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krock
Krockodile

Registered: 12/18/19
Posts: 154
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On agar; if the spores germinate will they grow past the staph?
#26389054 - 12/18/19 04:09 PM (4 years, 1 month ago) |
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Hi, I'm a long time lurker.
13 days ago I streaked two MEA plates with two different PC'd bamboo skewers. MEA recipe: (ME: 10g A: 10g Water: 500ml)
After a day or two the staph (i think) became visible. And the staph grew for a few days but growth of staph stagnated after 5 days.
Agar has been kept in temperature 19.5 to 20 C
I got the ms syringe (am+ from a smartshop) 8 months ago. It is slightly old because I wanted to wait until I got some more experience with cultivation before using it. I've been through clone -> agar -> rye grain -> wood shavings with a store bought king oyster that was starting to rot, which unfortunately only fruited really mutated rosecomb blobs. But I feel ready to do spores now.
My question is: If the spores were to germinate (which I hope they do soon), will the staph stop the mycelium from spreading?
And also if they don't, do you think trying again on activated charcoal agar would be a good idea?
I saw RR suggested about 10 g charcoal to 1 liter. So I would probably try something like that.
Here are some pics:

Edited by krock (12/18/19 04:18 PM)
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LadysKnight
Hello Ladies


Registered: 10/09/15
Posts: 1,670
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Re: On agar; if the spores germinate will they grow past the staph? [Re: krock]
#26389229 - 12/18/19 05:38 PM (4 years, 1 month ago) |
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In my experience, if the spores don't germinate around the same time as the contamination, any contam, then they won't at all.
I've used activated charcoal agar, with poor results. IME, the activated charcoal inhibits mycelial growth and spore germination.
Brf plates or pucks is the answer to a clean culture. Use it like agar to inhibit contam growth.
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krock
Krockodile

Registered: 12/18/19
Posts: 154
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Re: On agar; if the spores germinate will they grow past the staph? [Re: LadysKnight]
#26389985 - 12/19/19 03:30 AM (4 years, 1 month ago) |
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Thanks for sharing about your experience with charcoal! I thought it could be a good idea for germinating old spores.
I don't have any brf right now. But I've also read about someone putting a single grain from a prepped and PC'd grain jar on agar and then spore solution onto the grain. Would that be a good alternative to the brf plates/pucks in this context?
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LadysKnight
Hello Ladies


Registered: 10/09/15
Posts: 1,670
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Re: On agar; if the spores germinate will they grow past the staph? [Re: krock]
#26390034 - 12/19/19 04:27 AM (4 years, 1 month ago) |
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That plan with the single grain has the same chance of failure as plain agar. You're placing a contaminated liquid on a nutritious medium that is prone to contamination. BRF is much less prone to contaminate than either agar or grain.
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krock
Krockodile

Registered: 12/18/19
Posts: 154
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Re: On agar; if the spores germinate will they grow past the staph? [Re: LadysKnight]
#26390133 - 12/19/19 06:15 AM (4 years, 1 month ago) |
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ok, thank you! I will obtain some brf and try that
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krock
Krockodile

Registered: 12/18/19
Posts: 154
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Re: On agar; if the spores germinate will they grow past the staph? [Re: krock]
#26691877 - 05/24/20 06:20 AM (3 years, 8 months ago) |
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Ok so it has been a while, but I have been trying to make progress on this project. With little luck.
First of all this is what the plates look like after 5 months:
 The staph hasn't grown at all, and still no sign of cube mycelium. One of the plates recently got contaminated. Probably in the last 3 weeks.
I tried making some pucks as advised.
However I think I made the mixture a bit too wet. So there hasn't been much growth, but there is something on the pucks that kind of looks like mycelium.

Do you think it would be a good move to transfer those circled spots to agar?
1 month ago I also tried my hand making some classic brf cakes that I inoculated with different amounts of spore solution. These are half filled half pint jars:
 Solutions: 0.1 ml, 0.2 ml, 0.2 ml and 0.3ml. I've read that one doesn't really need much solution to get brf going, but maybe I used too little?
 One of the cakes, the one with 0.1 ml solution got contaminated.
Finally one of the reasons I think why this doesn't work is that maybe I killed my spores?
 In my country spores are not legal to own for microscopy use... When I took the spore solution home from abroad I put it in a small vodka flask for better stealth. The flask contained 40% abv vodka that I emptied right before I put the spore solution in it (thinking that this would prevent contamination). This might've been a stupid move, but at the same time I've not read anywhere that 40% effectively kills spores. I've read that around 70% is what effectively kills spores. Still this solution has been resting with the remains of the bottle for over 1 year now.
What should I do now? I really want to grow some cubes!
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krock
Krockodile

Registered: 12/18/19
Posts: 154
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Re: On agar; if the spores germinate will they grow past the staph? [Re: krock]
#26694709 - 05/25/20 10:50 AM (3 years, 8 months ago) |
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Am I wasting time with something that will never come to life?
Would it be better to risk ordering a paper print and start over?
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Roger Clemency
Smile


Registered: 03/23/20
Posts: 2,005
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Re: On agar; if the spores germinate will they grow past the staph? [Re: krock] 1
#26694754 - 05/25/20 11:15 AM (3 years, 8 months ago) |
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I would absolutely order a print. I personally hate syringes. I have one right now because a variety I wanted wasn’t available in print, only syringe. My first test drop on two plates was pure bacteria so I know I’ll have some work to do so I’ve just left it be for now.
Prints are definitely the way.
-------------------- Sour grapes, sweet revenge Heaven starts right where hell ends
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krock
Krockodile

Registered: 12/18/19
Posts: 154
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Re: On agar; if the spores germinate will they grow past the staph? [Re: Roger Clemency]
#26695128 - 05/25/20 02:29 PM (3 years, 8 months ago) |
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Yeah this'll probably be the last time I try a spore syringe. I've done a few gourmet grows, cloning from supermarket fruits to agar with good contamination free results. I wonder why using a spore syringe has to be so hard. I hope using a print will work better for me. What agar hardness do you use for spore germination?
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Roger Clemency
Smile


Registered: 03/23/20
Posts: 2,005
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Re: On agar; if the spores germinate will they grow past the staph? [Re: krock]
#26695178 - 05/25/20 03:02 PM (3 years, 8 months ago) |
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The last batch I made was probably pretty standard but worked well. 10g agar, 10g LME to 500ml water is a recipe I see a lot. I have a 350 ml bottle I use and I went 8g agar I think and 6g lme. It’s in my journal under random info I think. I’m going to try a little thinner mix next time for LI plates but it might work even better for germ plates. Though 2-3 day germination is about as good as I could hope for anyway
-------------------- Sour grapes, sweet revenge Heaven starts right where hell ends
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