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Offlinehallmaker
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First Agar try, need help with results
    #26515604 - 03/03/20 10:50 PM (3 years, 11 months ago)

OK, so I pretty much went with C10's Agar guide. The only difference is that I did not have a microwave. Instead, I place the ingredients in a beaker and placed the beaker in boiling water for 15+ minutes. The mixture never did boil. I had 2 different recipes:
MEA 10g + Agar 10g + fill beaker to 500 ml water
PEA 10g potato flakes + 10g Agar + dash of sugar + fill to 500 ml water

I did this for 2 varieties: B+ (spores were in water\needle. I drained some into a small bowl and used a loop) and Jamaican Pans Cyans (spore were from print, just used a loop).

So the B+ looked promising right away with whites streaks where I used the loop, but then they seemed to stall and most got yellowish.

The Pans did not do anything at first and I kinda gave up on them. But now I see big white patches. Not sure if this is a contamination or growth?

The good thing is I do not see a lot of contamination, if at all. But then I am not 100% of what I am looking at. My dishes seem pretty clear, so that is a bonus.

I included pics of the potato flakes and malt I used.

Can someone help me as to why my B+ stalled, if my Pans are contams and if not microwaving my mixture had anything to do with the results? Thanks for the help.




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OfflineHaywire
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Re: First Agar try, need help with results [Re: hallmaker]
    #26515795 - 03/04/20 03:41 AM (3 years, 11 months ago)

to me it seems all your plates are contaminated.
did you sterilize your agar at all?


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OfflineRandalf the Grey
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Re: First Agar try, need help with results [Re: Haywire]
    #26515810 - 03/04/20 04:00 AM (3 years, 11 months ago)

:confused: :whathesaid:
May be a dumb question but did you PC and for how long?
All the B+ plates are loaded with bacterial contams for sure. The pan plates may have some myc growth (myc that you're looking for) but I'm 99% they have company in there as well. I'm not experienced with pans, trying to revive and old MSS myself, but that looks way too puffy to me.

Side note: no need to drain syringe into bowl. That's probably where all your bacteria came from. Just put a drop or two into your plate, then either tilt the plate, or spread it with the loop.


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OfflineNeowynd8
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Re: First Agar try, need help with results [Re: hallmaker]
    #26515926 - 03/04/20 06:15 AM (3 years, 11 months ago)

I think you can reliably sterilize agar by simply boiling it, but you mentioned your agar itself never came to a boil which is concerning. Many people sterilize for 30-45 mins even with a PC so you could up the cook time and make sure agar boils.

The contam pattern looks like its mostly concentrated in areas that you streaked with a loop? Which makes me think your inoculation loops wasn't sterile or your spore solution is straight bacteria lol.

I always let the needle cool a few secs after flame sterilizing and then drop 4-5 drops on the SAB floor assuming they may have gotten fried then straight to the plate.


Edited by Neowynd8 (03/04/20 06:22 AM)


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OfflineFrankRhizo
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Re: First Agar try, need help with results [Re: Randalf the Grey]
    #26515937 - 03/04/20 06:25 AM (3 years, 11 months ago)

I think I had problems after I sterilized my agar and microwaved it after.


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Offlinehallmaker
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Re: First Agar try, need help with results [Re: FrankRhizo]
    #26515946 - 03/04/20 06:36 AM (3 years, 11 months ago)

I pressure cooked at 10 psi for 1 hour. I sterilized my loop by flame and cooled it in the agar. I used a zig-zag motor to apply the spores. Are contams white? I am having a hard time telling what is contams and what is myc. Thanks for any help


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OfflineNeowynd8
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Re: First Agar try, need help with results [Re: hallmaker]
    #26515952 - 03/04/20 06:45 AM (3 years, 11 months ago)

Yes, contams mostly molds Id say can be white/green/prolly every color. Molds are fungi also and form mycelium as mushrooms do which is prolly why they are so deceptive. I'd say bacteria in general is quite distinctive from mold. There is a thread on what things should and shouldn't look like on here somewhere.


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OfflineThe Mycologist
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Re: First Agar try, need help with results [Re: hallmaker]
    #26515953 - 03/04/20 06:47 AM (3 years, 11 months ago)

The cloud looking stuff is mycelium. But there are nasties there too.

Its gonna take some transfers to get it clean.

If its not white or not cloud like the it is a contam.


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OfflineNeowynd8
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Re: First Agar try, need help with results [Re: The Mycologist]
    #26515962 - 03/04/20 06:54 AM (3 years, 11 months ago)

After reading your post again, I may have spotted a major error in your technique. Did you squirt spore solution in a bowl and then dip your loop in that to grab spores? If so, never do that. I'm betting you did cuz all the plates from solution are trash and ones from print are much better.

Drop a drop in the plate from syringe then streak with loop or drop a drop on the loop and take it to plate.


Edited by Neowynd8 (03/04/20 06:57 AM)


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OfflineMagic Mushpoon
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Re: First Agar try, need help with results [Re: Neowynd8]
    #26516671 - 03/04/20 02:55 PM (3 years, 11 months ago)

Anything that's not white are contams. Contams can also be white, but mycelium can only be white, so if it's not white it's contams. Like Neo said, you don't want to put the liquid from the syringe into anything else before you inoculate. If using a syringe to agar, go straight from the syringe or contams are inevitable.


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