70 replies to this topic

[HrVanker]

Finding myself w/ some extra time and spores on my hands, I decided to get into agar. The spores were Ecuadorian and Redspore.

I found these cool little 8oz meal prep containers at the grocery store. Each has 2 shallow 4oz sections, and they seem perfect for agar work. Each section was filled w/ 25ml of agar and PCd after setting.

5 containers were inoculated w/ one drop of EQ spore solution, the other 5 w/ the Redspore, wrapped tightly w/ a 1" strip of plastic wrap, and put in the cupboard to incubate.


Possible Vectors of Contamination:
As per usual, I didn't really follow a tek. Usually I have a good idea of how things are supposed to work, then tailor a solution that fits my needs. Sometimes mistakes are made, and that definitely happened.

My big mistake was using plastic wrap in the PC. The plastic mostly disintegrated, and there was lots of condensation in the containers. When I opened the PC to check on the containers (after the pressure subsided), I quickly re-wrapped them with plastic. While inoculating, I poured off the excess water and wiped the lids w/ some lightly ISO'd paper towels to remove the condensation.

Looking forward to how this progresses.

Edited by HrVanker, 11 December 2020 - 04:17 PM.

[cujoloki]

Holy smokes they didn't melt! Good find for a Petri proxy! I use ketchup cups exclusively. Looking forward to the content.

[HrVanker]

Holy smokes they didn't melt! Good find for a Petri proxy! I use ketchup cups exclusively. Looking forward to the content.

These are PP5, same material that the nicer restaurant togo containers are made of. Ziploc and Glad containers are made from the same stuff as well!

[rockyfungus]

Jewelry PP5 cases, thrift store tupperware, any shallow PP5 gets hoarded.

 

Holy smokes they didn't melt! Good find for a Petri proxy! I use ketchup cups exclusively. Looking forward to the content.

Do your lids melt or warp?
 

Edited by rockyfungus, 11 December 2020 - 06:49 PM.

[cujoloki]

Jewelry PP5 cases, thrift store tupperware, any shallow PP5 gets hoarded.
 

Holy smokes they didn't melt! Good find for a Petri proxy! I use ketchup cups exclusively. Looking forward to the content.

Do your lids melt or warp?

A few lids will be stubborn to snap when I open pour at 150 at times.

[rockyfungus]

As per usual, I didn't really follow a tek. Usually I have a good idea of how things are supposed to work, then tailor a solution that fits my needs. Sometimes mistakes are made, and that definitely happened.

My big mistake was using plastic wrap in the PC. The plastic mostly disintegrated, and there was lots of condensation in the containers. When I opened the PC to check on the containers (after the pressure subsided), I quickly re-wrapped them with plastic. While inoculating, I poured off the excess water and wiped the lids w/ some lightly ISO'd paper towels to remove the condensation.
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I wrap just the top with foil. Like where lid hits the bottom container.So your really just covering the junction. Most of the time I invert the agar immediately so it all condenses to lid then empty in a sterile spot.

Honestly, best solution for me was sterilizing containers for 15 mins. Or if it all fits along with your agar for 40 minutes. Then pouring in a sterile environment. Pour thin and cool and you'll have no condensation.
 

[HrVanker]

I wrap just the top with foil. Like where lid hits the bottom container.So your really just covering the junction. Most of the time I invert the agar immediately so it all condenses to lid then empty in a sterile spot.

Honestly, best solution for me was sterilizing containers for 15 mins. Or if it all fits along with your agar for 40 minutes. Then pouring in a sterile environment. Pour thin and cool and you'll have no condensation.

I am aware of sterilizing the agar, then pouring. That seems like a bit of a headache to me though. However the condensation might be an equivalent trade-off.

I'm starting to see what appears to be bacteria in my dishes. It's mostly in the dishes from the spore syringe. But one or two from the print has similar looking activity. It could be from the syringe, w/ a little cross contamination. But I'm wondering about my PC as well... my new stove is electric, and it had a difficult time bringing the PC to pressure. It took 45min for the res button to pop up, and the rocker never really got going (though some pressure was steadily escaping).

For now, I'll let them go and see what happens. But you're right, I may need to adjust my approach.

[mycro]

What agar recipe did you use?

[HrVanker]

What agar recipe did you use?

Thank you for asking, you actually helped me catch another mistake... I ordered the agar off of Amazon, and was under the impression that I was ordering Malt Extract agar. But upon further investigation, what I used is generic nutrient agar that seems to be designed more for bacteria than fungi. Ughhh...

 

So it looks like I may have to start over. I'll leave these plates going for now, just to see what happens. But I'll be ordering some more suitable agar ASAP.

[rockyfungus]

Easier to buy any agar for cooking and buy MEA or light MEA and mix it on your own.

[HrVanker]

Easier to buy any agar for cooking and buy MEA or light MEA and mix it on your own.

 

Easier or more cost effective? 500g pre-mixed MEA costs about $60 on Amazon, and a pound each of agar-agar and malt extract costs around $40. At the moment, I'm leaning towards 100g of pre-mixed MEA or PDA (PDA is slightly cheaper) because it will last a few rounds and it's cheaper than buying bulk ingredients at the moment.

[rockyfungus]

I've never noticed any difference in growth from PDA, MEA, or grain water agar.

If you are just going for cost-effective, that's a great deal.

 

No clue haven't compared cost in a year or two. Check out brewer supplies for the malt. Asian grocery for agar.

Feel like my 3lb bag of malt was under $20, and the agar is maybe 35 cents at the grocery store for 25g. (damn imperial, wish it was all in grams) I never did the math because it's never broke the bank. Feel like under $50 worth of supplies has lasted me 2 years with pouring 25 plates a week.

Edited by rockyfungus, 15 December 2020 - 10:23 PM.

[cujoloki]

I use PDA homemade. The mycelium loves it. I make a sawdust supplemented PDA for woodlovers. Its white oak. I saw it myself. Potatoes are a few cents. Pretty cost effective for me. Has to be that way b/c well it has to be for me to partake in this hobby.

[HrVanker]

So I ended up buying some agar, LME, and the short 4oz mason jars so that I don't have to pour. Hopefully I'll have some more updates by Monday.

[HrVanker]

And here we go with batch no. 2! PC'd 20 jars, each w/ 10ml of LME agar. After they came out of the PC and started to gel up, I gave them a swish which seems to have remedied the small amount of condensation on the walls of the jars, but the lids still have some. For now, I'll leave them upside-down and get rid of the excess moisture when I inoculate.

The recipe was equal parts agar and LME, the smallest bit of pressure extracted coffee grounds, and a tiny bit of gypsum powder (to offset any PH changes from the coffee).

I'm excited for this run, but I'm also excited for future agar recipes. I'm thinking about trying just a plain WBS and agar mix next. Maybe the runoff from boiling WBS, some finally powdered WBS mixed in, or maybe both to try side-by-side.

[jrh]

Oooh, pretty. What's going in there?

 

If you're experimenting with agar recipes, in my extremely limited experience, cubes like nutritional yeast. I use it at .1 g nutritional yeast per 1 g agar agar. For example, for poured agar I use 8 g agar agar, 8 g LME, .8 g nutritional yeast, and 400 ml water. Mine came in flakes like fish food, so I blitzed it into a powder. Be warned that it doesn't dissolve, though, so you won't end up with a perfectly clear agar like you just made.

[HrVanker]

I've heard about using nutritional yeast. And I may just do that one day. But I'm more curious about using Cattatives™ as the primary nutrition source for agar. The idea of allowing self-selection for the type of substrate that will be used when fruiting makes a lot of sense to me. Hence my desire to try using WBS based agar.

But we'll see what happens. I still have to use all these jars first!

[HrVanker]

Okay, here's an update:

Jars were 'nocced up on 12/21. The first bits of mycelium started to show in the last 2 days.

6 jars were made using a Redspore print from back in July. They are all starting to show some nice clean growth, the biggest is almost dime-sized. No visible contaminations yet.

I also made 9 jars from a reliable GT syringe I ordered back in March. Each jar made from the syringe has at least one bacterial contamination. However they all have some mycelium growing, and it looks like I'll be able to clean them up eventually.

When I grew out the Redspore back in June/July, each spawn jar also had some bacteria going on. Now I'm certain that it had nothing to do with my technique, since this GT from the same order also went bacterial and the print jars look fine. I still have an Ecuador and a Dutch King syringe to go, but I wish I had some Maz...

I also have a few experimental jars of WBS agar! I took some WBS and ran it through a blender until it was a pretty uniform consistency. I used the same recipe as the LME agar but substituted the LME w/ WBS. On my next batch I plan to strain the WBS water before adding the agar, and using half the amount of WBS. Here is a pic of GT WBS agar jar:

Edited by HrVanker, 30 December 2020 - 08:01 PM.

[rockyfungus]

The ones that went bacterial are from syringes?

Did you go from syringe directly to agar, or use a loop?

[HrVanker]

The ones that went bacterial are from syringes?

Did you go from syringe directly to agar, or use a loop?

Yeah, the only ones that contaminated were from the syringe. I did go directly from the syringe, some got a whole drop and some got less. I really don't know that a loop would make a difference since it would still transfer some bacteria from the syringe.

 

I'm thinking that in my next go, I'll add some fresh peroxide to the spore solution and see what happens.