 07:23 PM)


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Invisiblebrindle foxx
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Re: Issues with spores and swabs [Re: polaritymind]
    #26809952 - 07/07/20 09:31 PM (3 years, 7 months ago)

Quote:

polaritymind said:
Is this all one spore print youre talking about? Sounds a bit like old and dead spores to me, or maybe your tools are too hot from flame sterilizing and kill the spores. In that case cool them in the target plate first.
Or a method I recently learned about it using a swab in a shotglass with water covered in cling wrap, microwaved, cooled in the SAB and then use the wet swab, that also transports some more spores than physical rubbing. But tbh I usually found that spores are small enough that even if it looks like nothing is on the tool there still usually is growth after.

What I also used to do is scrape the spores onto the plate, letting the black flakes drop directly onto the oponed plate. This is very excessive and not neccesary though, just another idea.



I flame the loop and stick it in agar for a second to cool it down before scratching the print and we are talking about 4 different spores I’m trying so it must be my technique. I have the APE on a swab and am gonna try to pluck the cotton with flamed and cooled in the agar tweezers. Shouldn’t spores in theory be just fine falling on the agar dry or being on the surface dry and do their thing? I seem to be able to be fine with anything else but the spore. Agar culture,slants,syringes, tissue, LC but spore to agar seems to be my weakness.. I thought maybe my agar was the issue so I even ordered plates from elsewhere and same luck.


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Offlinepolaritymind
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Re: Issues with spores and swabs [Re: brindle foxx]
    #26810233 - 07/08/20 03:24 AM (3 years, 7 months ago)

Quote:

Shouldn’t spores in theory be just fine falling on the agar dry or being on the surface dry and do their thing?



I mean I think this depends on how dense or soft your agar is, like the previous posters say it needs to be soft enough so the water from the plate can soak into the spores, that seems intuitively obvious to me.
I honestly have never really varied my agar recipe, always use this one, maybe modifying it what would be a softer agar, how many g specifically? Gotta say I'd be woried just having a liquid slush ind the end and would be annoyed to have to do tthe experimentation first to find the numbers.
Quote:

broth from boiling 150 grams sliced potatoes
in 500ml water for 30 minutes(add water to 500ml)
(You can use 5 g of instant potato flakes instead)
9 g agar agar
7 grams dextrose( = glucose, or 10 ml honey or corn syrup )
1 gram brewers yeast or yeast-extract (optional)





--------------------
"to affirm life is to also affirm death"
-Albert hofmann


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