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Invisibleboom boom

Registered: 02/27/18
Posts: 429
Trouble getting germination on agar
    #25070870 - 03/17/18 12:43 PM (6 years, 13 days ago)

So I've done a number of experiments on agar at this point, and I'm struggling to get anything to grow. The few contaminants I've seen seem to stall out, which is making me think my agar isn't as "edible" as it should be. My first concern is the water content. There's enough liquid on top of the agar that tilting a plate in any direction leads to a puddle of water moving to the lowest point. This problem doesn't seem too terrible on agar plates I haven't inoculated with an MS syringe, but they still seem worse than most pictures I see of other people's agar. Just a couple drops of spore solution in this one and the agar is still swimming over a week later. I don't see how it could be possible to get spores to sit still and germinate when they're floating around in this mess. I've got some newer plates where I dug out a valley with a scalpel to put the solution in, but those valleys are completely submerged, so I'm not expecting much.



My ratio of premix to water is correct, and as far as I can tell in the next picture, the MEA has turned out crystal clear, which leads me to believe that it was mixed well.



This is an example of the only growth I've seen on any of my plates. Maybe one out of three plates so far has gotten it. It's a little black dot that sometimes seems to have some white. But they never change. This particular dot appeared over the course of a day, and now 4 days later, it still looks exactly the same. Very tiny.



It's possible (I think) that this is a contaminant that doesn't "colonize" agar in the same way that most seem to, with big, obvious growths. Or if it does, it's just not very fast. My personal theory though is that this is either a cluster of spores germinating, or a contaminant, and it just isn't getting what it needs from the agar to be able to thrive. Or it's too submerged in water to grow correctly. With that in mind, I've got a couple of ideas on how to move forward from here. If some people want to weigh in, I would appreciate it.

1. I'm looking into switching from premix to one of our agar recipe teks. RR says that he uses premix, so I figured it would be a noob friendly way to go, but I'm just not convinced it's going to work for me. I'm using jars of RAK media MEA if anyone wants to know. 1g of premix and 20ml of distilled water per plate. Maybe switching to a homemade recipe and tinkering with the ratios would be a more effective way to find something that works for me?

2. I'm using unmodified ball jars and lids, but I'm wondering if perhaps some gas exchange would allow some evaporation, which might help with the pooling issue. I could drill holes and add an SFD if that might help me out, but store bought Petri dishes don't seem to have much in the way of GE after they've been wrapped, so I can't see why it would be necessary for these jars.

3. It's possible my spore syringe froze in the mail, and the spores are no good. They were delivered into an indoor mailbox, but I don't know if they were outside in a truck or something like that while they were being shipped. It was like 10 degrees Fahrenheit here and they were shipped from Vermont, so it's possible they were exposed to some shitty weather. If that's what is up, I can order more syringes in a couple weeks when it's a little warmer and see if I get more luck with them. I'm about to dump the last bit of this syringe into grains to see if I can get more luck germinating going that route. Maybe this agar is fine, and established mycelium will have better luck colonizing the agar than the spores have had.

Beyond that I'm stumped. Any ideas?

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Invisibleaudiosnipez
Sir BIgglesworth
I'm a teapot User Gallery


Registered: 06/26/18
Posts: 166
Loc: West
Re: Trouble getting germination on agar [Re: boom boom]
    #25334386 - 07/18/18 08:10 PM (5 years, 8 months ago)

I also have had a very hard time getting any spores from my ms syringe to germinate. 

I tried premixed PDA like RR suggested and I also used a grocery store tek with honey agar and potato flakes. After weeks no germination but there is no contamination either.

I have been patient but nothing. So my next step is switching to MEA and using a lighter nutrient ratio because I heard that helps. Also I tried dropping and doing streaks.

Did you ever have any luck? I still have gotten nothing even though the same syringes did work on grains. Hmmm.

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Offlinescab eater2
Stranger

Registered: 05/02/18
Posts: 65
Last seen: 5 years, 8 months
Re: Trouble getting germination on agar [Re: audiosnipez]
    #25334494 - 07/18/18 09:05 PM (5 years, 8 months ago)

Here's a simple agar recipe that has worked for me.

1/2 cup water
1 teaspoon agar
2 teaspoons potato flakes
1 drop of karo.
1 drop food coloring.

Mix it in a little sauce pan on low until dissolved.

This makes me about 8 plates worth of agar.

I spoon a teaspoon and a half into the smallest sized jars i have. Or just enough to cover the bottom. Use the same lids for pftek and cover holes with micropore tape. Alternatively you could use the glad mini rounds in place of jars. Cover jars with foil and pc for 45 mins. I heard you can pc for 20 mins but i find 45 to work for me.

Remove from pc, remove foil and let the tops dry. inoculate with 1 drop from a syringe and wait about a week for growth.

Another trick you can do to help you out is to make 2 controls. Leave one agar plate alone untouched. Use this one to insure sterality. Take another agar plate cough in it or touch it a little. Use this plate to insure growth is possible.

1 more thing, i used this agar

https://www.amazon.com/gp/aw/d/B008SV6OLM/ref=mp_s_a_1_8_a_it?ie=UTF8&qid=1531969571&sr=8-8&keywords=Agar&dpPl=1&dpID=51MyvJjPgHL&ref=plSrch

Make sure the agar you are using does not have any added sugar or other ingredients.

Edited by scab eater2 (07/18/18 09:07 PM)

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Invisiblesandman420
Saint PP
Male


Registered: 06/17/04
Posts: 5,384
Trusted Cultivator
Re: Trouble getting germination on agar [Re: scab eater2]
    #25334515 - 07/18/18 09:14 PM (5 years, 8 months ago)

Germinating spore syringes on agar has always been a pain in my ass, I much prefer to germinate on grain and transfer to agar if all I have is a syringe. It's just way easier for me and my gorilla hands and it works every single time.


--------------------
- Sandbag Tek - How To Sterilize Spawn Bags - All About Static Pressure / Pressure Drop for DIY Flow Hoods - Sandman's LC Tek-

Marijuanaut escapes earth to cultivate - Grow-room is church temple of the new stoner breed

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Invisibleboom boom

Registered: 02/27/18
Posts: 429
Re: Trouble getting germination on agar [Re: audiosnipez]
    #25334516 - 07/18/18 09:15 PM (5 years, 8 months ago)

Quote:

audiosnipez said:
I also have had a very hard time getting any spores from my ms syringe to germinate. 

I tried premixed PDA like RR suggested and I also used a grocery store tek with honey agar and potato flakes. After weeks no germination but there is no contamination either.

I have been patient but nothing. So my next step is switching to MEA and using a lighter nutrient ratio because I heard that helps. Also I tried dropping and doing streaks.

Did you ever have any luck? I still have gotten nothing even though the same syringes did work on grains. Hmmm.




My syringe in this case was contaminated. I couldn't get it to work on grains either. Got some new syringes and suddenly had 100% germinate.

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